U.S. patent application number 15/365239 was filed with the patent office on 2017-06-01 for methods and compositions of chondrisomes.
The applicant listed for this patent is FLAGSHIP VENTURES MANAGEMENT, INC.. Invention is credited to David Chess, Jacob Feala, Kiana Mahdaviani, Michael Mee, John Miles Milwid, Jacob Rosenblum Rubens, Kyle Trudeau, Geoffrey A. von Maltzahn.
Application Number | 20170151287 15/365239 |
Document ID | / |
Family ID | 58777827 |
Filed Date | 2017-06-01 |
United States Patent
Application |
20170151287 |
Kind Code |
A1 |
von Maltzahn; Geoffrey A. ;
et al. |
June 1, 2017 |
METHODS AND COMPOSITIONS OF CHONDRISOMES
Abstract
Therapeutic chondrisome compositions and related methods are
described.
Inventors: |
von Maltzahn; Geoffrey A.;
(Somerville, MA) ; Milwid; John Miles;
(Winchester, MA) ; Mee; Michael; (Boston, MA)
; Rubens; Jacob Rosenblum; (Cambridge, MA) ;
Chess; David; (Waltham, MA) ; Trudeau; Kyle;
(Boston, MA) ; Mahdaviani; Kiana; (Chestnut Hill,
MA) ; Feala; Jacob; (Franklin, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FLAGSHIP VENTURES MANAGEMENT, INC. |
Cambridge |
MA |
US |
|
|
Family ID: |
58777827 |
Appl. No.: |
15/365239 |
Filed: |
November 30, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62261157 |
Nov 30, 2015 |
|
|
|
62261169 |
Nov 30, 2015 |
|
|
|
62261170 |
Nov 30, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/14 20130101;
A61K 35/34 20130101; A61K 35/35 20130101; C12N 15/87 20130101; G01N
33/15 20130101; A61K 35/12 20130101; A61K 38/1709 20130101; A61K
35/33 20130101; A61P 3/00 20180101; A61K 35/19 20130101; A61K
9/0029 20130101 |
International
Class: |
A61K 35/35 20060101
A61K035/35; A61K 35/14 20060101 A61K035/14; A61K 35/19 20060101
A61K035/19; C12N 15/87 20060101 C12N015/87; A61K 38/17 20060101
A61K038/17; A61K 48/00 20060101 A61K048/00; A61K 35/33 20060101
A61K035/33; G01N 33/15 20060101 G01N033/15; A61K 35/34 20060101
A61K035/34; A61K 47/46 20060101 A61K047/46 |
Claims
1. A pharmaceutical composition comprising a preparation of
chondrisomes isolated from a mitochondrial source and having one or
more of the following characteristics: a. the chondrisomes of the
preparation have a mean average size between 150-1500 nm; b. the
chondrisomes of the preparation have a polydispersity (D90/D10)
between 1.1 to 6; c. outer chondrisome membrane integrity wherein
the preparation exhibits <20% increase in oxygen consumption
rate over state 4 rate following addition of reduced cytochrome c;
d. complex I level of 1-8 mOD/ug total protein; e. complex II level
of 0.05-5 mOD/ug total protein; f. complex III level of 1-30 mOD/ug
total protein; g. complex IV level of 4-50 mOD/ug total protein; h.
genomic concentration 0.001-2 mtDNA ug/mg protein; i. membrane
potential of the preparation is between -5 to -200 mV.
2. A pharmaceutical composition comprising a preparation of
chondrisomes isolated from a mitochondrial source and having one or
more of the following characteristics: a. a protein carbonyl level
of less than 100 nmol carbonyl/mg chondrisome protein. b. <20%
mol/mol ER proteins c. >5% mol/mol mitochondrial proteins
(MitoCarta); d. >0.05% mol/mol of MT-CO2, MT-ATP6, MT-ND5 and
MT-ND6 protein; e. Genetic quality >80%; f. Relative ratio
mtDNA/nuclear DNA >1000; g. Endotoxin level <0.2 EU/ug
protein; h. Substantially absent exogenous non-human serum.
3. A pharmaceutical composition comprising a preparation of
chondrisomes isolated from a mitochondrial source and having one or
more of the following characteristics: a. Glutamate/malate RCR 3/2
of 1-15; b. Glutamate/malate RCR 3/4o of 1-30; c.
Succinate/rotenone RCR 3/2 of 1-15; d. Succinate/rotenone RCR 3/4o
of 1-30; e. complex I activity of 0.05-100 nmol/min/mg total
protein; f. complex II activity of 0.05-50 nmol/min/mg total
protein; g. complex III activity of 0.05-20 nmol/min/mg total
protein; h. complex IV activity of 0.1-50 nmol/min/mg total
protein; i. complex V activity of 1-500 nmol/min/mg total protein;
j. reactive oxygen species (ROS) production level of 0.01-50 pmol
H2O2/ug protein/hr; k. Citrate Synthase activity of 0.05-5
mOD/min/ug total protein; l. Alpha ketoglutarate dehydrogenase
activity of 0.05-10 mOD/min/ug total protein; m. Creatine Kinase
activity of 0.1-100 mOD/min/ug total protein; n. Pyruvate
dehydrogenase activity of 0.1-10 mOD/min/ug total protein; o.
Aconitase activity of 0.1-50 mOD/min/ug total protein; p. Maximal
fatty acid oxidation level of 0.05-50 pmol O2/min/ug chondrisome
protein; q. Palmitoyl carnitine & Malate RCR3/2 state 3/state 2
respiratory control ratio (RCR 3/2) of 1-10; r. electron transport
chain efficiency of 1-1000 nmol O2/min/mg protein/.DELTA.GATP (in
kcal/mol)
4. A pharmaceutical composition comprising a preparation of
chondrisomes isolated from a mitochondrial source and having one or
more of the following characteristics: a. total lipid content of
50,000-2,000,000 pmol/mg; b. double bonds/total lipid ratio of
0.8-8 pmol/pmol; c. phospholipid/total lipid ratio of 50-100
100*pmol/pmol; d. phosphosphingolipid/total lipid ratio of 0.2-20
100*pmol/pmol; e. ceramide content 0.05-5 100*pmol/pmol total
lipid; f. cardiolipin content 0.05-25 100*pmol/pmol total lipid; g.
lyso-phosphatidylcholine (LPC) content of 0.05-5 100*pmol/pmol
total lipid; h. Lyso-Phosphatidylethanolamine (LPE) content of
0.005-2 100*pmol/pmol total lipid; i. Phosphatidylcholine (PC)
content of 10-80 100*pmol/pmol total lipid; j.
Phosphatidylcholine-ether (PC O-) content 0.1-10 100*pmol/pmol
total lipid; k. Phosphatidylethanolamine (PE) content 1-30
100*pmol/pmol total lipid; l. Phosphatidylethanolamine-ether (PE
O-) content 0.05-30 100*pmol/pmol total lipid; m.
Phosphatidylinositol (PI) content 0.05-15 100*pmol/pmol total
lipid; n. Phosphatidylserine (PS) content 0.05-20 100*pmol/pmol
total lipid; o. Sphingomyelin (SM) content 0.01-20 100*pmol/pmol
total lipid; p. Triacylglycerol (TAG) content 0.005-50
100*pmol/pmol total lipid; q. PE:LPE ratio 30-350; r. PC:LPC ratio
30-700; s. PE 18:n (n>0) content 0.5-20% pmol AA/pmol lipid
class; t. PE 20:4 content 0.05-20% pmol AA/pmol lipid class; u. PC
18:n (n>0) content 5-50% pmol AA/pmol lipid class; v. PC 20:4
content 1-20%
5. A pharmaceutical composition comprising a preparation of
chondrisomes isolated from a mitochondrial source and having one or
more of the following characteristics: a. Increases basal
respiration of recipient cells at least 10%; b. Chondrisomes of the
preparation are taken up by at least 1% of recipient cells; c.
Chondrisomes of the preparation are taken up and maintain membrane
potential in recipient cells; d. Chondrisomes of the preparation
persist in recipient cells at least 6 hours; e. Decrease cellular
lipid levels of recipient cells at least 5%; f. increases uncoupled
respiration of recipient cells at least 5%; g. decreases
mitochondrial permeability transition pore (MPTP) formation in
recipient cells at least 5% and does not increase more than 10%; h.
increases Akt levels in recipient cells at least 10%; i. decreases
total NAD/NADH ratio in recipient cells at least 5%; j. Reduces ROS
levels in recipient cells at least 5%.
6. A pharmaceutical preparation described herein, further having
one or more of the following characteristics: a. Increases
fractional shortening in subject with cardiac ischemia at least 5%;
b. Increases end diastolic volume in subject with cardiac ischemia
at least 5%; c. decreases end systolic volume in subject with
cardiac ischemia at least 5%; d. decreases infarct area of ischemic
heart at least 5%; e. increases stroke volume in subject with
cardiac ischemia at least 5%; f. increases ejection fraction in
subject with cardiac ischemia at least 5%; g. increases cardia
output in subject with cardiac ischemia at least 5%; h. increases
cardiac index in subject with cardiac ischemia at least 5%; i.
decreases serum CKNB levels in subject with cardiac ischemia at
least 5%; j. decreases serum cTnI levels in subject with cardiac
ischemia at least 5%; k. decreases serum hydrogen peroxide in
subject with cardiac ischemia at least 5%.
7. A pharmaceutical preparation comprising isolated, modified
chondrisomes derived from a cellular source of mitochondria.
8. The pharmaceutical preparation of claim 7, wherein the
modification arises from exposing the cellular source of
mitochondria to an external stress condition or agent.
9. The pharmaceutical preparation of claim 7, wherein the
chondrisomes are engineered to overexpress or knock-down or
knock-out an endogenous gene product.
10. The pharmaceutical preparation of claim 7, wherein the
chondrisomes are engineered to express a heterologous gene
product.
11. The pharmaceutical preparation of claim 7, wherein the
chondrisomes are loaded with a heterologous cargo agent.
12. The pharmaceutical preparation of claim 7, wherein the
chondrisomes are engineered to express a protein at least 85%, 90%,
95%, 97%, 98%, 99%, 100% identical to the sequence of human UCP1
(SEQ ID NO:1), UCP2 (SEQ ID NO:2), UCP3 (SEQ ID NO:3), UCP4 (SEQ ID
NO:4), UCP5 (SEQ ID NO:5), SIRT3 (SEQ ID NO:7), pyruvate
dehydrogenase kinase (SEQ ID NO:8).
13. The pharmaceutical preparation of claim 7, wherein the
chondrisomes are loaded with an exogenous agent selected from a
nucleic acid, a polypeptide, or chemical compound.
14. The pharmaceutical composition of any one of claims 7-13,
wherein the composition has one or more of the following properties
recited in claims 1-6:
15. A pharmaceutical composition formulated for administration to a
mammal, comprising a preparation of chondrisomes isolated from a
mitochondrial source, wherein the chondrisomes of the preparation
express an uncoupling protein.
16. A pharmaceutical preparation described in any preceding claim,
wherein the preparation does not produce an unwanted immune
response in a recipient mammal.
17. A pharmaceutical preparation described in any preceding claim,
wherein the preparation is stable for at least 6 hours, 12 hours,
24 hours, 48 hours, 72 hours, 96 hours, 5 days, 7 days, 10 days, 14
days, 21 days, 30 days at a temperature of 4.degree. C. or
less.
18. A pharmaceutical preparation described in any preceding claim,
wherein the chondrisomes are encapsulated.
19. A pharmaceutical preparation described in any preceding claim,
wherein the source of the mitochondria is selected from epithelial,
connective, muscular, or nervous tissue.
20. A pharmaceutical preparation described in any preceding claim,
configured for: parenteral delivery.
21. A method of preparing a chondrisome preparation, comprising:
(a) providing a tissue or cellular source of mitochondria; (b)
manipulating (e.g., dissociating or stimulating) the tissue or
cellular source to produce a subcellular composition; (c)
separating the subcellular composition into a cellular debris
fraction and a chondrisome enriched fraction, (d) separating the
chondrisome-enriched fraction into a fraction containing
chondrisomes and a fraction substantially lacking chondrisomes, (e)
suspending the fraction containing chondrisomes in a solution,
thereby preparing a chondrisome preparation.
22. The method of claim 21, wherein the dissociating comprises
applying to the tissue or cellular source a first shear force
followed by a second, higher shear force.
23. The method of claim 21, wherein the first shear force is
applied by douncing and the second shear force is applied by
passing the homogenate through a needle.
24. The method of claim 21, wherein one or both of the separating
steps (c) and (d) comprises differential centrifugation.
25. The method of claim 21, wherein one or both of the separating
steps (c) and (d) comprises differential size filtration.
26. The method of claim 21, wherein the dissociating step is
performed in no more than 10_fold the volume of buffer relative to
the volume of the tissue or cellular source.
27. The method of claim 21, wherein the fraction containing
mitochondria is suspended in no more than 10-fold the volume of
buffer relative to the volume of the solid fraction containing
mitochondria.
28. The method of claim 21, wherein the dissociating is performed
in the absence of an exogenous protease.
29. The method of claim 21, wherein the yield of the preparation is
>0.05 ug protein/10E6 cells.
30. The method of claim 21, wherein the yield of the preparation is
>100 ug protein/g tissue.
31. The method of claim 21, wherein the yield of the preparation is
1E9 to 9E12 particles/mg total protein.
32. The method of claim 21, wherein the solution of step (e)
comprises one or more of: an osmotic modulator, a pH buffer, a
salt.
33. The method of claim 21, wherein the tissue or cellular source
of mitochondria is exposed to one or more modulator before or
during the preparation.
34. The method of claim 33, wherein the modulator is a
mitochondrial biogenesis agent.
35. The method of claim 33, wherein the modulator is a modulator of
metabolic activity.
36. The method of claim 33, wherein the modulator is hypoxia or
hyperoxia.
37. The method of claim 33, wherein the modulator is one or more
of: a temperature change, a drug, a metabolite, an energy source, a
stressor.
38. A pharmaceutical composition made by a method of any one of
claims 21-37.
39. A method of delivering a chondrisome preparation to a subject
in need thereof, comprising: administering to the subject a
pharmaceutical composition or chondrisome preparation of any one of
claims 1-20.
40. The method of claim 39, wherein the administration is for a
time and in an amount sufficient to enhance a cell or tissue
function in the subject; for a time and in an amount sufficient to
improve function of an injured or diseased cell or tissue in the
subject; for a time and in an amount sufficient to increase
mitochondrial content and/or activity in a cell or tissue of the
subject.
41. The method of claim 39, wherein the administration is for a
time and in an amount sufficient to effect a biological function
described herein.
42. A method of delivering a chondrisome preparation to a mammalian
cell or tissue ex vivo, comprising contacting the cell or tissue
with a pharmaceutical composition or chondrisome preparation of any
one of claims 1-20.
43. The method of claim 42, wherein the preparation is delivered to
cultured cells, to an isolated or cultured tissue, or to an
isolated or cultured organ.
44. The method of claim 42, wherein the contacting is for a time
and in an amount sufficient to enhance the cell or tissue function;
for a time and in an amount sufficient to improve function of an
injured or diseased cell or tissue; for a time and in an amount
sufficient to increase mitochondrial content and/or activity in the
cell or tissue.
45. The method of claim 42, wherein the administration is for a
time and in an amount sufficient to effect a biological function
described herein.
46. A method of enhancing function of a target cell or tissue,
comprising delivering to the target cell or tissue a pharmaceutical
composition or chondrisome preparation of any one of claims
1-20.
47. The method of claim 46, wherein the target cell or tissue is in
an injured state.
48. The method of claim 46, wherein the composition is delivered to
the target cell or tissue in-vivo in a human subject.
49. The method of claim 46, wherein the composition is delivered to
the target cell or tissue ex-vivo.
50. A method of delivering a mitochondrial preparation to a subject
in-vivo, comprising delivering to the subject a pharmaceutical
composition or chondrisome preparation of any one of claims
1-20.
51. A method of delivering a mitochondrial preparation to a cell or
tissue ex-vivo, comprising delivering to the target cell or tissue
a pharmaceutical composition or chondrisome preparation of any one
of claims 1-20.
52. A method of increasing mitochondrial content and/or activity in
a target cell or tissue, comprising delivering to the target cell
or tissue a pharmaceutical composition or chondrisome preparation
of any one of claims 1-20.
53. The method of claim 52, wherein the composition is delivered to
the target cell or tissue in-vivo in a human subject.
54. The method of claim 52, wherein the composition is delivered
ex-vivo to a human target cell or tissue.
55. A method of increasing tissue ATP levels, comprising delivering
to a target cell or tissue a pharmaceutical composition or
chondrisome preparation of any one of claims 1-20.
56. The method of claim 55, wherein the composition is delivered to
the target cell or tissue in-vivo in a human subject.
57. The method of claim 55, wherein the composition is delivered
ex-vivo to a human target cell or tissue.
58. A method of delivering a payload to a subject in need thereof,
comprising administering to the subject a pharmaceutical
composition or chondrisome preparation described herein, wherein
the chondrisomes of the composition comprise the payload.
59. The method of claim 58, wherein the payload is a nucleic acid,
a polypeptide or a small molecule (e.g., an agent listed in Table
4).
60. A method of increasing thermogenesis in a target cell or
tissue, comprising delivering to the target cell or tissue a
pharmaceutical composition or chondrisome preparation of any one of
claims 1-20.
61. The method of claim 60, wherein the target cell or tissue is
adipocyte tissue of a mammalian subject.
62. The method of claim 60, wherein the adipocyte tissue is white
adipocyte tissue.
63. A method of reducing fat tissue mass in the fat tissue of a
subject, comprising: administering to the fat tissue of the subject
a pharmaceutical composition or chondrisome preparation of any one
of claims 1-20.
64. A method of decreasing triglyceride or serum cholesterol levels
in a subject in need thereof, comprising: administering to the
subject a pharmaceutical composition or chondrisome preparation of
any one of claims 1-20.
65. The method of claim 60, 63 or 64, wherein the chondrisomes of
the composition express a protein at least 85% identical to UCP1
(SEQ ID NO:1).
66. The method of claim 60, 63 or 64, wherein the chondrisomes of
the composition are isolated from brown adipocytes.
67. The method of claim 60, 63 or 64, wherein the chondrisomes of
the composition are autologous to the target tissue or subject.
68. The method of claim 60, 63 or 64, wherein the chondrisomes of
the composition are allogeneic to the target tissue or subject.
69. The method of claim 61, 63 or 64, wherein the subject is a
human.
70. The method of claim 61, 63 or 64, wherein the subject is an
agriculturally important animal
71. The method of claim 60, 63 or 64, wherein the composition is
administered to white adipocytes.
72. A method of treating a mitochondrial disease or a disease or
condition associated with mitochondrial function in a subject in
need thereof, comprising administering to the subject
pharmaceutical composition or chondrisome preparation of any one of
claims 1-20.
73. The method of claim 72, wherein the mitochondrial disease
comprises a mutation in the mitochondrial genome.
74. The method of claim 72, wherein the mitochondrial disease
comprises a mutation in a nuclear gene associated with
mitochondrial structure or function.
75. A method of treating a subject for a disease or condition
described herein, comprising administering to the subject a
pharmaceutical composition or chondrisome preparation of any one of
claims 1-20.
76. The method of any one of claims 39-75, wherein at least 5% of
the chondrisomes of the composition are internalized into the
target tissue or cell.
77. The method of any one of claims 39-75, wherein the target
tissue or cell is selected from the group consisting of:
epithelial, connective, muscular, and nervous tissue or cell.
78. The method of any one of claims 39-75, wherein the chondrisomes
of the composition are obtained from a source cell type different
than the target tissue or cell type.
79. The method of any one of claims 39-75, wherein the chondrisomes
of the composition are obtained from the same cell type as the
target tissue or cell type.
80. The method of any one of claims 39-75, wherein the target
tissue or cell is in the digestive system, the endocrine system,
the excretory system, the lymphatic system, the skin, muscle, the
nervous system, the reproductive system, the respiratory system, or
the skeletal system.
81. The method of any one of claims 39-75, wherein the chondrisomes
of the composition are encapsulated.
82. The method of any one of claims 39-75, wherein the chondrisomes
of the composition are autologous to the target cell or tissue.
83. The method of any one of claims 39-75, wherein the chondrisomes
of the composition are allogeneic to the subject.
84. The method of any one of claims 39-75, wherein the target cell
or tissue is from, or the subject is, a subject who has or is at
risk for: ischemia; a mitochondrial disease; an infectious disease
(e.g. a viral infection (e.g., HIV, HCV, RSV), a bacterial
infection, a fungal infection, sepsis); cardiovascular disorder
(e.g., atherosclerosis, hypercholesterolemia, thrombosis, clotting
disorder, angiogenic disorder such as macular degeneration); an
autoimmune disorder (e.g. diabetes, lupus, multiple sclerosis,
psoriasis, rheumatoid arthritis); an inflammatory disorder (e.g.
arthritis, pelvic inflammatory disease); a neurological disorder
(e.g. Alzheimer's disease, Huntington's disease; autism; Duchenne
muscular dystrophy); a proliferative disorder (e.g. cancer, benign
neoplasms); a respiratory disorder (e.g. chronic obstructive
pulmonary disease); a digestive disorder (e.g. inflammatory bowel
disease, ulcer); a musculoskeletal disorder (e.g. fibromyalgia,
arthritis); an endocrine, metabolic, or nutritional disorder (e.g.
diabetes, osteoporosis); an urological disorder (e.g. renal
disease); a psychological disorder (e.g. depression,
schizophrenia); a skin disorder (e.g. wounds, eczema); or a blood
or lymphatic disorder (e.g. anemia, hemophilia).
85. The method of any one of claims 39-75, wherein the chondrisomes
of the composition are isolated from a source selected from the
group consisting of: circulating cells (e.g., platelets,
leukocytes), muscle cells (e.g., cardiomyocytes, skeletal muscle,
smooth muscle), epidermal cells (e.g., keratinocytes, melanocytes),
dermal cells, hypodermal cells (e.g., fibroblasts, brown
adipocytes), stem cells (e.g., mesenchymal stem cells), liver
cells, kidney cells, pancreas cells, spleen cells).
86. The method of any one of claims 39-75, wherein the chondrisomes
are modified, e.g.: (a) genetically engineered to overexpress or
knock-down or knock-out an endogenous gene product (e.g., an
endogenous mitochondrial or nuclear gene product); (b) engineered
to express a heterologous gene product (e.g., a heterologous, e.g.,
allogeneic or xenogeneic, mitochondrial or nuclear gene product),
or (c) loaded with a heterologous cargo agent, such as a
polypeptide, nucleic acid or small molecule (e.g., a dye, a drug, a
metabolite).
87. The method of any one of claims 39-75, wherein the composition
is administered in an amount and for a time sufficient to effect a
biological function described herein.
88. A method of making a pharmaceutical preparation suitable for
administration to a human subject, comprising: a. providing a
parent cell or tissue source, b. isolating a preparation of
chondrisomes from the parent cell or tissue source, and c.
evaluating (e.g., testing or measuring) a sample of the preparation
for one or more characteristics described herein; and d.
formulating the preparation for administration to a human subject
if one or more (2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the
characteristics meet a pre-determined reference value, thereby
making a pharmaceutical preparation suitable for administration to
a human subject.
89. The method of claim 88, wherein the one or more of
characteristics is a release test for the pharmaceutical
composition.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Nos. 62/261,157, 62/261,169, and 62/261,170, all filed
on Nov. 30, 2015, the contents of which are hereby incorporated
herein by reference in their entirety.
BACKGROUND
[0002] Mitochondria are membrane bound subcellular structures found
in eukaryotic cells. Sometimes described as the power plants of
cells, mitochondria generate most of the energy of the cell in the
form of adenosine triphosphate (ATP) through respiration. Damage
and subsequent dysfunction of mitochondria are important factors in
a range of human diseases.
SUMMARY OF THE INVENTION
[0003] Described herein are novel preparations of chondrisomes
derived from mitochondria, and related methods, that have
advantageous and surprising qualities for use in human
pharmaceutical and in veterinary applications. Chondrisome
preparations and methods described herein have beneficial
structural characteristics, yield, concentration, stability,
viability, integrity, or function, e.g., a bioenergetic or
biological function, for use in therapeutic applications.
[0004] Accordingly, in one aspect, the invention features a
pharmaceutical preparation comprising isolated chondrisomes,
derived from a cellular source of mitochondria. In one embodiment,
the preparation (or the chondrisomes of the preparation) has one or
more (2, 3, 4, 5, 6 or more) of the following characteristics:
[0005] the chondrisomes of the preparation have a mean average size
between 150-1500 nm, e.g., between 200-1200 nm, e.g., between
500-1200 nm, e.g., 175-950 nm;
[0006] the chondrisomes of the preparation have a polydispersity
(D90/D10) between 1.1 to 6, e.g., between 1.5-5. In embodiments,
chondrisomes of a preparation from a cultured cell source (e.g.,
cultured fibroblasts) have a polydispersity (D90/D10) between 2-5,
e.g., between 2.5-5;
[0007] outer chondrisome membrane integrity wherein the preparation
exhibits <20% (e.g., <15%, <10%, <5%, <4 , <3%,
<2%, <1%) increase in oxygen consumption rate over state 4
rate following addition of reduced cytochrome c;
[0008] complex I level of 1-8 mOD/ug total protein, e.g., 3-7
mOD/ug total protein, 1-5 mOD/ug total protein. In embodiments,
chondrisomes of a preparation from a cultured cell source (e.g.,
cultured fibroblasts) have a complex I level of 1-5 mOD/ug total
protein;
[0009] complex II level of 0.05-5 mOD/ug total protein, e.g., 0.1-4
mOD/ug total protein, e.g., 0.5-3 mOD/ug total protein. In
embodiments, chondrisomes of a preparation from a cultured cell
source (e.g., cultured fibroblasts) have a complex II level of
0.05-1 mOD/ug total protein;
[0010] complex III level of 1-30 mOD/ug total protein, e.g., 2-30,
5-10, 10-30 mOD/ug total protein. In embodiments, chondrisomes of a
preparation from a cultured cell source (e.g., cultured
fibroblasts) have a complex III level of 1-5 mOD/ug total
protein;
[0011] complex IV level of 4-50 mOD/ug total protein, e.g., 5-50,
e.g., 10-50, 20-50 mOD/ug total protein. In embodiments,
chondrisomes of a preparation from a cultured cell source (e.g.,
cultured fibroblasts) have a complex IV level of 3-10 mOD/ug total
protein;
[0012] genomic concentration 0.001-2 (e.g., 0.001-1, 0.01-1,
0.01-.1, 0.01-.05, 0.1-.2) mtDNA ug/mg protein;
[0013] membrane potential of the preparation is between -5 to -200
mV, e.g., between -100 to -200 mV, -50 to -200 mV, -50 to -75 mV,
-50 to -100 mV. In some embodiments, membrane potential of the
preparation is less than -150 mV, less than -100 mV, less than -75
mV, less than -50 mV, e.g., -5 to -20 mV;
[0014] a protein carbonyl level of less than 100 nmol carbonyl/mg
chondrisome protein (e.g., less than 90 nmol carbonyl/mg
chondrisome protein, less than 80 nmol carbonyl/mg chondrisome
protein, less than 70 nmol carbonyl/mg chondrisome protein, less
than 60 nmol carbonyl/mg chondrisome protein, less than 50 nmol
carbonyl/mg chondrisome protein, less than 40 nmol carbonyl/mg
chondrisome protein, less than 30 nmol carbonyl/mg chondrisome
protein, less than 25 nmol carbonyl/mg chondrisome protein, less
than 20 nmol carbonyl/mg chondrisome protein, less than 15 nmol
carbonyl/mg chondrisome protein, less than 10 nmol carbonyl/mg
chondrisome protein, less than 5 nmol carbonyl/mg chondrisome
protein, less than 4 nmol carbonyl/mg chondrisome protein, less
than 3 nmol carbonyl/mg chondrisome protein;
[0015] <20% mol/mol ER proteins (e.g., >15%, >10%, >5%,
>3%, >2%, >1%) mol/mol ER proteins;
[0016] >5% mol/mol mitochondrial proteins (proteins identified
as mitochondrial in the MitoCarta database (Calvo et al., NAR 20151
doi:10.1093/nar/gkv1003)), e.g., >10%, >15%, >20%,
>25%, >30%, >35%, >40%; >50%, >55%, >60%,
>65%, >70%, >75%, >80%; >90% mol/mol mitochondrial
proteins);
[0017] >0.05% mol/mol of MT-CO2, MT-ATP6, MT-ND5 and MT-ND6
protein (combined) (e.g., >0.1%; >05%, >1%, >2%,
>3%, >4%, >5%, >7, >8%, >9%, >10, >15%
mol/mol of MT-CO2, MT-ATP6, MT-ND5 and MT-ND6 protein);
[0018] Genetic quality >80%, e.g., >85%, >90%, >95%,
>97%, >98%, >99%;
[0019] Relative ratio mtDNA/nuclear DNA is >1000 (e.g.,
>1,500, >2000, >2,500, >3,000, >4,000, >5000,
>10,000, >25,000, >50,000, >100,000, >200,000,
>500,000);
[0020] Endotoxin level <0.2 EU/ug protein (e.g., <0.1, 0.05,
0.02, 0.01 EU/ug protein);
[0021] Substantially absent exogenous non-human serum;
[0022] Glutamate/malate RCR 3/2 of 1-15, e.g., 2-15, 5-15, 2-10,
2-5, 10-15;
[0023] Glutamate/malate RCR 3/4o of 1-30, 1-20, 2-20, 5-20, 3-15,
10-30;
[0024] Succinate/rotenone RCR 3/2 of 1-15, 2-15, 5-15, 1-10,
10-15;
[0025] Succinate/rotenone RCR 3/4o of 1-30, 1-20, 2-20, 5-20, 3-15,
10-30;
[0026] complex I activity of 0.05-100 nmol/min/mg total protein
(e.g., 0.05-50, 0.05-20, 0.5-10, 0.1-50, 1-50, 2-50, 5-100, 1-20
nmol/min/mg total protein);
[0027] complex II activity of 0.05-50 nmol/min/mg total protein
(e.g., 0.05-50, 0.05-20, 0.5-10, 0.1-50, 1-50, 2-50, 5-50, 1-20
nmol/min/mg total protein);
[0028] complex III activity of 0.05-20 nmol/min/mg total protein
(e.g., 0.05-50, 0.05-20, 0.5-10, 0.1-50, 1-50, 2-50, 5-100, 1-20
nmol/min/mg total protein);
[0029] complex IV activity of 0.1-50 nmol/min/mg total protein
(e.g., 0.05-50, 0.05-20, 0.5-10, 0.1-50, 1-50, 2-50, 5-50, 1-20
nmol/min/mg total protein);
[0030] complex V activity of 1-500 nmol/min/mg total protein (e.g.,
10-500, 10-250, 10-200, 100-500 nmol/min/mg total protein);
[0031] reactive oxygen species (ROS) production level of 0.01-50
pmol H2O2/ug protein/hr (e.g., 0.05-40, 0.05-25, 1-20, 2-20,
0.05-20, 1-20 pmol H2O2/ug protein/hr);
[0032] Citrate Synthase activity of 0.05-5 (e.g., 0.5-5, 0.5-2,
1-5, 1-4) mOD/min/ug total protein;
[0033] Alpha ketoglutarate dehydrogenase activity of 0.05-10 (e.g.,
0.1-10, 0.1-8, 0.5-8, 0.1-5, 0.5-5, 0.5-3, 1-3) mOD/min/ug total
protein;
[0034] Creatine Kinase activity of 0.1-100 (e.g., 0.5-50, 1-100,
1-50, 1-25, 1-15, 5-15) mOD/min/ug total protein;
[0035] Pyruvate dehydrogenase activity of 0.1-10 (e.g., 0.5-10,
0.5-8, 1-10, 1-8, 1-5, 2-3) mOD/min/ug total protein;
[0036] Aconitase activity of 0.1-50 (e.g., 5-50, 0.1-2, 0.1-20,
0.5-30) mOD/min/ug total protein. In embodiments, aconitase
activity in a chondrisome preparation from platelets is between
0.5-5 mOD/min/ug total protein. In embodiments, aconitase activity
in a chondrisome preparation from cultured cells, e.g.,
fibroblasts, is between 5-50 mOD/min/ug total protein;
[0037] Maximal fatty acid oxidation level of 0.05-50 (e.g.,
0.05-40, 0.05-30, 0.05-10, 0.5-50, 0.5-25, 0.5-10, 1-5) pmol
O2/min/ug chondrisome protein;
[0038] Palmitoyl carnitine & Malate RCR3/2 state 3/state 2
respiratory control ratio (RCR 3/2) of 1-10 (e.g., 1-5);
[0039] electron transport chain efficiency of 1-1000 (e.g.,
10-1000, 10-800, 10-700, 50-1000, 100-1000, 500-1000, 10-400,
100-800) nmol O2/min/mg protein/.DELTA.GATP (in kcal/mol);
[0040] total lipid content of 50,000-2,000,000 pmol/mg (e.g.,
50,000-1,000,000; 50,000-500,000 pmol/mg);
[0041] double bonds/total lipid ratio of 0.8-8 (e.g., 1-5, 2-5,
1-7, 1-6) pmol/pmol;
[0042] phospholipid/total lipid ratio of 50-100 (e.g., 60-80,
70-100, 50-80) 100*pmol/pmol;
[0043] phosphosphingolipid/total lipid ratio of 0.2-20 (e.g.,
0.5-15, 0.5-10, 1-10, 0.5-10, 1-5, 5-20) 100*pmol/pmol;
[0044] ceramide content 0.05-5 (e.g., 0.1-5, 0.1-4, 1-5, 0.05-3)
100*pmol/pmol total lipid;
[0045] cardiolipin content 0.05-25 (0.1-20, 0.5-20, 1-20, 5-20,
5-25, 1-25, 10-25, 15-25) 100*pmol/pmol total lipid;
[0046] lyso-phosphatidylcholine (LPC) content of 0.05-5 (e.g.,
0.1-5, 1-5, 0.1-3, 1-3, 0.05-2) 100*pmol/pmol total lipid;
[0047] Lyso-Phosphatidylethanolamine (LPE) content of 0.005-2
(e.g., 0.005-1, 0.05-2, 0.05-1) 100*pmol/pmol total lipid;
[0048] Phosphatidylcholine (PC) content of 10-80 (e.g., 20-60,
30-70, 20-80, 10-60 m 30-50) 100*pmol/pmol total lipid;
[0049] Phosphatidylcholine-ether (PC O-) content 0.1-10 (e.g.,
0.5-10, 1-10, 2-8, 1-8) 100*pmol/pmol total lipid;
[0050] Phosphatidylethanolamine (PE) content 1-30 (e.g., 2-20,
1-20, 5-20) 100*pmol/pmol total lipid;
[0051] Phosphatidylethanolamine-ether (PE O-) content 0.05-30
(e.g., 0.1-30, 0.1-20, 1-20, 0.1-5, 1-10, 5-20) 100*pmol/pmol total
lipid;
[0052] Phosphatidylinositol (PI) content 0.05-15 (e.g., 0.1-15,
0.1-10, 1-10, 0.1-5, 1-10, 5-15) 100*pmol/pmol total lipid;
[0053] Phosphatidylserine (PS) content 0.05-20 (e.g., 0.1-15,
0.1-20, 1-20, 1-10, 0.1-5, 1-10, 5-15) 100*pmol/pmol total
lipid;
[0054] Sphingomyelin (SM) content 0.01-20 (e.g., 0.01-15, 0.01-10,
0.5-20, 0.5-15, 1-20, 1-15, 5-20) 100*pmol/pmol total lipid;
[0055] Triacylglycerol (TAG) content 0.005-50 (e.g., 0.01-50,
0.1-50, 1-50, 5-50, 10-50, 0.005-30, 0.01-25, 0.1-30) 100*pmol/pmol
total lipid;
[0056] PE:LPE ratio 30-350 (e.g., 50-250, 100-200, 150-300);
[0057] PC:LPC ratio 30-700 (e.g., 50-300, 50-250, 100-300, 400-700,
300-500, 50-600, 50-500, 100-500, 100-400);
[0058] PE 18:n (n>0) content 0.5-20% (e.g., 1-20%, 1-10%, 5-20%,
5-10%, 3-9%) pmol AA/pmol lipid class;
[0059] PE 20:4 content 0.05-20% (e.g., 1-20%, 1-10%, 5-20%, 5-10%)
pmol AA/pmol lipid class;
[0060] PC 18:n (n>0) content 5-50% (e.g., 5-40%, 5-30%, 20-40%,
20-50%) pmol AA/pmol lipid class;
[0061] PC 20:4 content 1-20% (e.g., 2-20%, 2-15%, 5-20%, 5-15%)
pmol AA/pmol lipid class.
[0062] In certain embodiments, the preparation or composition has
one or more of the following characteristics upon administration to
a recipient cell, tissue or subject (a control may be a negative
control (e.g., a control tissue or subject that has not been
treated or administered a preparation), or a baseline prior to
administration, e.g., a cell, tissue or subject prior to
administration of the preparation or composition):
[0063] Increases basal respiration of recipient cells at least 10%
(e.g., >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0064] Chondrisomes of the preparation are taken up by at least 1%
(e.g., at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%) of
recipient cells;
[0065] Chondrisomes of the preparation are taken up and maintain
membrane potential in recipient cells;
[0066] Chondrisomes of the preparation persist in recipient cells
at least 6 hours, e.g., at least 12 hours, 18 hours, 24 hours, 2
days, 3 days, 4 days, a week, 2 weeks, a month, 2 months, 3 months,
6 months;
[0067] increase ATP levels in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0068] decrease apotosis in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0069] decrease cellular lipid levels in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0070] increase membrane potential in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0071] increase uncoupled respiration in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0072] increase PI3K activity in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0073] reduce reductive stress in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0074] decrease reactive oxygen species (e.g. H2O.sub.2) in the
cell, tissue of subject (e.g., in serum of a target subject) (e.g.,
by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 509%, 60%,
70%, 80%, 90%, or more, e.g., compared to a reference value, e.g.,
a control value, e.g., an untreated control);
[0075] Decrease cellular lipid levels of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0076] increases uncoupled respiration of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0077] decrease mitochondrial permeability transition pore (MPTP)
formation in recipient cells at least 5% and does not increase more
than 10% relative to a control;
[0078] increase Akt levels in recipient cells at least 10% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0079] decrease total NAD/NADH ratio in recipient cells at least 5%
(e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0080] Reduce ROS levels in recipient cells at least 5% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0081] Increase fractional shortening in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control;
[0082] Increase end diastolic volume in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control;
[0083] decrease end systolic volume in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control;
[0084] decrease infarct area of ischemic heart at least 5% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0085] increase stroke volume in subject with cardiac ischemia at
least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0086] increase ejection fraction in subject with cardiac ischemia
at least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0087] increase cardia output in subject with cardiac ischemia at
least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0088] increase cardiac index in subject with cardiac ischemia at
least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0089] decrease serum CKNB levels in subject with cardiac ischemia
at least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0090] decrease serum cTnI levels in subject with cardiac ischemia
at least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0091] decrease serum hydrogen peroxide in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control;
[0092] decrease serum cholesterol levels and/or triglycerides in a
subject at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control.
[0093] In embodiments, the pharmaceutical preparation is stable for
at least 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours,
5 days, 7 days, 10 days, 14 days, 21 days, 30 days, 45 days, 60
days, 90 days, 120 days, 180 days, or longer (for example, at
4.degree. C., 0.degree. C., -4.degree. C., or -20.degree. C.,
-80.degree. C.).
[0094] In embodiments, the chondrisomes in the preparation may be
encapsulated, e.g., in a natural, synthetic or engineered
encapsulation material such as a lipid based material, e.g., a
micelle, synthetic or natural vesicle, exosome, lipid raft,
clathrin coated vesicle, or platelet (mitoparticle), MSC or
astrocyte microvesicle membrane.
[0095] In embodiments, the preparation may be configured for
systemic or local delivery, e.g., for enteral, parenteral (e.g.,
IV, SC, IM), or transdermal delivery.
[0096] In embodiments, the concentration of the preparation or
composition is between 150-20,000 ug protein/ml; between 150-15,000
ug/ml; 200-15,000 ug/ml; 300-15,000 ug/ml; 500-15,000 ug/ml;
200-10,000 ug/ml; 200-5,000 ug/ml; 300-10,000 ug/ml; >200 ug/ml;
>250 ug/ml; >300 ug/ml; >350 ug/ml; >400 ug/ml; >450
ug/ml; >500 ug/ml; >600 ug/ml; >700 ug/ml; >800 ug/ml;
>900 ug/ml; >1 mg/ml; >2 mg/ml; >3 mg/ml; >4 mg/ml;
>5 mg/ml; >6 mg/ml; >7 mg/ml; >8 mg/ml; >9 mg/ml;
>10 mg/ml; >11 mg/ml; >12 mg/ml; >14 mg/ml; >15
mg/ml (and, e.g., <20 mg/ml).
[0097] In embodiments, the preparation does not produce an
undesirable immune response in a recipient animal, e.g., a
recipient mammal such as a human (e.g., does not significantly
increase levels of IL-1-beta, IL-6, GM-CSF, TNF-alpha, or lymph
node size, in the recipient).
[0098] In certain embodiments, the chondrisomes of the preparation
express a metabolite transporter, e.g., UCP1, UCP2, UCP3, UCP4 or
UCP5. The expressed transporter may be endogenous or heterologous
to the source mitochondria (e.g., the transporter may be naturally
expressed, or the mitochondria or chondrisomes may be modified
(e.g., genetically modified or loaded) to express or over-express
the transporter. In one embodiment, the chondrisomes are engineered
to express a protein at least 85%, 90%, 95%, 97%, 98%, 100%
identical to the sequence of human UCP1 (SEQ ID NO:1), human UCP2
(SEQ ID NO:2), human UCP3 (SEQ ID NO:3), human UCP4 (SEQ ID NO:4)
or human UCP5 (SEQ ID NO:5), wherein the protein has transporter
activity.
[0099] In other embodiments, the mitochondria of the preparation
have reduced expression, or lack expression, of a metabolite
transporter, e.g., UCP1, UCP2, UCP3, UCP4 or UCP5. The transporter
may be knocked down or knocked-out in the source mitochondria
and/or in the chondrisomes, e.g., using routine methods in the art,
such as CRISPR or RNAi.
[0100] In embodiments, the source of the mitochondria is selected
from epithelial, connective, muscular, or nervous tissue.
[0101] In embodiments, the cellular source of mitochondria is a
plurality of tissue types or cell types.
[0102] In some embodiments, the preparation is made using a method
of making a pharmaceutical composition described herein.
[0103] In certain embodiments, the chondrisomes of the preparation
are modified, e.g., the source mitochondria or chondrisomes are (a)
genetically engineered to overexpress or knock-down or knock-out an
endogenous gene product (e.g., an endogenous mitochondrial or
nuclear gene product); (b) engineered to express a heterologous
gene product (e.g., a heterologous, e.g., allogeneic or xenogeneic,
mitochondrial or nuclear gene product), or (c) loaded with a
heterologous cargo agent, such as a polypeptide, nucleic acid or
small molecule (e.g., a dye, a drug, a metabolite) or an agent
listed in Table 4. In embodiments, the chondrisomes of the
preparation are modified as described herein.
[0104] In another aspect, the invention features a pharmaceutical
preparation comprising isolated, modified chondrisomes derived from
a cellular source of mitochondria. Chondrisomes may be modified by
a modification made to the source mitochondria (e.g., a
modification to the cell, tissue or subject that provides the
source mitochondria), or by a modification made to the chondrisome
preparation after isolation from the source. For example, the
source mitochondria or chondrisomes of the preparation are (a)
subjected to or combined with an external condition or agent (e.g.,
a stress condition or agent that induces one or more mitochondrial
activity to compensate), (b) genetically engineered to overexpress
or knock-down or knock-out an endogenous gene product (e.g., an
endogenous mitochondrial or nuclear gene product, e.g., an
endogenous mitochondrial or nuclear gene product described herein);
(c) engineered to express a heterologous gene product (e.g., a
heterologous, e.g., allogeneic or xenogeneic, mitochondrial or
nuclear gene product, e.g., an exogenous mitochondrial or nuclear
gene product described herein), or (d) loaded with a heterologous
cargo agent, such as a polypeptide, nucleic acid or small molecule
(e.g., a dye, a drug, a metabolite or other cargo described
herein), or an agent listed in Table 4.
[0105] In embodiments, the source of mitochondria is modified. For
example, the source is subjected to an external condition or agent,
such as a stress condition or agent. In embodiments, the source of
mitochondria is subjected to a temperature change. In embodiments,
the source of mitochondria is subjected to hypoxia or hyperoxia. In
another embodiment, chondrisomes are obtained from a source exposed
to stressed nutrient conditions, e.g., lack of glucose or other
sugar substrate, amino acids, or a combination thereof. In another
embodiment, the source of mitochondria is exposed to different
concentrations of one or more nutrients, e.g., reduced
concentration of glucose or other sugar substrate, amino acids, or
a combination thereof. In another embodiment, chondrisomes are
obtained from a source exposed to osmotic stress, e.g., increase or
decrease in solute concentration. In some embodiments, chondrisomes
are obtained from a source that has been injured or a source
undergoing a wound healing process. In embodiments, the source of
mitochondria may be treated with a toxin, e.g., metformin. In
another embodiment, a source of mitochondria may be treated with
one or more infectious agents, such as a virus or bacteria (e.g.,
hepatitis C virus (HCV) and hepatitis B virus (HBV)).
[0106] In some embodiments, the source mitochondrial genome is
engineered, e.g., to express, overexpress or knock-down or
knock-out a mitochondrial gene, e.g., a gene listed in Table 2 or
any other gene described herein.
[0107] In some embodiments, the source may be engineered to express
a cytosolic enzyme (e.g., a protease, phosphatase, kinase,
demethylase, methyltransferase, acetylase) that targets a
mitochondrial protein. For example, the source mitochondria or
chondrisomes are engineered to express a protein at least 85%, 90%,
95%, 97%, 98%, 99%, 100% identical to the sequence of human SIRT3
(SEQ ID NO:7). For example, the source mitochondria or chondrisomes
are engineered to express a protein at least 85%, 90%, 95%, 97%,
98%, 99%, 100% identical to the sequence of human pyruvate
dehydrogenase kinase (SEQ ID NO:8). For example, the source
mitochondria or chondrisomes are engineered to express a protein at
least 85%, 90%, 95%, 97%, 98%, 99%, 100% identical to the sequence
of human O-GlcNAc transferase (SEQ ID NO:9) or to an alternative
splice variant thereof (e.g., comprising amino acid 177-1046 of SEQ
ID NO:9; amino acid 23-1046 of SEQ ID NO:9; or amino acid 382-1046
of SEQ ID NO:9).
[0108] In some embodiments, the source is modified to modulate a
mitochondrial transporter, e.g., by phosphorylation, e.g., the
mitochondrial source is treated with dephosphorylated pyruvate
dehydrogenase to catabolize glucose and gluconeogenesis precursors.
In another embodiment, the source is treated with phosphorylated
pyruvate dehydrogenase to shift metabolism toward fat
utilization.
[0109] In some embodiments, the source of mitochondria has altered
distribution and/or quantity of nuclear encoded mitochondrial
targeted proteins. For example, the source is engineered to express
a mitochondrial import signal appended to an RNA encoding a target
protein, or a fusion that includes a protein mitochondrial import
signal and a non-mitochondrial target protein. In other examples,
the source may be modified to target cytosolic proteins, such as
proteases or enzymes, to the source mitochondria. Import into
mitochondria can be effected by N-terminal targeting sequences
(presequences) or internal targeting sequences.
[0110] In embodiments, the isolated chondrisomes are modified. For
example, a chondrisome preparation comprises an exogenous agent,
e.g., has been loaded with an exogenous agent such as a nucleic
acid (e.g., DNA, RNA), protein, or chemical compound. In some
embodiments, the exogenous agent is a cargo or payload, e, g., a
payload for administration to a cell, tissue or subject, e.g., an
agent listed in Table 4.
[0111] In some embodiments, the exogenous agent is a modified
protein, e.g., a modified protein described herein.
[0112] In embodiments, the modified chondrisome preparation has one
or more of the following characteristics:
[0113] the chondrisomes of the preparation have a mean average size
between 150-1500 nm, e.g., between 200-1200 nm, e.g., between
500-1200 nm, e.g., 175-950 nm;
[0114] the chondrisomes of the preparation have a polydispersity
(D90/D10) between 1.1 to 6, e.g., between 1.5-5. In embodiments,
chondrisomes of a preparation from a cultured cell source (e.g.,
cultured fibroblasts) have a polydispersity (D90/D10) between 2-5,
e.g., between 2.5-5;
[0115] outer chondrisome membrane integrity wherein the preparation
exhibits <20% (e.g., <15%, <10%, <5%, <4 , <3%,
<2%, <1%) increase in oxygen consumption rate over state 4
rate following addition of reduced cytochrome c;
[0116] complex I level of 1-8 mOD/ug total protein, e.g., 3-7
mOD/ug total protein, 1-5 mOD/ug total protein. In embodiments,
chondrisomes of a preparation from a cultured cell source (e.g.,
cultured fibroblasts) have a complex I level of 1-5 mOD/ug total
protein;
[0117] complex II level of 0.05-5 mOD/ug total protein, e.g., 0.1-4
mOD/ug total protein, e.g., 0.5-3 mOD/ug total protein. In
embodiments, chondrisomes of a preparation from a cultured cell
source (e.g., cultured fibroblasts) have a complex II level of
0.05-1 mOD/ug total protein;
[0118] complex III level of 1-30 mOD/ug total protein, e.g., 2-30,
5-10, 10-30 mOD/ug total protein. In embodiments, chondrisomes of a
preparation from a cultured cell source (e.g., cultured
fibroblasts) have a complex III level of 1-5 mOD/ug total
protein;
[0119] complex IV level of 4-50 mOD/ug total protein, e.g., 5-50,
e.g., 10-50, 20-50 mOD/ug total protein. In embodiments,
chondrisomes of a preparation from a cultured cell source (e.g.,
cultured fibroblasts) have a complex IV level of 3-10 mOD/ug total
protein;
[0120] genomic concentration 0.001-2 (e.g., 0.001-1, 0.01-1,
0.01-.1, 0.01-.05, 0.1-.2) mtDNA ug/mg protein;
[0121] membrane potential of the preparation is between -5 to -200
mV, e.g., between -100 to -200 mV, -50 to -200 mV, -50 to -75 mV,
-50 to -100 mV. In some embodiments, membrane potential of the
preparation is less than -150 mV, less than -100 mV, less than -75
mV, less than -50 mV, e.g., -5 to -20 mV;
[0122] a protein carbonyl level of less than 100 nmol carbonyl/mg
chondrisome protein (e.g., less than 90 nmol carbonyl/mg
chondrisome protein, less than 80 nmol carbonyl/mg chondrisome
protein, less than 70 nmol carbonyl/mg chondrisome protein, less
than 60 nmol carbonyl/mg chondrisome protein, less than 50 nmol
carbonyl/mg chondrisome protein, less than 40 nmol carbonyl/mg
chondrisome protein, less than 30 nmol carbonyl/mg chondrisome
protein, less than 25 nmol carbonyl/mg chondrisome protein, less
than 20 nmol carbonyl/mg chondrisome protein, less than 15 nmol
carbonyl/mg chondrisome protein, less than 10 nmol carbonyl/mg
chondrisome protein, less than 5 nmol carbonyl/mg chondrisome
protein, less than 4 nmol carbonyl/mg chondrisome protein, less
than 3 nmol carbonyl/mg chondrisome protein;
[0123] <20% mol/mol ER proteins (e.g., >15%, >10%, >5%,
>3%, >2%, >1%) mol/mol ER proteins;
[0124] >5% mol/mol mitochondrial proteins (proteins identified
as mitochondrial in the MitoCarta database (Calvo et al., NAR 20151
doi:10.1093/nar/gkv1003)), e.g., >10%, >15%, >20%,
>25%, >30%, >35%, >40%; >50%, >55%, >60%,
>65%, >70%, >75%, >80%; >90% mol/mol mitochondrial
proteins);
[0125] >0.05% mol/mol of MT-CO2, MT-ATP6, MT-ND5 and MT-ND6
protein (e.g., >0.1%; >05%, >1%, >2%, >3%, >4%,
>5%, >7, >8%, >9%, >10, >15% mol/mol of MT-CO2,
MT-ATP6, MT-ND5 and MT-ND6 protein);
[0126] Genetic quality >80%, e.g., >85%, >90%, >95%,
>97%, >98%, >99%; Relative ratio mtDNA/nuclear DNA is
>1000 (e.g., >1,500, >2000, >2,500, >3,000,
>4,000, >5000, >10,000, >25,000, >50,000,
>100,000, >200,000, >500,000);
[0127] Endotoxin level <0.2 EU/ug protein (e.g., <0.1, 0.05,
0.02, 0.01 EU/ug protein);
[0128] Substantially absent exogenous non-human serum;
[0129] Glutamate/malate RCR 3/2 of 1-15, e.g., 2-15, 5-15, 2-10,
2-5, 10-15;
[0130] Glutamate/malate RCR 3/4o of 1-30, 1-20, 2-20, 5-20, 3-15,
10-30;
[0131] Succinate/rotenone RCR 3/2 of 1-15, 2-15, 5-15, 1-10,
10-15;
[0132] Succinate/rotenone RCR 3/4o of 1-30, 1-20, 2-20, 5-20, 3-15,
10-30;
[0133] complex I activity of 0.05-100 nmol/min/mg total protein
(e.g., 0.05-50, 0.05-20, 0.5-10, 0.1-50, 1-50, 2-50, 5-100, 1-20
nmol/min/mg total protein);
[0134] complex II activity of 0.05-50 nmol/min/mg total protein
(e.g., 0.05-50, 0.05-20, 0.5-10, 0.1-50, 1-50, 2-50, 5-50, 1-20
nmol/min/mg total protein);
[0135] complex III activity of 0.05-20 nmol/min/mg total protein
(e.g., 0.05-50, 0.05-20, 0.5-10, 0.1-50, 1-50, 2-50, 5-100, 1-20
nmol/min/mg total protein);
[0136] complex IV activity of 0.1-50 nmol/min/mg total protein
(e.g., 0.05-50, 0.05-20, 0.5-10, 0.1-50, 1-50, 2-50, 5-50, 1-20
nmol/min/mg total protein);
[0137] complex V activity of 1-500 nmol/min/mg total protein (e.g.,
10-500, 10-250, 10-200, 100-500 nmol/min/mg total protein);
[0138] reactive oxygen species (ROS) production level of 0.01-50
pmol H2O2/ug protein/hr (e.g., 0.05-40, 0.05-25, 1-20, 2-20,
0.05-20, 1-20 pmol H2O2/ug protein/hr);
[0139] Citrate Synthase activity of 0.05-5 (e.g., 0.5-5, 0.5-2,
1-5, 1-4) mOD/min/ug total protein; Alpha ketoglutarate
dehydrogenase activity of 0.05-10 (e.g., 0.1-10, 0.1-8, 0.5-8,
0.1-5, 0.5-5, 0.5-3, 1-3) mOD/min/ug total protein;
[0140] Creatine Kinase activity of 0.1-100 (e.g., 0.5-50, 1-100,
1-50, 1-25, 1-15, 5-15) mOD/min/ug total protein;
[0141] Pyruvate dehydrogenase activity of 0.1-10 (e.g., 0.5-10,
0.5-8, 1-10, 1-8, 1-5, 2-3) mOD/min/ug total protein;
[0142] Aconitase activity of 0.1-50 (e.g., 5-50, 0.1-2, 0.1-20,
0.5-30) mOD/min/ug total protein. In embodiments, aconitase
activity in a chondrisome preparation from platelets is between
0.5-5 mOD/min/ug total protein. In embodiments, aconitase activity
in a chondrisome preparation from cultured cells, e.g.,
fibroblasts, is between 5-50 mOD/min/ug total protein;
[0143] Maximal fatty acid oxidation level of 0.05-50 (e.g.,
0.05-40, 0.05-30, 0.05-10, 0.5-50, 0.5-25, 0.5-10, 1-5) pmol
O2/min/ug chondrisome protein;
[0144] Palmitoyl carnitine & malate RCR3/2 state 3/state 2
respiratory control ratio (RCR 3/2) of 1-10 (e.g., 1-5);
[0145] electron transport chain efficiency of 1-1000 (e.g.,
10-1000, 10-800, 10-700, 50-1000, 100-1000, 500-1000, 10-400,
100-800) nmol O2/min/mg protein/.DELTA.GATP (in kcal/mol);
[0146] total lipid content of 50,000-2,000,000 pmol/mg (e.g.,
50,000-1,000,000; 50,000-500,000 pmol/mg);
[0147] double bonds/total lipid ratio of 0.8-8 (e.g., 1-5, 2-5,
1-7, 1-6) pmol/pmol;
[0148] phospholipid/total lipid ratio of 50-100 (e.g., 60-80,
70-100, 50-80) 100*pmol/pmol;
[0149] phosphosphingolipid/total lipid ratio of 0.2-20 (e.g.,
0.5-15, 0.5-10, 1-10, 0.5-10, 1-5, 5-20) 100*pmol/pmol;
[0150] ceramide content 0.05-5 (e.g., 0.1-5, 0.1-4, 1-5, 0.05-3)
100*pmol/pmol total lipid;
[0151] cardiolipin content 0.05-25 (0.1-20, 0.5-20, 1-20, 5-20,
5-25, 1-25, 10-25, 15-25) 100*pmol/pmol total lipid;
[0152] lyso-phosphatidylcholine (LPC) content of 0.05-5 (e.g.,
0.1-5, 1-5, 0.1-3, 1-3, 0.05-2) 100*pmol/pmol total lipid;
[0153] Lyso-Phosphatidylethanolamine (LPE) content of 0.005-2
(e.g., 0.005-1, 0.05-2, 0.05-1) 100*pmol/pmol total lipid;
[0154] Phosphatidylcholine (PC) content of 10-80 (e.g., 20-60,
30-70, 20-80, 10-60 m 30-50) 100*pmol/pmol total lipid;
[0155] Phosphatidylcholine-ether (PC O-) content 0.1-10 (e.g.,
0.5-10, 1-10, 2-8, 1-8) 100*pmol/pmol total lipid;
[0156] Phosphatidylethanolamine (PE) content 1-30 (e.g., 2-20,
1-20, 5-20) 100*pmol/pmol total lipid;
Phosphatidylethanolamine-ether (PE O-) content 0.05-30 (e.g.,
0.1-30, 0.1-20, 1-20, 0.1-5, 1-10, 5-20) 100*pmol/pmol total
lipid;
[0157] Phosphatidylinositol (PI) content 0.05-15 (e.g., 0.1-15,
0.1-10, 1-10, 0.1-5, 1-10, 5-15) 100*pmol/pmol total lipid;
[0158] Phosphatidylserine (PS) content 0.05-20 (e.g., 0.1-15,
0.1-20, 1-20, 1-10, 0.1-5, 1-10, 5-15) 100*pmol/pmol total
lipid;
[0159] Sphingomyelin (SM) content 0.01-20 (e.g., 0.01-15, 0.01-10,
0.5-20, 0.5-15, 1-20, 1-15, 5-20) 100*pmol/pmol total lipid;
[0160] Triacylglycerol (TAG) content 0.005-50 (e.g., 0.01-50,
0.1-50, 1-50, 5-50, 10-50, 0.005-30, 0.01-25, 0.1-30) 100*pmol/pmol
total lipid;
[0161] PE:LPE ratio 30-350 (e.g., 50-250, 100-200, 150-300);
[0162] PC:LPC ratio 30-700 (e.g., 50-300, 50-250, 100-300, 400-700,
300-500, 50-600, 50-500, 100-500, 100-400);
[0163] PE 18:n (n>0) content 0.5-20% (e.g., 1-20%, 1-10%, 5-20%,
5-10%, 3-9%) pmol AA/pmol lipid class;
[0164] PE 20:4 content 0.05-20% (e.g., 1-20%, 1-10%, 5-20%, 5-10%)
pmol AA/pmol lipid class;
[0165] PC 18:n (n>0) content 5-50% (e.g., 5-40%, 5-30%, 20-40%,
20-50%) pmol AA/pmol lipid class;
[0166] PC 20:4 content 1-20% (e.g., 2-20%, 2-15%, 5-20%, 5-15%)
pmol AA/pmol lipid class.
[0167] In certain embodiments, the preparation or composition has
one or more of the following characteristics upon administration to
a recipient cell, tissue or subject (a control may be a negative
control (e.g., a control tissue or subject that has not been
treated or administered a preparation), or a baseline prior to
administration, e.g., a cell, tissue or subject prior to
administration of the preparation or composition):
[0168] Increases basal respiration of recipient cells at least 10%
(e.g., >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0169] Chondrisomes of the preparation are taken up by at least 1%
(e.g., at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%) of
recipient cells;
[0170] Chondrisomes of the preparation are taken up and maintain
membrane potential in recipient cells;
[0171] Chondrisomes of the preparation persist in recipient cells
at least 6 hours, e.g., at least 12 hours, 18 hours, 24 hours, 2
days, 3 days, 4 days, a week, 2 weeks, a month, 2 months, 3 months,
6 months;
[0172] increase ATP levels in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0173] decrease apotosis in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0174] decrease cellular lipid levels in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0175] increase membrane potential in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0176] increase uncoupled respiration in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0177] increase PI3K activity in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0178] reduce reductive stress in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0179] decrease reactive oxygen species (e.g. H2O.sub.2) in the
cell, tissue of subject (e.g., in serum of a target subject) (e.g.,
by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 509%, 60%,
70%, 80%, 90%, or more, e.g., compared to a reference value, e.g.,
a control value, e.g., an untreated control);
[0180] Decrease cellular lipid levels of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0181] increases uncoupled respiration of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0182] decrease mitochondrial permeability transition pore (MPTP)
formation in recipient cells at least 5% and does not increase more
than 10% relative to a control;
[0183] increase Akt levels in recipient cells at least 10% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0184] decrease total NAD/NADH ratio in recipient cells at least 5%
(e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0185] Reduce ROS levels in recipient cells at least 5% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0186] Increase fractional shortening in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control;
[0187] Increase end diastolic volume in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control;
[0188] decrease end systolic volume in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control;
[0189] decrease infarct area of ischemic heart at least 5% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0190] increase stroke volume in subject with cardiac ischemia at
least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0191] increase ejection fraction in subject with cardiac ischemia
at least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0192] increase cardia output in subject with cardiac ischemia at
least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0193] increase cardiac index in subject with cardiac ischemia at
least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0194] decrease serum CKNB levels in subject with cardiac ischemia
at least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0195] decrease serum cTnI levels in subject with cardiac ischemia
at least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0196] decrease serum hydrogen peroxide in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control;
[0197] decrease serum cholesterol levels and/or triglycerides in a
subject at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control.
[0198] In another aspect, the invention features a process of
making a chondrisome preparation. The process includes obtaining or
providing a tissue or cellular source of mitochondria (e.g., fresh
tissue, frozen tissue, suspension tissue such as blood or blood
products, cultured cells, frozen cultured cells, from a living or
cadaver donor); manipulating the tissue (e.g., dissociating or
activating the tissue or cellular source) to produce a subcellular
composition; separating the subcellular composition into a cellular
debris fraction and a chondrisome enriched fluid fraction;
separating the chondrisome enriched fraction into a fraction
containing chondrisomes and a fraction substantially lacking
chondrisomes; and suspending the fraction containing chondrisomes
in a solution, thereby preparing a chondrisome preparation. The
solution may be, e.g., a storage buffer or a formulation for
administration. In some embodiments, the preparation may be stored
in storage solution for a period of time and changed into a
formulation for administration before use. A storage or formulation
solution may include, e.g., an osmotic regulator, e.g., a sugar
such as mannitol, sucrose, trehalose; a physiological salt, e.g., a
salt of sodium, chloride or potassium; a pH buffer).
[0199] In embodiments, the dissociating comprises applying to the
tissue or cellular source a plurality of different shear forces,
e.g., a first shear force (e.g., with a dounce device) followed by
a second, higher shear force (e.g., passing through a needle). The
separating steps may be performed by, e.g., differential
centrifugation or differential size filtration.
[0200] In embodiments, the dissociating step is performed in no
more than 10 fold (no more than 8-fold, 6-fold, 5-fold, 4-fold,
3-fold, 2-fold) the volume of buffer relative to the volume of the
tissue or cellular source. Likewise, the final fraction containing
mitochondria is suspended in no more than 10-fold (no more than
8-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold) the volume of
buffer relative to the volume of the fraction containing
mitochondria.
[0201] In embodiments, the dissociating and subsequent steps are
performed in the absence of an exogenous protease.
[0202] In certain embodiments, the yield of the preparation is
>0.05 (e.g., >0.1, >0.2, >0.5, >1, >2, >3,
>5, >6, >7, >8, >8, >10, >20, >30, >40,
>50, >60, >80, >90, >100, >150, >200, >300)
ug protein/10E6 cells. In certain embodiments, the yield of the
preparation is >100 (e.g., >200, >300, >400, >500,
>600, >700, >800, >900, >1,000, >2,000,
>3,000, >5,000, >7000, >10,000) ug protein/g tissue. In
embodiments, the yield is 1E9 to 9E12 (e.g., >1E9, >5E9,
>1E10, >5E10, >1E11, >5E11, >1E12, >5E12)
particles/mg total protein.
[0203] The tissue or cellular source may be exposed to one or more
modulator before or during the preparation. The modulator may be,
e.g., a mitochondrial biogenesis agent (e.g., a mitochondrial
biogenesis agent described herein); a modulator of metabolic
activity (e.g., modulator of metabolic activity described herein);
an environmental modulator such as hypoxia, a temperature
change.
[0204] In another aspect, the invention features a preparation of
chondrisomes made by a process described herein.
[0205] In another aspect, the invention features a method of
delivering a chondrisome preparation to a subject in-vivo, e.g., to
a human in need thereof. The method includes administering to the
subject a pharmaceutical composition or chondrisome preparation
described herein.
[0206] In embodiments the composition or preparation is
administered locally to a target tissue of the subject. In other
embodiments, the composition or preparation is administered
systemically. The composition may be configured for local
administration, or for systemic administration.
[0207] In embodiments, the administration may be for a time and in
an amount sufficient to enhance a cell or tissue function in the
subject; for a time and in an amount sufficient to improve function
of an injured or diseased cell or tissue in the subject; for a time
and in an amount sufficient to increase mitochondrial content
and/or activity in a cell or tissue of the subject; for a time and
in an amount sufficient to induce or decrease (e.g., block)
cellular differentiation, de-differentiation, or
trans-differentiation of the cell or tissue of the subject. The
administration may be for a time and in an amount sufficient to
effect one or more of:
[0208] Increases basal respiration of recipient cells at least 10%
(e.g., >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0209] Chondrisomes of the preparation are taken up by at least 1%
(e.g., at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%) of
recipient cells;
[0210] Chondrisomes of the preparation are taken up and maintain
membrane potential in recipient cells;
[0211] Chondrisomes of the preparation persist in recipient cells
at least 6 hours, e.g., at least 12 hours, 18 hours, 24 hours, 2
days, 3 days, 4 days, a week, 2 weeks, a month, 2 months, 3 months,
6 months;
[0212] increase ATP levels in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0213] decrease apotosis in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0214] decrease cellular lipid levels in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0215] increase membrane potential in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0216] increase uncoupled respiration in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0217] increase PI3K activity in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0218] reduce reductive stress in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0219] decrease reactive oxygen species (e.g. H2O.sub.2) in the
cell, tissue of subject (e.g., in serum of a target subject) (e.g.,
by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 509%, 60%,
70%, 80%, 90%, or more, e.g., compared to a reference value, e.g.,
a control value, e.g., an untreated control)
[0220] Decrease cellular lipid levels of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0221] increases uncoupled respiration of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0222] decrease mitochondrial permeability transition pore (MPTP)
formation in recipient cells at least 5% and does not increase more
than 10% relative to a control;
[0223] increase Akt levels in recipient cells at least 10% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0224] decrease total NAD/NADH ratio in recipient cells at least 5%
(e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0225] Reduce ROS levels in recipient cells at least 5% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0226] Increase fractional shortening in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control;
[0227] Increase end diastolic volume in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control;
[0228] decrease end systolic volume in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control;
[0229] decrease infarct area of ischemic heart at least 5% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0230] increase stroke volume in subject with cardiac ischemia at
least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0231] increase ejection fraction in subject with cardiac ischemia
at least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0232] increase cardia output in subject with cardiac ischemia at
least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0233] increase cardiac index in subject with cardiac ischemia at
least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0234] decrease serum CKNB levels in subject with cardiac ischemia
at least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0235] decrease serum cTnI levels in subject with cardiac ischemia
at least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a
control;
[0236] decrease serum hydrogen peroxide in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control;
[0237] decrease serum cholesterol levels and/or triglycerides in a
subject at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control.
[0238] In another aspect, the invention features a method of
delivering a chondrisome preparation to a cell or tissue ex-vivo.
The method includes contacting the cell or tissue with a
pharmaceutical composition or chondrisome preparation described
herein. The composition or preparation may be delivered to an
isolated or cultured cell or a population thereof (e.g., a cell
therapy preparation), an isolated or cultured tissue (e.g., a
tissue explant or tissue for transplantation, e.g., a human vein, a
musculoskeletal graft such as bone or tendon, cornea, skin, heart
valves, nerves), an isolated or cultured organ (e.g., an organ to
be transplanted into a human, e.g., a human heart, liver, lung,
kidney, pancreas, intestine, thymus, eye).
[0239] In embodiments, the contacting may be for a time and in an
amount sufficient to enhance a function of the cell or tissue; for
a time and in an amount sufficient to improve function of an
injured or diseased cell or tissue; for a time and in an amount
sufficient to improve or enhance viability (e.g., reduce cell
death, e.g., reduce apoptosis or ferroptosis) of the cell or
tissue; for a time and in an amount sufficient to increase
mitochondrial content and/or activity in the cell or tissue; for a
time and in an amount sufficient to induce or decrease (e.g.,
block) cellular differentiation, de-differentiation, or
trans-differentiation of the cell or tissue. The administration may
be for a time and in an amount sufficient to modulate one or more
of these parameters in the subject, e.g., at least 5%, 10%, 15%,
20%, 30%, 40%, 50%, 60%, 70%, 80% or greater, e.g., compared to a
reference (e.g., compared to a control cell or tissue, or compared
to prior to the administration).
[0240] The contacting may be for a time and in an amount sufficient
to modulate, e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%,
70%, 80% or greater, e.g., compared to a reference (e.g., compared
to a control cell or tissue, or compared to prior to the
administration), one or more of:
[0241] Increases basal respiration of recipient cells at least 10%
(e.g., >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0242] Chondrisomes of the preparation are taken up by at least 1%
(e.g., at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%) of
recipient cells;
[0243] Chondrisomes of the preparation are taken up and maintain
membrane potential in recipient cells;
[0244] Chondrisomes of the preparation persist in recipient cells
at least 6 hours, e.g., at least 12 hours, 18 hours, 24 hours, 2
days, 3 days, 4 days, a week, 2 weeks, a month, 2 months, 3 months,
6 months;
[0245] increase ATP levels in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0246] decrease apotosis in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0247] decrease cellular lipid levels in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0248] increase membrane potential in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0249] increase uncoupled respiration in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0250] increase PI3K activity in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0251] reduce reductive stress in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0252] decrease reactive oxygen species (e.g. H2O.sub.2) in the
cell, tissue of subject (e.g., in serum of a target subject) (e.g.,
by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 509%, 60%,
70%, 80%, 90%, or more, e.g., compared to a reference value, e.g.,
a control value, e.g., an untreated control)
[0253] Decrease cellular lipid levels of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0254] increases uncoupled respiration of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0255] decrease mitochondrial permeability transition pore (MPTP)
formation in recipient cells at least 5% and does not increase more
than 10% relative to a control;
[0256] increase Akt levels in recipient cells at least 10% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0257] decrease total NAD/NADH ratio in recipient cells at least 5%
(e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0258] Reduce ROS levels in recipient cells at least 5% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0259] In another aspect, the invention features a method of
enhancing function (e.g., enhancing respiratory function, enhancing
viability), of a target cell or tissue. The method includes
delivering or administering to the target cell or tissue a
composition described herein. The target cell or tissue may be in
an injured state, e.g., from trauma or disease. The composition may
be delivered to the target cell or tissue ex-vivo, in vitro, or
in-vivo in a human subject.
[0260] In embodiments, the contacting may be for a time and in an
amount sufficient to enhance a cell or tissue function; for a time
and in an amount sufficient to improve function of an injured or
diseased cell or tissue; for a time and in an amount sufficient to
increase mitochondrial content and/or activity in the cell or
tissue; for a time and in an amount sufficient to induce or
decrease (e.g., block) cellular differentiation,
de-differentiation, or trans-differentiation of the cell or tissue.
The administration may be for a time and in an amount sufficient to
modulate one or more of these parameters in the subject, e.g., at
least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or greater,
e.g., compared to a reference (e.g., compared to a control subject,
or compared to prior to the administration). The contacting may be
for a time and in an amount sufficient to modulate, e.g., at least
5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or greater, e.g.,
compared to a reference (e.g., compared to a control subject, or
compared to prior to the administration) one or more of:
[0261] Increases basal respiration of recipient cells at least 10%
(e.g., >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0262] Chondrisomes of the preparation are taken up by at least 1%
(e.g., at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%) of
recipient cells;
[0263] Chondrisomes of the preparation are taken up and maintain
membrane potential in recipient cells;
[0264] Chondrisomes of the preparation persist in recipient cells
at least 6 hours, e.g., at least 12 hours, 18 hours, 24 hours, 2
days, 3 days, 4 days, a week, 2 weeks, a month, 2 months, 3 months,
6 months;
[0265] increase ATP levels in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0266] decrease apotosis in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0267] decrease cellular lipid levels in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0268] increase membrane potential in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0269] increase uncoupled respiration in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0270] increase PI3K activity in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0271] reduce reductive stress in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0272] decrease reactive oxygen species (e.g. H2O.sub.2) in the
cell, tissue of subject (e.g., in serum of a target subject) (e.g.,
by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 509%, 60%,
70%, 80%, 90%, or more, e.g., compared to a reference value, e.g.,
a control value, e.g., an untreated control)
[0273] Decrease cellular lipid levels of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0274] increases uncoupled respiration of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0275] decrease mitochondrial permeability transition pore (MPTP)
formation in recipient cells at least 5% and does not increase more
than 10% relative to a control;
[0276] increase Akt levels in recipient cells at least 10% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0277] decrease total NAD/NADH ratio in recipient cells at least 5%
(e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0278] Reduce ROS levels in recipient cells at least 5% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0279] In another aspect, the invention features a method of
increasing mitochondrial content and/or activity in a target cell
or tissue, comprising delivering to the target cell or tissue a
composition described herein. The composition may be delivered to
the target cell or tissue in-vivo in a human subject, or ex-vivo to
a human target cell or tissue. Mitochondrial content and/or
activity may be increased e.g., at least 5%, 10%, 15%, 20%, 30%,
40%, 50%, 60%, 70%, 80% or greater, e.g., compared to a reference
(e.g., compared to a control subject, or compared to prior to the
administration).
[0280] In embodiments, the contacting may be for a time and in an
amount sufficient to enhance a cell or tissue function; for a time
and in an amount sufficient to improve function of an injured or
diseased cell or tissue; for a time and in an amount sufficient to
increase mitochondrial content and/or activity in the cell or
tissue; for a time and in an amount sufficient to induce or
decrease (e.g., block) cellular differentiation,
de-differentiation, or trans-differentiation of the cell or tissue.
The administration may be for a time and in an amount sufficient to
modulate one or more of these parameters in the subject, e.g., at
least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or greater,
e.g., compared to a reference (e.g., compared to a control subject,
or compared to prior to the administration). The contacting may be
for a time and in an amount sufficient to modulate, e.g., at least
5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or greater, e.g.,
compared to a reference (e.g., compared to a control subject, or
compared to prior to the administration):
[0281] Increases basal respiration of recipient cells at least 10%
(e.g., >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0282] Chondrisomes of the preparation are taken up by at least 1%
(e.g., at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%) of
recipient cells;
[0283] Chondrisomes of the preparation are taken up and maintain
membrane potential in recipient cells;
[0284] Chondrisomes of the preparation persist in recipient cells
at least 6 hours, e.g., at least 12 hours, 18 hours, 24 hours, 2
days, 3 days, 4 days, a week, 2 weeks, a month, 2 months, 3 months,
6 months;
[0285] increase ATP levels in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0286] decrease apotosis in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0287] decrease cellular lipid levels in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0288] increase membrane potential in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0289] increase uncoupled respiration in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0290] increase PI3K activity in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0291] reduce reductive stress in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0292] decrease reactive oxygen species (e.g. H2O.sub.2) in the
cell, tissue of subject (e.g., in serum of a target subject) (e.g.,
by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 509%, 60%,
70%, 80%, 90%, or more, e.g., compared to a reference value, e.g.,
a control value, e.g., an untreated control);
[0293] Decrease cellular lipid levels of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0294] increases uncoupled respiration of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0295] decrease mitochondrial permeability transition pore (MPTP)
formation in recipient cells at least 5% and does not increase more
than 10% relative to a control;
[0296] increase Akt levels in recipient cells at least 10% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0297] decrease total NAD/NADH ratio in recipient cells at least 5%
(e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0298] Reduce ROS levels in recipient cells at least 5% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control.
[0299] In another aspect, the invention features a method of
increasing tissue ATP levels, comprising delivering to a target
cell or tissue a composition described herein. The composition may
be delivered to the target cell or tissue in-vivo in a human
subject, or ex-vivo to a human target cell or tissue. Tissue ATP
levels may be increased e.g., at least 5%, 10%, 15%, 20%, 30%, 40%,
50%, 60%, 70%, 80% or greater, e.g., compared to a reference (e.g.,
compared to a control subject, or compared to prior to the
administration).
[0300] In embodiments, the contacting may be for a time and in an
amount sufficient to enhance a cell or tissue function; for a time
and in an amount sufficient to improve function of an injured or
diseased cell or tissue; for a time and in an amount sufficient to
increase mitochondrial content and/or activity in the cell or
tissue; for a time and in an amount sufficient to induce or
decrease (e.g., block) cellular differentiation,
de-differentiation, or trans-differentiation of the cell or tissue.
The administration may be for a time and in an amount sufficient to
modulate one or more of these parameters in the subject, e.g., at
least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or greater,
e.g., compared to a reference (e.g., compared to a control subject,
or compared to prior to the administration). The contacting may be
for a time and in an amount sufficient to modulate, e.g., at least
5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or greater, e.g.,
compared to a reference (e.g., compared to a control subject, or
compared to prior to the administration):
[0301] Increases basal respiration of recipient cells at least 10%
(e.g., >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0302] Chondrisomes of the preparation are taken up by at least 1%
(e.g., at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%) of
recipient cells;
[0303] Chondrisomes of the preparation are taken up and maintain
membrane potential in recipient cells;
[0304] Chondrisomes of the preparation persist in recipient cells
at least 6 hours, e.g., at least 12 hours, 18 hours, 24 hours, 2
days, 3 days, 4 days, a week, 2 weeks, a month, 2 months, 3 months,
6 months;
[0305] increase ATP levels in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0306] decrease apotosis in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0307] decrease cellular lipid levels in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0308] increase membrane potential in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0309] increase uncoupled respiration in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0310] increase PI3K activity in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0311] reduce reductive stress in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0312] decrease reactive oxygen species (e.g. H2O.sub.2) in the
cell, tissue of subject (e.g., in serum of a target subject) (e.g.,
by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 509%, 60%,
70%, 80%, 90%, or more, e.g., compared to a reference value, e.g.,
a control value, e.g., an untreated control)
[0313] Decrease cellular lipid levels of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0314] increases uncoupled respiration of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0315] decrease mitochondrial permeability transition pore (MPTP)
formation in recipient cells at least 5% and does not increase more
than 10% relative to a control;
[0316] increase Akt levels in recipient cells at least 10% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0317] decrease total NAD/NADH ratio in recipient cells at least 5%
(e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0318] Reduce ROS levels in recipient cells at least 5% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control.
[0319] In another aspect, the invention features a method of
delivering a payload or cargo to a subject in need thereof. The
method includes administering to the subject a composition
described herein, wherein the chondrisomes of the composition
comprise the payload. A payload may be a nucleic acid, a small
molecule, a polypeptide, e.g., an agent listed in Table 4.
[0320] In embodiments, the contacting may be for a time and in an
amount sufficient to enhance a cell or tissue function; for a time
and in an amount sufficient to improve function of an injured or
diseased cell or tissue; for a time and in an amount sufficient to
increase mitochondrial content and/or activity in the cell or
tissue; for a time and in an amount sufficient to induce or
decrease (e.g., block) cellular differentiation,
de-differentiation, or trans-differentiation of the cell or tissue.
The administration may be for a time and in an amount sufficient to
modulate one or more of these parameters in the subject, e.g., at
least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or greater,
e.g., compared to a reference (e.g., compared to a control subject,
or compared to prior to the administration). The contacting may be
for a time and in an amount sufficient to modulate, e.g., at least
5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or greater, e.g.,
compared to a reference (e.g., compared to a control subject, or
compared to prior to the administration):
[0321] Increases basal respiration of recipient cells at least 10%
(e.g., >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0322] Chondrisomes of the preparation are taken up by at least 1%
(e.g., at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%) of
recipient cells;
[0323] Chondrisomes of the preparation are taken up and maintain
membrane potential in recipient cells;
[0324] Chondrisomes of the preparation persist in recipient cells
at least 6 hours, e.g., at least 12 hours, 18 hours, 24 hours, 2
days, 3 days, 4 days, a week, 2 weeks, a month, 2 months, 3 months,
6 months;
[0325] increase ATP levels in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0326] decrease apotosis in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0327] decrease cellular lipid levels in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0328] increase membrane potential in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0329] increase uncoupled respiration in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0330] increase PI3K activity in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0331] reduce reductive stress in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0332] decrease reactive oxygen species (e.g. H2O.sub.2) in the
cell, tissue of subject (e.g., in serum of a target subject) (e.g.,
by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 509%, 60%,
70%, 80%, 90%, or more, e.g., compared to a reference value, e.g.,
a control value, e.g., an untreated control)
[0333] Decrease cellular lipid levels of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0334] increases uncoupled respiration of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0335] decrease mitochondrial permeability transition pore (MPTP)
formation in recipient cells at least 5% and does not increase more
than 10% relative to a control;
[0336] increase Akt levels in recipient cells at least 10% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0337] decrease total NAD/NADH ratio in recipient cells at least 5%
(e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0338] Reduce ROS levels in recipient cells at least 5% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control.
[0339] In another aspect, the invention features a method of
treating a subject, e.g., a human, in need thereof. The method
includes administering to the subject (e.g., a subject identified
as having, or diagnosed with, a condition or disease described
herein) a pharmaceutical composition or chondrisome preparation
described herein.
[0340] In one embodiment, the subject is treated for a
mitochondrial disease, e.g., a mitochondrial disease characterized
by a mutation in the mitochondrial genome, or a mitochondrial
disease characterized by a mutation in a nuclear gene associated
with mitochondrial structure or function. In some embodiments, the
subject is treated for a disease or condition associated with
mitochondrial function.
[0341] In one embodiment, the subject is treated for a metabolic
disease or condition, e.g., metabolic syndrome, high blood pressure
(e.g., 130/80 or higher), high blood sugar, excess body fat,
obesity, high cholesterol (e.g., HDL cholesterol 50 mg/dl or lower
in men or 40 mg/dl or lower in women) or triglyceride levels (e.g.,
serum triglycerides 150 mg/dl or above).
[0342] In one embodiment, the subject is treated for a
cardiovascular disorder (e.g. atherosclerosis,
hypercholesterolemia, thrombosis, clotting disorder, myocardial
infarction, sudden cardiac arrest, heart failure, angiogenic
disorder such as macular degeneration, pulmonary hypertension,
critical limb ischemia, critical organ ischemia (e.g. liver, lung,
heart, spleen, pancreas, mesentery, brain), or traumatic brain
injury).
[0343] In one embodiment, the subject is treated for a
neurodegenerative disorder (e.g. Alzheimer's disease, Huntington's
disease, Parkinson's disease, Friedreich's ataxia and other
ataxias, amyotrophic lateral sclerosis (ALS) and other motor neuron
diseases, autism, Duchenne muscular dystrophy);
[0344] In one embodiment, the subject is treated for a
neuropsychiatric disease (e.g., bipolar disorder, depression,
schizophrenia, Rett's syndrome).
[0345] In one embodiment, the subject is treated for a neuropathy
or myopathy, such as Leber's hereditary optic neuropathy (LHON),
encephalopathy, lactacidosis, myoclonic epilepsy with ragged red
fibers (MERFF); epilepsy; and mitochondrial myopathy.
[0346] In one embodiment, the subject is treated for an infectious
disease (e.g. a viral infection (e.g., HIV, HCV, RSV), a bacterial
infection, a fungal infection, sepsis).
[0347] In other embodiments, the subject is treated for an
autoimmune disorder (e.g. diabetes, lupus, multiple sclerosis,
psoriasis, rheumatoid arthritis); an inflammatory disorder (e.g.
arthritis, pelvic inflammatory disease); a proliferative disorder
(e.g. cancer, benign neoplasms); a respiratory disorder (e.g.
chronic obstructive pulmonary disease); a digestive disorder (e.g.
inflammatory bowel disease, ulcer); a musculoskeletal disorder
(e.g. fibromyalgia, arthritis); an endocrine, metabolic, or
nutritional disorder (e.g. diabetes, osteoporosis); an urological
disorder (e.g. renal disease); a psychological disorder (e.g.
depression, schizophrenia); a skin disorder (e.g. wounds, eczema);
or a blood or lymphatic disorder (e.g. anemia, hemophilia); an
optical disorder (e.g., glaucoma, optic neuropathy).
[0348] In each of the above embodiments, the method includes
administering the pharmaceutical composition or chondrisome
preparation in combination with a second therapeutic agent, e.g., a
standard-of-care agent for treatment of the disease or
condition.
[0349] The administration may be for a time and in an amount
sufficient to enhance a cell or tissue function in the subject. The
administration may be for a time and in an amount sufficient to
improve function of an injured cell or diseased tissue in the
subject. The administration may be for a time and in an amount
sufficient to increase mitochondrial content and/or activity in a
cell or tissue of the subject. The administration may be for a time
and in an amount sufficient to increase tissue ATP levels in the
subject. The administration may be for a time and in an amount
sufficient to induce or decrease (e.g., block) cellular
differentiation, de-differentiation, or trans-differentiation.
[0350] The administration may be for a time and in an amount
sufficient to effect one or more of:
[0351] Increases basal respiration of recipient cells at least 10%
(e.g., >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0352] Chondrisomes of the preparation are taken up by at least 1%
(e.g., at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%) of
recipient cells;
[0353] Chondrisomes of the preparation are taken up and maintain
membrane potential in recipient cells;
[0354] Chondrisomes of the preparation persist in recipient cells
at least 6 hours, e.g., at least 12 hours, 18 hours, 24 hours, 2
days, 3 days, 4 days, a week, 2 weeks, a month, 2 months, 3 months,
6 months;
[0355] increase ATP levels in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0356] decrease apotosis in a recipient cell, tissue or subject
(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, 90%, or more, e.g., compared to a reference value,
e.g., a control value, e.g., an untreated control);
[0357] decrease cellular lipid levels in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0358] increase membrane potential in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0359] increase uncoupled respiration in a recipient cell, tissue
or subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0360] increase PI3K activity in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0361] reduce reductive stress in a recipient cell, tissue or
subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 509%, 60%, 70%, 80%, 90%, or more, e.g., compared to a
reference value, e.g., a control value, e.g., an untreated
control);
[0362] decrease reactive oxygen species (e.g. H2O.sub.2) in the
cell, tissue of subject (e.g., in serum of a target subject) (e.g.,
by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 509%, 60%,
70%, 80%, 90%, or more, e.g., compared to a reference value, e.g.,
a control value, e.g., an untreated control)
[0363] Decrease cellular lipid levels of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0364] increases uncoupled respiration of recipient cells at least
5% (e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0365] decrease mitochondrial permeability transition pore (MPTP)
formation in recipient cells at least 5% and does not increase more
than 10% relative to a control;
[0366] increase Akt levels in recipient cells at least 10% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0367] decrease total NAD/NADH ratio in recipient cells at least 5%
(e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control;
[0368] Reduce ROS levels in recipient cells at least 5% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control;
[0369] In one aspect, the invention features a pharmaceutical
composition comprising a preparation of chondrisomes expressing an
exogenous protein. In another aspect, the invention features a
pharmaceutical composition comprising a preparation of chondrisomes
expressing an uncoupling protein.
[0370] In one embodiment, the pharmaceutical preparation of
chondrisomes expresses an uncoupling protein, e.g., UCP1, UCP2,
UCP3, UCP4 or UCP5. The uncoupling protein may be endogenous or
heterologous (exogenous) to the source mitochondria (e.g., the
uncoupling protein may be naturally expressed, or the source
mitochondria or chondrisomes may be modified (e.g., genetically
modified or loaded) to express or over-express the protein. The
source mitochondria or chondrisomes may be genetically modified by,
e.g., the introduction of modified RNA, or DNA, encoding the
protein.
[0371] In one embodiment, the source mitochondria or chondrisomes
are engineered to express a protein at least 85%, 90%, 95%, 97%,
98%, 100% identical to the sequence of human UCP1 (SEQ ID NO:1),
human UCP2 (SEQ ID NO:2), human UCP3 (SEQ ID NO:3), human UCP4 (SEQ
ID NO:4) or human UCP5 (SEQ ID NO:5), wherein the protein has
transporter activity in the chondrisomes.
[0372] In one embodiment, the chondrisomes are derived from brown
fat of a subject, e.g., a human. In one embodiment, the
chondrisomes are derived from a non-brown fat source.
[0373] In another aspect, the invention features a method of
increasing thermogenesis in a subject, reducing fat tissue mass in
a subject, and/or increasing mitochondrial number or function in
the fat tissue of a subject (e.g., in a target cell or tissue of a
subject). The method includes delivering to the target cell or
tissue a composition described herein. The composition may be
delivered to the target cell or tissue in-vivo, e.g., in a human
subject, or ex-vivo to a human target cell or tissue.
[0374] In embodiments, the target cell or tissue is white
adipocytes.
[0375] In embodiments, the mitochondria of the composition express
a transporter, e.g., UCP1, UCP2, UCP3, UCP4 or UCP5. The expressed
transporter may be endogenous or heterologous to the source
mitochondria (e.g., the transporter may be naturally expressed, or
the mitochondria may be modified (e.g., genetically modified or
loaded) to express or over-express the transporter. In one
embodiment, the mitochondria are engineered to express a protein at
least 85%, 90%, 95%, 97%, 98%, 100% identical to the sequence of
human UCP1 (SEQ ID NO:1), human UCP2 (SEQ ID NO:2), human UCP3 (SEQ
ID NO:3), human UCP4 (SEQ ID NO:4) or human UCP5 (SEQ ID NO:5),
wherein the protein has transporter activity in the
chondrisomes.
[0376] In embodiments, the chondrisomes of the composition are
isolated at least in part from brown adipocytes, e.g., from a live
or deceased donor subject. In other embodiments, the chondrisomes
of the composition are isolated at least in part from brown
adipocytes in culture (e.g., a primary culture of brown adipocytes,
or an immortalized brown adipocyte cell line).
[0377] In embodiments, the source mitochondria or chondrisomes of
the composition are autologous to the subject. In other
embodiments, the source mitochondria or chondrisomes of the
composition are allogeneic.
[0378] In embodiments, the composition is administered to fat
tissue, e.g., to white adipocytes, of the subject.
[0379] In embodiments, thermogenesis may be increased, and/or fat
tissue mass or volume is reduced, and/or mitochondrial number or
function may increase e.g., at least 5%, 10%, 15%, 20%, 30%, 40%,
50%, 60%, 70%, 80% or greater compared to a reference (e.g.,
compared to a control subject, or compared to prior to the
administration).
[0380] The administration may be for a time and in an amount
sufficient to promote weight loss in the subject.
[0381] In another aspect, the invention features a method of
modulating one or more serum composition, e.g., modulating one or
more serum metabolites, e.g., decreasing serum cholesterol and/or
triglycerides in a subject in need thereof. The method includes
administering to the subject a pharmaceutical composition described
herein. The method includes delivering to the target cell or tissue
a composition described herein. The composition may be delivered to
the target cell or tissue in-vivo, e.g., in a human subject, or
ex-vivo to a human target cell or tissue.
[0382] In embodiments, the target cell or tissue is a fat tissue of
the subject, e.g., white adipocytes.
[0383] In embodiments, the source mitochondria or chondrisomes of
the composition express a transporter, e.g., UCP1, UCO2, UCP3, UCP4
or UCP5. The expressed transporter may be endogenous or
heterologous to the source mitochondria (e.g., the transporter may
be naturally expressed, or the mitochondria may be modified (e.g.,
genetically modified or loaded) to express or over-express the
transporter. In one embodiment, the mitochondria are engineered to
express a protein at least 85%, 90%, 95%, 97%, 98%, 100% identical
to the sequence of human UCP1 (SEQ ID NO:1), human UCP2 (SEQ ID
NO:2), human UCP3 (SEQ ID NO:3), human UCP4 (SEQ ID NO:4) or human
UCP5 (SEQ ID NO:5), wherein the protein has transporter activity in
the mitochondria.
[0384] In embodiments, the chondrisomes of the composition are
isolated at least in part from brown adipocytes.
[0385] In embodiments, the source mitochondria or chondrisomes of
the composition are autologous to the subject. In other
embodiments, the source mitochondria or chondrisomes of the
composition are allogeneic.
[0386] In embodiments, the composition is administered to a fat
tissue, e.g., white adipocytes of the subject.
[0387] In embodiments, serum cholesterol in the subject may be
reduced e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,
80% or greater compared to a reference (e.g., compared to a control
subject, or compared to prior to the administration).
[0388] In embodiments, serum triglycerides may be reduced in the
subject, e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,
80% or greater compared to a reference (e.g., compared to a control
subject, or compared to prior to the administration).
[0389] In another aspect, the invention features a method of making
a pharmaceutical preparation suitable for administration to a human
subject, comprising:
[0390] (a) providing a parent cell or tissue source of
mitochondria,
[0391] (b) isolating a preparation of chondrisomes from the parent
cell or tissue source (e.g., as described herein), and
[0392] (c) evaluating (e.g., testing or measuring) a sample of the
preparation for one or more of the following characteristics:
[0393] the chondrisomes of the preparation have a mean average size
between 150-1500 nm, e.g., between 200-1200 nm, e.g., between
500-1200 nm, e.g., 175-950 nm;
[0394] the chondrisomes of the preparation have a polydispersity
(D90/D10) between 1.1 to 6, e.g., between 1.5-5. In embodiments,
chondrisomes of a preparation from a cultured cell source (e.g.,
cultured fibroblasts) have a polydispersity (D90/D10) between 2-5,
e.g., between 2.5-5;
[0395] outer chondrisome membrane integrity wherein the preparation
exhibits <20% (e.g., <15%, <10%, <5%, <4 , <3%,
<2%, <1%) increase in oxygen consumption rate over state 4
rate following addition of reduced cytochrome c;
[0396] complex I level of 1-8 mOD/ug total protein, e.g., 3-7
mOD/ug total protein, 1-5 mOD/ug total protein. In embodiments,
chondrisomes of a preparation from a cultured cell source (e.g.,
cultured fibroblasts) have a complex I level of 1-5 mOD/ug total
protein;
[0397] complex II level of 0.05-5 mOD/ug total protein, e.g., 0.1-4
mOD/ug total protein, e.g., 0.5-3 mOD/ug total protein. In
embodiments, chondrisomes of a preparation from a cultured cell
source (e.g., cultured fibroblasts) have a complex II level of
0.05-1 mOD/ug total protein;
[0398] complex III level of 1-30 mOD/ug total protein, e.g., 2-30,
5-10, 10-30 mOD/ug total protein. In embodiments, chondrisomes of a
preparation from a cultured cell source (e.g., cultured
fibroblasts) have a complex III level of 1-5 mOD/ug total
protein;
[0399] complex IV level of 4-50 mOD/ug total protein, e.g., 5-50,
e.g., 10-50, 20-50 mOD/ug total protein. In embodiments,
chondrisomes of a preparation from a cultured cell source (e.g.,
cultured fibroblasts) have a complex IV level of 3-10 mOD/ug total
protein;
[0400] genomic concentration 0.001-2 (e.g., 0.001-1, 0.01-1,
0.01-.1, 0.01-.05, 0.1-.2) mtDNA ug/mg protein;
[0401] membrane potential of the preparation is between -5 to -200
mV, e.g., between -100 to -200 mV, -50 to -200 mV, -50 to -75 mV,
-50 to -100 mV. In some embodiments, membrane potential of the
preparation is less than -150 mV, less than -100 mV, less than -75
mV, less than -50 mV, e.g., -5 to -20 mV;
[0402] a protein carbonyl level of less than 100 nmol carbonyl/mg
chondrisome protein (e.g., less than 90 nmol carbonyl/mg
chondrisome protein, less than 80 nmol carbonyl/mg chondrisome
protein, less than 70 nmol carbonyl/mg chondrisome protein, less
than 60 nmol carbonyl/mg chondrisome protein, less than 50 nmol
carbonyl/mg chondrisome protein, less than 40 nmol carbonyl/mg
chondrisome protein, less than 30 nmol carbonyl/mg chondrisome
protein, less than 25 nmol carbonyl/mg chondrisome protein, less
than 20 nmol carbonyl/mg chondrisome protein, less than 15 nmol
carbonyl/mg chondrisome protein, less than 10 nmol carbonyl/mg
chondrisome protein, less than 5 nmol carbonyl/mg chondrisome
protein, less than 4 nmol carbonyl/mg chondrisome protein, less
than 3 nmol carbonyl/mg chondrisome protein;
[0403] <20% mol/mol ER proteins (e.g., >15%, >10%, >5%,
>3%, >2%, >1%) mol/mol ER proteins;
[0404] >5% mol/mol mitochondrial proteins (proteins identified
as mitochondrial in the MitoCarta database (Calvo et al., NAR 20151
doi:10.1093/nar/gkv1003)), e.g., >10%, >15%, >20%,
>25%, >30%, >35%, >40%; >50%, >55%, >60%,
>65%, >70%, >75%, >80%; >90% mol/mol mitochondrial
proteins);
[0405] >0.05% mol/mol of MT-CO2, MT-ATP6, MT-ND5 and MT-ND6
protein (e.g., >0.1%; >05%, >1%, >2%, >3%, >4%,
>5%, >7, >8%, >9%, >10, >15% mol/mol of MT-CO2,
MT-ATP6, MT-ND5 and MT-ND6 protein);
[0406] Genetic quality >80%, e.g., >85%, >90%, >95%,
>97%, >98%, >99%;
[0407] Relative ratio mtDNA/nuclear DNA is >1000 (e.g.,
>1,500, >2000, >2,500, >3,000, >4,000, >5000,
>10,000, >25,000, >50,000, >100,000, >200,000,
>500,000);
[0408] Endotoxin level <0.2 EU/ug protein (e.g., <0.1, 0.05,
0.02, 0.01 EU/ug protein);
[0409] Substantially absent exogenous non-human serum;
[0410] Glutamate/malate RCR 3/2 of 1-15, e.g., 2-15, 5-15, 2-10,
2-5, 10-15;
[0411] Glutamate/malate RCR 3/4o of 1-30, 1-20, 2-20, 5-20, 3-15,
10-30;
[0412] Succinate/rotenone RCR 3/2 of 1-15, 2-15, 5-15, 1-10,
10-15;
[0413] Succinate/rotenone RCR 3/4o of 1-30, 1-20, 2-20, 5-20, 3-15,
10-30;
[0414] complex I activity of 0.05-100 nmol/min/mg total protein
(e.g., 0.05-50, 0.05-20, 0.5-10, 0.1-50, 1-50, 2-50, 5-100, 1-20
nmol/min/mg total protein);
[0415] complex II activity of 0.05-50 nmol/min/mg total protein
(e.g., 0.05-50, 0.05-20, 0.5-10, 0.1-50, 1-50, 2-50, 5-50, 1-20
nmol/min/mg total protein);
[0416] complex III activity of 0.05-20 nmol/min/mg total protein
(e.g., 0.05-50, 0.05-20, 0.5-10, 0.1-50, 1-50, 2-50, 5-100, 1-20
nmol/min/mg total protein);
[0417] complex IV activity of 0.1-50 nmol/min/mg total protein
(e.g., 0.05-50, 0.05-20, 0.5-10, 0.1-50, 1-50, 2-50, 5-50, 1-20
nmol/min/mg total protein);
[0418] complex V activity of 1-500 nmol/min/mg total protein (e.g.,
10-500, 10-250, 10-200, 100-500 nmol/min/mg total protein);
[0419] reactive oxygen species (ROS) production level of 0.01-50
pmol H2O2/ug protein/hr (e.g., 0.05-40, 0.05-25, 1-20, 2-20,
0.05-20, 1-20 pmol H2O2/ug protein/hr);
[0420] Citrate Synthase activity of 0.05-5 (e.g., 0.5-5, 0.5-2,
1-5, 1-4) mOD/min/ug total protein;
[0421] Alpha ketoglutarate dehydrogenase activity of 0.05-10 (e.g.,
0.1-10, 0.1-8, 0.5-8, 0.1-5, 0.5-5, 0.5-3, 1-3) mOD/min/ug total
protein;
[0422] Creatine Kinase activity of 0.1-100 (e.g., 0.5-50, 1-100,
1-50, 1-25, 1-15, 5-15) mOD/min/ug total protein;
[0423] Pyruvate dehydrogenase activity of 0.1-10 (e.g., 0.5-10,
0.5-8, 1-10, 1-8, 1-5, 2-3) mOD/min/ug total protein;
[0424] Aconitase activity of 0.1-50 (e.g., 5-50, 0.1-2, 0.1-20,
0.5-30) mOD/min/ug total protein. In embodiments, aconitase
activity in a chondrisome preparation from platelets is between
0.5-5 mOD/min/ug total protein. In embodiments, aconitase activity
in a chondrisome preparation from cultured cells, e.g.,
fibroblasts, is between 5-50 mOD/min/ug total protein;
[0425] Maximal fatty acid oxidation level of 0.05-50 (e.g.,
0.05-40, 0.05-30, 0.05-10, 0.5-50, 0.5-25, 0.5-10, 1-5) pmol
O2/min/ug chondrisome protein;
[0426] Palmitoyl carnitine & Malate RCR3/2 state 3/state 2
respiratory control ratio (RCR 3/2) of 1-10 (e.g., 1-5);
[0427] electron transport chain efficiency of 1-1000 (e.g.,
10-1000, 10-800, 10-700, 50-1000, 100-1000, 500-1000, 10-400,
100-800) nmol O2/min/mg protein/.DELTA.GATP (in kcal/mol)
[0428] total lipid content of 50,000-2,000,000 pmol/mg (e.g.,
50,000-1,000,000; 50,000-500,000 pmol/mg);
[0429] double bonds/total lipid ratio of 0.8-8 (e.g., 1-5, 2-5,
1-7, 1-6) pmol/pmol;
[0430] phospholipid/total lipid ratio of 50-100 (e.g., 60-80,
70-100, 50-80) 100*pmol/pmol;
[0431] phosphosphingolipid/total lipid ratio of 0.2-20 (e.g.,
0.5-15, 0.5-10, 1-10, 0.5-10, 1-5, 5-20) 100*pmol/pmol.
[0432] ceramide content 0.05-5 (e.g., 0.1-5, 0.1-4, 1-5, 0.05-3)
100*pmol/pmol total lipid;
[0433] cardiolipin content 0.05-25 (0.1-20, 0.5-20, 1-20, 5-20,
5-25, 1-25, 10-25, 15-25) 100*pmol/pmol total lipid;
[0434] lyso-phosphatidylcholine (LPC) content of 0.05-5 (e.g.,
0.1-5, 1-5, 0.1-3, 1-3, 0.05-2) 100*pmol/pmol total lipid;
[0435] Lyso-Phosphatidylethanolamine (LPE) content of 0.005-2
(e.g., 0.005-1, 0.05-2, 0.05-1) 100*pmol/pmol total lipid;
[0436] Phosphatidylcholine (PC) content of 10-80 (e.g., 20-60,
30-70, 20-80, 10-60 m 30-50) 100*pmol/pmol total lipid;
[0437] Phosphatidylcholine-ether (PC O-) content 0.1-10 (e.g.,
0.5-10, 1-10, 2-8, 1-8) 100*pmol/pmol total lipid;
[0438] Phosphatidylethanolamine (PE) content 1-30 (e.g., 2-20,
1-20, 5-20) 100*pmol/pmol total lipid;
Phosphatidylethanolamine-ether (PE O-) content 0.05-30 (e.g.,
0.1-30, 0.1-20, 1-20, 0.1-5, 1-10, 5-20) 100*pmol/pmol total
lipid;
[0439] Phosphatidylinositol (PI) content 0.05-15 (e.g., 0.1-15,
0.1-10, 1-10, 0.1-5, 1-10, 5-15) 100*pmol/pmol total lipid;
[0440] Phosphatidylserine (PS) content 0.05-20 (e.g., 0.1-15,
0.1-20, 1-20, 1-10, 0.1-5, 1-10, 5-15) 100*pmol/pmol total
lipid;
[0441] Sphingomyelin (SM) content 0.01-20 (e.g., 0.01-15, 0.01-10,
0.5-20, 0.5-15, 1-20, 1-15, 5-20) 100*pmol/pmol total lipid;
[0442] Triacylglycerol (TAG) content 0.005-50 (e.g., 0.01-50,
0.1-50, 1-50, 5-50, 10-50, 0.005-30, 0.01-25, 0.1-30) 100*pmol/pmol
total lipid;
[0443] PE:LPE ratio 30-350 (e.g., 50-250, 100-200, 150-300);
[0444] PC:LPC ratio 30-700 (e.g., 50-300, 50-250, 100-300, 400-700,
300-500, 50-600, 50-500, 100-500, 100-400);
[0445] PE 18:n (n>0) content 0.5-20% (e.g., 1-20%, 1-10%, 5-20%,
5-10%, 3-9%) pmol AA/pmol lipid class;
[0446] PE 20:4 content 0.05-20% (e.g., 1-20%, 1-10%, 5-20%, 5-10%)
pmol AA/pmol lipid class;
[0447] PC 18:n (n>0) content 5-50% (e.g., 5-40%, 5-30%, 20-40%,
20-50%) pmol AA/pmol lipid class;
[0448] PC 20:4 content 1-20% (e.g., 2-20%, 2-15%, 5-20%, 5-15%)
pmol AA/pmol lipid class; and
[0449] (d) processing the preparation for administration to a human
subject if one or more (2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the
characteristics meet a pre-determined reference value (e.g., a
reference value recited above), thereby making a pharmaceutical
preparation suitable for administration to a human subject.
[0450] In some embodiments, processing includes formulating,
packaging, labeling or selling for human use.
[0451] In some embodiments, the pre-determined reference value is a
quality control potency assay. In some embodiments, the
pre-determined reference value is a quality control identity assay.
In some embodiments, the pre-determined reference value is a
manufacturing release assay.
[0452] In another aspect, the invention features methods of
delivering a composition or preparation described herein to an
ischemic tissue or subject, and methods of reducing, improving or
treating ischemia in a tissue or subject.
[0453] In one embodiment, the methods include: providing to the
tissue or subject (e.g., in-vivo or ex-vivo) a pharmaceutical
composition comprising a preparation of chondrisomes described
herein.
[0454] In one embodiment, the methods include: providing to the
tissue or subject (e.g., in-vivo or ex-vivo) a pharmaceutical
composition comprising a preparation of chondrisomes having at
least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) of the following
characteristics: [0455] (a) the chondrisomes of the preparation
have a polydispersity (D90/D10) between 1.1 to 6, e.g., between
1.5-5, between 2-5, e.g., between 2.5-5; [0456] (b) outer
chondrisome membrane integrity wherein the preparation exhibits
<20% (e.g., <15%, <10%, <5%, <4 , <3%, <2%,
<1%) increase in oxygen consumption rate over state 4 rate
following addition of reduced cytochrome c; [0457] (c) a protein
carbonyl level of less than 100 nmol carbonyl/mg chondrisome
protein (e.g., less than 90 nmol carbonyl/mg chondrisome protein,
less than 80 nmol carbonyl/mg chondrisome protein, less than 70
nmol carbonyl/mg chondrisome protein, less than 60 nmol carbonyl/mg
chondrisome protein, less than 50 nmol carbonyl/mg chondrisome
protein, less than 40 nmol carbonyl/mg chondrisome protein, less
than 30 nmol carbonyl/mg chondrisome protein, less than 25 nmol
carbonyl/mg chondrisome protein, less than 20 nmol carbonyl/mg
chondrisome protein, less than 15 nmol carbonyl/mg chondrisome
protein, less than 10 nmol carbonyl/mg chondrisome protein, less
than 5 nmol carbonyl/mg chondrisome protein, less than 4 nmol
carbonyl/mg chondrisome protein, less than 3 nmol carbonyl/mg
chondrisome protein; [0458] (d) cardiolipin content 0.05-25
(0.1-20, 0.5-20, 1-20, 5-20, 5-25, 1-25, 10-25, 15-25)
100*pmol/pmol total lipid; [0459] (e) Sphingomyelin (SM) content
0.01-20 (e.g., 0.01-15, 0.01-10, 0.5-20, 0.5-15, 1-20, 1-15, 5-20)
100*pmol/pmol total lipid; [0460] (f) substantially lacks
detectable amounts of endotoxin, infectious agent, and exogenous
serum.
[0461] In one embodiment, the methods include providing to the
tissue or subject (e.g., in-vivo or ex-vivo) a pharmaceutical
composition comprising a preparation of chondrisomes in an amount
and for a time sufficient to modulate (e.g., decrease) a cardiac
protein in the tissue or subject (e.g., in-vivo or ex-vivo)
comprising: contacting the cell with a composition comprising a
preparation of chondrisomes in an amount and for a time sufficient
to modulate (e.g., decrease) the cardiac protein in the cell. In
embodiments, the cardiac protein is troponin I, troponin T,
creatine kinase (CK-MB) or combinations thereof. In embodiments,
the composition further decreases at least one selected from the
group consisting of myoglobin, B-type natriuretic peptide, and
high-sensitivity C-reactive protein (hs-CRP). The composition is
provided for a time and in an amount sufficient to reduce or
improve ischemia in the tissue or subject.
[0462] In one embodiment, the methods include providing to the
tissue or subject, a pharmaceutical composition comprising a
composition or preparation of chondrisomes described herein in an
amount and for a time sufficient to decrease apoptosis and/or
ferroptosis in the cell or subject.
[0463] In one embodiment, the methods include providing to the
tissue or subject, a pharmaceutical composition comprising a
composition or preparation of chondrisomes described herein in an
amount and for a time sufficient to decrease reactive oxygen
species (e.g. H2O.sub.2) in the tissue or subject.
[0464] In one embodiment, the chondrisomes of the composition have
a bioenergetic characteristic selected from the group consisting
of:
[0465] electron transport chain efficiency of 1-1000 (e.g.,
10-1000, 10-800, 10-700, 50-1000, 100-1000, 500-1000, 10-400,
100-800) nmol O2/min/mg protein/.DELTA.GATP (in kcal/mol)
[0466] Alpha ketoglutarate dehydrogenase activity of 0.05-10 (e.g.,
0.1-10, 0.1-8, 0.5-8, 0.1-5, 0.5-5, 0.5-3, 1-3) mOD/min/ug total
protein;
[0467] Maximal fatty acid oxidation level of 0.05-50 (e.g.,
0.05-40, 0.05-30, 0.05-10, 0.5-50, 0.5-25, 0.5-10, 1-5) pmol
O2/min/ug chondrisome protein;
[0468] Pyruvate dehydrogenase activity of 0.1-10 (e.g., 0.5-10,
0.5-8, 1-10, 1-8, 1-5, 2-3) mOD/min/ug total protein;
[0469] In embodiments where the subject or tissue has cardiac
ischemia, the composition is administered in an amount and for a
time sufficient to: [0470] (a) Reduce ROS levels in recipient cells
at least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a control;
[0471] (b) Increase fractional shortening in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control; [0472] (c) Increase end diastolic volume in subject with
cardiac ischemia at least 5% (e.g., >10%, >15%, >20%,
>30%, >40%, >50%, >60%, >70%, >80%, >90%)
relative to a control; [0473] (d) decrease end systolic volume in
subject with cardiac ischemia at least 5% (e.g., >10%, >15%,
>20%, >30%, >40%, >50%, >60%, >70%, >80%,
>90%) relative to a control; [0474] (e) increase stroke volume
in subject with cardiac ischemia at least 5% (e.g., >10%,
>15%, >20%, >30%, >40%, >50%, >60%, >70%,
>80%, >90%) relative to a control; [0475] (f) increase
ejection fraction in subject with cardiac ischemia at least 5%
(e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control; [0476]
(g) increase cardia output in subject with cardiac ischemia at
least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a control;
[0477] (h) increase cardiac index in subject with cardiac ischemia
at least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a control;
[0478] (i) decrease serum CKNB levels in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control; [0479] (j) decrease serum cTnI levels in subject with
cardiac ischemia at least 5% (e.g., >10%, >15%, >20%,
>30%, >40%, >50%, >60%, >70%, >80%, >90%)
relative to a control; [0480] (k) decrease serum hydrogen peroxide
in subject with cardiac ischemia at least 5% (e.g., >10%,
>15%, >20%, >30%, >40%, >50%, >60%, >70%,
>80%, >90%) relative to a control; [0481] (l) decrease serum
cholesterol levels and/or triglycerides in a subject at least 5%
(e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control. [0482]
(m) improve (e.g., increase) ejection fraction (e.g., at least 5%,
10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or greater compared to
a reference (e.g., compared to a control subject, or compared to
prior to the administration)); [0483] (n) decrease infarcted area
(% IR/AAR) (e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%,
70%, 80% or greater compared to a reference (e.g., compared to a
control subject, or compared to prior to the administration));
[0484] (o) decrease blood creatine kinase (e.g., CK-MB) levels
(e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or
greater compared to a reference (e.g., compared to a control
subject, or compared to prior to the administration)) and/or [0485]
(p) decrease blood cTnI levels (e.g., at least 5%, 10%, 15%, 20%,
30%, 40%, 50%, 60%, 70%, 80% or greater compared to a reference
(e.g., compared to a control subject, or compared to prior to the
administration)
[0486] For all aspects described herein:
[0487] In embodiments of the compositions or methods described
herein, chondrisomes of the preparation are associated with, e.g.,
internalized into, or partially fused with, the target tissue or
cell. In some embodiments, chondrisomes of the preparation are
internalized into the cytosol of a target cell (e.g., >5% e.g.,
>10%, >20%, >30%, >40%, >50%, >60%, >70%,
>80% or >90% of associated chondrisomes are internalized into
the cytosol of the target cell). In some embodiments, chondrisomes
of the preparation are associated with the endogenous mitochondrial
network of a target cell (e.g., >5% e.g., >10%, >20%,
>30%, >40%, >50%, >60%, >70%, >80% or >90% of
associated chondrisomes are internalized into the endogenous
mitochondrial network of the target cell). In some embodiments,
chondrisomes of the preparation are internalized into the lysosomes
of a target cell (e.g., between 1-90%, e.g., <90%, <80%,
<70%, <60%, <50%, <40%, <30%, <20%, <10% of
associated chondrisomes are internalized into the cytosol of the
target cell). In some embodiments, chondrisomes of the preparation
are associated with the mitochondrial outer membrane of a target
cell (e.g., >5% e.g., >10%, >20%, >30%, >40%,
>50%, >60%, >70%, >80% or >90% of associated
chondrisomes are associated with the mitochondrial outer membrane
of the target cell).
[0488] In embodiments of the compositions or methods described
herein, greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or
more of the chondrisomes of a preparation are internalized into the
target tissue or cell. In other embodiments, chondrisomes of the
preparation are internalized into the cytosol of a target cell,
e.g., greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or
more of the chondrisomes are internalized into the cytosol. In some
embodiments, less than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%
of the chondrisomes are internalized into lysosomes of the target
cells.
[0489] In embodiments of the compositions or methods described
herein, the target tissue or cell is selected from the group
consisting of: epithelial, connective, muscular, and nervous tissue
or cell.
[0490] In embodiments of the methods described herein, the
chondrisome composition or preparation is delivered over a period
of time, e.g., over a period of hours or days. In some embodiments,
a chondrisome composition or preparation is contacted with a target
cell, tissue or subject over a period of time, during which
association of the chondrisomes with the target cell, tissue or
subject increases over time, e.g., as measured by increased
detection of a cargo or payload delivered to the target cell,
tissue or subject by the preparation, over time.
[0491] In embodiments of the methods described herein, the
chondrisome composition or preparation is treated with an agent,
and/or administered in combination with an agent, to modulate
subcellular targeting of the administered preparation. In
embodiments, the agent enables endosomal/lysosomal escape and/or
enhances cytosolic or non-lysosomal delivery of the preparation. In
embodiments, the agent is a peptide or protein that enhances
cytosolic or non-lysosomal delivery of the preparation, e.g.,
haemagglutinin, diINF-7, penton base, gp41, gp41/polyethylenimine,
TAT, L2 from Papillomavirus, envelope protein (E) of West Nile
virus, listeriolysin O (LLO), Pneumococcal pneumolysin (PLO),
Streptococcal streptolysin O (SLO), Diphtheria toxin (DT),
Pseudomonas aeruginosa exotoxin A (ETA), Shiga toxin, cholera
toxin, ricin, saporin, gelonin, human calcitonin derived peptide,
fibroblast growth factors receptor (FGFR3), melittin, (R-Ahx-R)(4)
AhxB, glycoprotein H (gpH) from herpes simplex, KALA, GALA, a
synthetic surfactant, penetratin (pAntp), R6-Penetratin with
arginine-residues, EB1, bovine prion protein (bPrPp), Poly
(L-histidine), Sweet Arrow Peptide (SAP). In other embodiments, the
agent is a chemical that enhances cytosolic or non-lysosomal
delivery of the preparation, e.g., polyethylenimine (PEI),
Poly(amidoamine)s (PAAs), poly(propylacrylic acid) (PPAA), ammonium
chloride, chloroquine, methylamine.
[0492] In embodiments of the compositions or methods described
herein, the target tissue or cell is in the digestive system, the
endocrine system, the excretory system, the lymphatic system, the
skin, muscle, the nervous system, the reproductive system, the
respiratory system, or the skeletal system.
[0493] In embodiments of the compositions or methods described
herein, the chondrisomes are obtained from a human cell or tissue
source, or from human cells in culture.
[0494] In embodiments of the compositions or methods described
herein, the chondrisomes are obtained from a cell type different
than the target tissue or cell type.
[0495] In embodiments of the compositions or methods described
herein, the chondrisomes are obtained from the same cell type as
the target tissue or cell type.
[0496] In any composition, preparation, or method described herein,
the chondrisomes may be encapsulated.
[0497] In embodiments of the methods described herein, the
chondrisomes are autologous to the subject. In other embodiments of
the methods described herein, the chondrisomes are allogeneic to
the subject. The subject may be an animal, e.g., a mammal, e.g., a
human.
[0498] In embodiments of the methods described herein, the
compositions or preparations are administered locally to a tissue
or organ of a subject (e.g., by local injection or perfusion). In
other embodiments, the compositions or preparations are
administered systemically to a subject.
[0499] In embodiments of the compositions or methods described
herein, the parent cell source is selected from the group
consisting of: circulating cells (e.g., platelets, leukocytes),
muscle cells (e.g., cardiomyocytes, skeletal muscle, smooth
muscle), epidermal cells (e.g., keratinocytes, melanocytes), dermal
cells, hypodermal cells (e.g., fibroblasts, brown adipocytes), stem
cells (e.g., mesenchymal stem cells, hemapoietic stem cells, neural
stem cells), liver cells, kidney cells, pancreas cells, spleen
cells.
[0500] In embodiments of the compositions or methods described
herein, the target cell or tissue is from, or the subject is, a
subject who has or is at risk for: ischemia; a mitochondrial
disease (e.g., a genetic mitochondrial disease); an infectious
disease (e.g. a viral infection (e.g., HIV, HCV, RSV), a bacterial
infection, a fungal infection, sepsis); cardiovascular disorder
(e.g. atherosclerosis, hypercholesterolemia, thrombosis, clotting
disorder, angiogenic disorder such as macular degeneration); an
autoimmune disorder (e.g. diabetes, lupus, multiple sclerosis,
psoriasis, rheumatoid arthritis); an inflammatory disorder (e.g.
arthritis, pelvic inflammatory disease); a neurological disorder
(e.g. Alzheimer's disease, Huntington's disease; autism; Duchenne
muscular dystrophy); a proliferative disorder (e.g. cancer, benign
neoplasms); a respiratory disorder (e.g. chronic obstructive
pulmonary disease); a digestive disorder (e.g. inflammatory bowel
disease, ulcer); a musculoskeletal disorder (e.g. fibromyalgia,
arthritis); an endocrine, metabolic, or nutritional disorder (e.g.
diabetes, osteoporosis); an urological disorder (e.g. renal
disease); a psychological disorder (e.g. depression,
schizophrenia); a skin disorder (e.g. wounds, eczema); or a blood
or lymphatic disorder (e.g. anemia, hemophilia).
[0501] In embodiments of the compositions or methods described
herein, the chondrisomes are modified, e.g.: (a) subjected to or
combined with an external condition or agent (e.g., a stress
condition or agent that induces one or more mitochondrial activity
to compensate), (b) genetically engineered to overexpress or
knock-down or knock-out an endogenous gene product (e.g., an
endogenous mitochondrial or nuclear gene product); (c) engineered
to express a heterologous gene product (e.g., a heterologous, e.g.,
allogeneic or xenogeneic, mitochondrial or nuclear gene product),
or (d) loaded with a heterologous cargo agent, such as a
polypeptide, nucleic acid or small molecule (e.g., a dye, a drug, a
metabolite) e.g., an agent listed in Table 4.
[0502] In embodiments of the methods described herein, the
composition is administered in an amount and for a time sufficient
to effect, in the target cell, tissue or subject, one or more
(e.g., 2, 3, 4, 5, 6, 7 or more) of the following: [0503] a.
delivery of cargo to target cells from the administered
mitochondria of the preparation (e.g., UCP1) following delivery of
the mitochondrial preparation; [0504] b. Increases basal
respiration of recipient cells at least 10% (e.g., >15%,
>20%, >30%, >40%, >50%, >60%, >70%, >80%,
>90%) relative to a control; [0505] c. Chondrisomes of the
preparation are taken up by at least 1% (e.g., at least 2%, 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%) of recipient cells; [0506] d.
Chondrisomes of the preparation are taken up and maintain membrane
potential in recipient cells; [0507] e. Chondrisomes of the
preparation persist in recipient cells at least 6 hours, e.g., at
least 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, a week,
2 weeks, a month, 2 months, 3 months, 6 months; [0508] f. increase
ATP levels in a recipient cell, tissue or subject (e.g., by at
least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,
80%, 90%, or more, e.g., compared to a reference value, e.g., a
control value, e.g., an untreated control); [0509] g. decrease
apotosis in a recipient cell, tissue or subject (e.g., by at least
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%,
90%, or more, e.g., compared to a reference value, e.g., a control
value, e.g., an untreated control); [0510] h. decrease cellular
lipid levels in a recipient cell, tissue or subject (e.g., by at
least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,
80%, 90%, or more, e.g., compared to a reference value, e.g., a
control value, e.g., an untreated control); [0511] i. increase
membrane potential in a recipient cell, tissue or subject (e.g., by
at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,
80%, 90%, or more, e.g., compared to a reference value, e.g., a
control value, e.g., an untreated control); [0512] j. increase
uncoupled respiration in a recipient cell, tissue or subject (e.g.,
by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 509%, 60%,
70%, 80%, 90%, or more, e.g., compared to a reference value, e.g.,
a control value, e.g., an untreated control); [0513] k. increase
PI3K activity in a recipient cell, tissue or subject (e.g., by at
least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 509%, 60%, 70%,
80%, 90%, or more, e.g., compared to a reference value, e.g., a
control value, e.g., an untreated control); [0514] l. reduce
reductive stress in a recipient cell, tissue or subject (e.g., by
at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 509%, 60%,
70%, 80%, 90%, or more, e.g., compared to a reference value, e.g.,
a control value, e.g., an untreated control); [0515] m. decrease
reactive oxygen species (e.g. H2O.sub.2) in the cell, tissue of
subject (e.g., in serum of a target subject) (e.g., by at least 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 509%, 60%, 70%, 80%, 90%,
or more, e.g., compared to a reference value, e.g., a control
value, e.g., an untreated control); [0516] n. Decrease cellular
lipid levels of recipient cells at least 5% (e.g., >10%,
>15%, >20%, >30%, >40%, >50%, >60%, >70%,
>80%, >90%) relative to a control; [0517] o. increases
uncoupled respiration of recipient cells at least 5% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control; [0518] p.
decrease mitochondrial permeability transition pore (MPTP)
formation in recipient cells at least 5% and does not increase more
than 10% relative to a control; [0519] q. increase Akt levels in
recipient cells at least 10% (e.g., >10%, >15%, >20%,
>30%, >40%, >50%, >60%, >70%, >80%, >90%)
relative to a control; [0520] r. decrease total NAD/NADH ratio in
recipient cells at least 5% (e.g., >10%, >15%, >20%,
>30%, >40%, >50%, >60%, >70%, >80%, >90%)
relative to a control; [0521] s. Reduce ROS levels in recipient
cells at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control; [0522] t. Increase fractional shortening in subject with
cardiac ischemia at least 5% (e.g., >10%, >15%, >20%,
>30%, >40%, >50%, >60%, >70%, >80%, >90%)
relative to a control; [0523] u. Increase end diastolic volume in
subject with cardiac ischemia at least 5% (e.g., >10%, >15%,
>20%, >30%, >40%, >50%, >60%, >70%, >80%,
>90%) relative to a control; [0524] v. decrease end systolic
volume in subject with cardiac ischemia at least 5% (e.g., >10%,
>15%, >20%, >30%, >40%, >50%, >60%, >70%,
>80%, >90%) relative to a control; [0525] w. decrease infarct
area of ischemic heart at least 5% (e.g., >10%, >15%,
>20%, >30%, >40%, >50%, >60%, >70%, >80%,
>90%) relative to a control; [0526] x. increase stroke volume in
subject with cardiac ischemia at least 5% (e.g., >10%, >15%,
>20%, >30%, >40%, >50%, >60%, >70%, >80%,
>90%) relative to a control; [0527] y. increase ejection
fraction in subject with cardiac ischemia at least 5% (e.g.,
>10%, >15%, >20%, >30%, >40%, >50%, >60%,
>70%, >80%, >90%) relative to a control; [0528] z.
increase cardia output in subject with cardiac ischemia at least 5%
(e.g., >10%, >15%, >20%, >30%, >40%, >50%,
>60%, >70%, >80%, >90%) relative to a control; [0529]
aa. increase cardiac index in subject with cardiac ischemia at
least 5% (e.g., >10%, >15%, >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%) relative to a control;
[0530] bb. decrease serum CKNB levels in subject with cardiac
ischemia at least 5% (e.g., >10%, >15%, >20%, >30%,
>40%, >50%, >60%, >70%, >80%, >90%) relative to a
control; [0531] cc. decrease serum cTnI levels in subject with
cardiac ischemia at least 5% (e.g., >10%, >15%, >20%,
>30%, >40%, >50%, >60%, >70%, >80%, >90%)
relative to a control; [0532] dd. decrease serum hydrogen peroxide
in subject with cardiac ischemia at least 5% (e.g., >10%,
>15%, >20%, >30%, >40%, >50%, >60%, >70%,
>80%, >90%) relative to a control; [0533] ee. decrease serum
cholesterol levels in a subject at least 5% (e.g., >10%,
>15%, >20%, >30%, >40%, >50%, >60%, >70%,
>80%, >90%) relative to a control.
BRIEF DESCRIPTION OF THE FIGURES
[0534] The figures are meant to be illustrative of one or more
features, aspects, or embodiments of the invention and are not
intended to be limiting.
[0535] FIG. 1 is a graph showing quantification of lipid droplet
content using nile red average integrated intensity in control INS1
cells and INS1 cells that received 10 or 40 .mu.g BAT chondrisome
protein/100K cells. Bar graphs represent average.+-.SD of percent
change form control group
[0536] FIG. 2 is a graph showing quantification of UCP1 protein
levels normalized to their corresponding loading control (GAPDH) in
HEPG2 cells 24 h and 48 h after receiving 20 .mu.g of BAT
chondrisome protein/100K cells. Bar graphs represent
average.+-.SD.
[0537] FIG. 3 is a graph showing quantification of uncoupled
mitochondrial respiration after oligomycin injection in control
HEPG2 cells and HEPG2 cells that received 4 to 90 .mu.g BAT
chondrisome protein/100K cells. Bar graphs represent
average.+-.SD.
[0538] FIG. 4 is an image showing Leigh fibroblasts treated with 8
or 32 ug of human fibroblast or human platelet chondrisomes
(chondr.) and assessed for phospho-GSK-3.alpha. and total Akt
levels with Akt activity assay kit and western blot analysis.
Representative bands for phospho-GSK-3.alpha. and total Akt are
shown for the indicated conditions.
[0539] FIG. 5 is a table showing the concentration of plasma
cytokines in mice 24 hours after intravenous (IV) or subcutaneous
(SC) treatment with chondrisomes or Vehicle. The concentration of
each cytokine is below the limit of detection.
[0540] FIG. 6 is a panel of images showing representative
hematoxylin and eosin (H&E) stained slides of the liver, lung,
and spleen from animals treated intravenously with chondrisomes or
vehicle. There is no elevated level of immune cell infiltration in
the chondrisome treatment compared to the vehicle treatment in any
of the three organs.
[0541] FIG. 7 is a graph showing the ratio of the weights of the
treated to untreated perigondal fat pads of animals that received
chondrisomes or vehicle injections. The ratio is significantly
lower in animals that received chondrisome treatment as assessed
via an unpaired t test.
[0542] FIG. 8 is a graph showing the serum concentration of total
cholesterol in animals that received chondrisomes or vehicle
injections. The ratio is significantly lower in animals that
received chondrisome treatment as assessed via Welch's t test.
[0543] FIG. 9 is a graph showing the serum concentration of total
triglycerides in animals that received chondrisomes or vehicle
injections. The ratio is trending to be lower in animals that
received chondrisome treatment.
[0544] FIG. 10 is a graph showing the ratio of the weight of
chondrisome-injected to vehicle-injected paw lymph nodes for
animals that received a single or multiple treatments of syngeneic
chondrisomes or allogeneic chondrisomes.
DETAILED DESCRIPTION
[0545] The invention describes chondrisome preparations and
pharmaceutical compositions that have beneficial characteristics
suitable for administration to a target tissue or cell (e.g., ex
vivo or in vivo), useful in methods to modify (e.g., modify the
metabolic or cellular state of) a target tissue or cell (e.g., ex
vivo or in vivo), and/or to treat a subject (e.g., a mammal such as
a human) The preparations and compositions described herein may
also be modified, e.g., may include a heterologous function or
activity, e.g., may include a payload such as an effector molecule,
a drug, a targeting agent; may overexpress or under express an
endogenous mitochondrial or nuclear gene; may express a
heterologous mitochondrial or nuclear gene.
Chondrisome Preparations
[0546] Chondrisome preparations of the invention may be produced
from any eukaryotic source, e.g., from yeast, plant, mammalian
(e.g., human or agricultural animal) tissue.
Tissue Sources
[0547] Mitochondrial preparations can be isolated from any
mammalian (e.g., human) tissue or cell, e.g., from epithelial,
connective, muscular, or nervous tissue or cells, and combinations
thereof. Mitochondrial preparations can be isolated from any
eukaryotic (e.g., mammalian) organ system, for example, from the
cardiovascular system (heart, vasculature); digestive system
(esophagus, stomach, liver, gallbladder, pancreas, intestines,
colon, rectum and anus); endocrine system (hypothalamus, pituitary
gland, pineal body or pineal gland, thyroid, parathyroids, adrenal
glands); excretory system (kidneys, ureters, bladder); lymphatic
system (lymph, lymph nodes, lymph vessels, tonsils, adenoids,
thymus, spleen); integumentary system (skin, hair, nails); muscular
system (e.g., skeletal muscle); nervous system (brain, spinal cord,
nerves); reproductive system (ovaries, uterus, mammary glands,
testes, vas deferens, seminal vesicles, prostate); respiratory
system (pharynx, larynx, trachea, bronchi, lungs, diaphragm);
skeletal system (bone, cartilage), and combinations thereof.
[0548] In embodiments, the source tissue is a source of stem cells
or progenitor cells, e.g., bone marrow stromal cells,
marrow-derived adult progenitor cells (MAPCs), endothelial
progenitor cells (EPC), blast cells, intermediate progenitor cells
formed in the subventricular zone, neural stem cells, muscle stem
cells, satellite cells, liver stem cells, hematopoietic stem cells,
bone marrow stromal cells, epidermal stem cells, embryonic stem
cells, mesenchymal stem cells, umbilical cord stem cells, precursor
cells, muscle precursor cells, myoblast, cardiomyoblast, neural
precursor cells, glial precursor cells, neuronal precursor cells,
hepatoblasts.
[0549] In some embodiments, the source tissue is a tissue sample
from a mammal, e.g., a human. The tissue sample may be, e.g., from
a living human or from a cadaver. The tissue may be fresh (used
within days of harvest, typically stored at .ltoreq.4.degree. C.),
or may be frozen.
[0550] In embodiments, the tissue sample is from a highly mitotic
tissue (e.g., a highly mitotic healthy tissue, such as epithelium,
embryonic tissue, bone marrow, intestinal crypts). In embodiments,
the tissue sample is a highly metabolic tissue (e.g., skeletal
tissue, neural tissue, cardiomyocytes).
[0551] In some embodiments, the source tissue is from a young
donor, e.g., a donor under 25 years, 20 years, 18 years, 16 years,
12 years, 10 years, 8 years of age or less.
[0552] In certain embodiments, the cells of the tissue have
telomeres of average size greater than 3000, 4000, 5000, 6000,
7000, 8000, 9000, or 10000 nucleotides in length (e.g., between
4,000-10,000 nucleotides in length, between 6,000-10,000
nucleotides in length).
[0553] In certain embodiments, the mitochondrial mutation load of
the source tissue is low, e.g., fewer than 0.001/17,000,
0.01/17,000, 0.1/17,000, 1/17,000, 2/17,000, 5/17,000, 10/17,000,
50/17,000, 100/17,000 of the genetic content deviates from the
reference haplotype mitochondrial sequence of the source.
Blood Product Sources
[0554] Mitochondrial preparations of the invention are isolated
from blood or blood fractions, e.g., whole blood, platelets,
platelet mitoparticles, peripheral blood mononuclear cells (PBMCs),
platelet rich plasma, or platelet free plasma.
[0555] Human blood can be generally obtained from healthy human
volunteers under approved protocols, from blood banks, or form
commercial sources.
[0556] Platelets are typically isolated from blood, e.g., by
differential centrifugation. Briefly, platelet rich plasma (PRP) is
prepared from whole blood through centrifugation at low g force,
wherein the PRP remains in the supernatant and red blood cells and
white blood cells pellet, followed by centrifugation at higher g
force to pellet the platelets in the PRP. In some embodiments,
platelets are activated before isolation of mitochondria or
mitoparticles. In some embodiments, one or more platelet activation
inhibitors may be used during isolation.
[0557] Activated platelets release mitochondria, both within
membrane encapsulated microvesicles (referred to herein as
"mitoparticles") and as free organelles. (See Boudreau et al. 2014.
Platelets release mitochondria serving as substrate for
bactericidal group IIA-secreted phospholipase A2 to promote
inflammation. Blood. Vol. 24 No. 14: 2173-2183.) Such mitoparticles
may be isolated from platelets (e.g., through differential
centrifugation or filtration), concentrated, and surprisingly may
be used as a source of mitochondrial activity or chondrisomes in
the methods described herein.
[0558] Platelet free plasma (PFP) may also be a source of
mitochondria in the compositions and methods described herein.
[0559] The most common method for isolation of PBMCs, e.g.,
lymphocytes and monocytes, from blood is through a density gradient
medium (e.g., Ficoll) based on the principle of differential
migration of blood cells through the media during the
centrifugation stage of the procedure. In brief, either
anticoagulant or defibrinated blood specimens are layered on top of
the gradient (e.g., Ficoll) solution, then briefly centrifuged to
form different layers containing different types of cells. The
bottom layer is made up of red blood cells (erythrocytes) which are
collected or aggregated by the medium and sink completely through
to the bottom. The next layer up from the bottom is primarily
granulocytes, which also migrate down through the solution. The
next layer toward to top is the lymphocytes, which are typically at
the interface between the plasma and the Ficoll solution, along
with monocytes and platelets. To recover the lymphocytes, this
layer is carefully recovered, washed with a salt solution to remove
platelets, Ficoll, and plasma, then centrifuged again.
[0560] The source of mitochondria may be an apheresis product,
e.g., apheresis derived plasma, e.g., fresh frozen plasma; red
blood cells; platelets; leukocytes.
Cultured Cell Sources
[0561] Preparations of chondrisomes are isolated from cells in
culture, for example cultured mammalian cells, e.g., cultured human
cells. The cells may be progenitor cells or non-progenitor (e.g.,
differentiated) cells. The cells may be primary cells or cell lines
(e.g., a mammalian, e.g., human, cell line described herein).
[0562] In embodiments, the cells are from a highly mitotic tissue
(e.g., a highly mitotic healthy tissue, such as epithelium,
embryonic tissue, bone marrow, intestinal crypts). In embodiments,
the tissue sample is a highly metabolic tissue (e.g., skeletal
tissue, neural tissue, cardiomyocytes).
[0563] In some embodiments, the cells are from a young donor, e.g.,
a donor 25 years, 20 years, 18 years, 16 years, 12 years, 10 years,
8 years of age, 5 years of age, 1 year of age, or less. In some
embodiments, the cells are from fetal tissue.
[0564] In certain embodiments, the cells have telomeres of average
size greater than 3000, 4000, 5000, 6000, 7000, 8000, 9000, or
10000 nucleotides in length (e.g., between 4,000-10,000 nucleotides
in length, between 6,000-10,000 nucleotides in length).
[0565] In certain embodiments, the mitochondrial mutation load of
the source tissue is low, e.g., fewer than 0.001/17,000,
0.01/17,000, 0.1/17,000, 1/17,000, 2/17,000, 5/17,000, 10/17,000,
50/17,000, 100/17,000 of the genetic content deviates from the
reference haplotype mitochondrial sequence of the source.
[0566] Chondrisomes may be prepared from the cultured cells after 1
or more passages. In some embodiments, chondrisomes are prepared
from cells having undergone no more than 20, no more than 15
passages in culture, e.g., between 1-15 passages, between 1-10
passages, between 1-8 passages, between 1-6 passages, between 1-4
passages.
[0567] In embodiments, the cells are cultured induced pluripotent
stem cells (iPS cells), bone marrow stromal cells, marrow-derived
adult progenitor cells (MAPCs), endothelial progenitor cells (EPC),
blast cells, intermediate progenitor cells formed in the
subventricular zone, neural stem cells, muscle stem cells,
satellite cells, liver stem cells, hematopoietic stem cells, bone
marrow stromal cells, epidermal stem cells, embryonic stem cells,
mesenchymal stem cells, umbilical cord stem cells, precursor cells,
muscle precursor cells, myoblast, cardiomyoblast, neural precursor
cells, glial precursor cells, neuronal precursor cells,
hepatoblasts.
[0568] In one embodiment, the cells are iPS derived, e.g., iPS
derived cardiomyocytes or neural stem cells.
[0569] In embodiments, chondrisomes are prepared from a human cell
line. The production of permanent human cell lines is described,
e.g., in U.S. Pat. No. 9,315,773. In embodiments, the cell line is
HeLa or HEK293. In embodiments, chondrisomes are prepared from a
cell line described in Table 1.
[0570] In embodiments, chondrisomes are prepared from cultured
primary cells, e.g., cultured primary fibroblasts, skeletal muscle
cells (e.g., cardiomyocytes), keratinocytes, epithelial cells,
hepatocytes, neuronal, glial, or neural stem cells, skeletal
myoblasts, smooth muscle cells, adipose tissue (e.g., brown adipose
tissue) (see, e.g., Nechad et al. 1983. Development of brown fat
cells in monolayer culture Exp Cell Res. 149(1):105-18).
TABLE-US-00001 TABLE 1 Cell lines for chondrisome production
Species Cell Type Tissue/Cell line type ATCC Number Human Cancerous
tissue Bladder Cancer TCP-1020 .TM. Bone Cancer TCP-1009 .TM.
Breast Cancer TCP-1004 .TM. Brain Cancer TCP-1018 .TM. Colon Cancer
TCP-1006 .TM. Ovarian Cancer TCP-1021 .TM. Leukemia TCP-1010 .TM.
Lymphoma TCP-1025 .TM. Liver Cancer TCP-1011 .TM. Lung Cancer
TCP-1016 .TM. Melanoma TCP-1014 .TM. Pancreatic Cancer TCP-1026
.TM. Non-cancerous tissue Epithelial Cornea CRL-11515 .TM. Mammary
Gland CRL-8798 .TM. Colon CRL-2302 .TM. Kidney CRL-2190 .TM. Liver
CRL-11233 .TM. Blood B lymphoblast CRL-1980 .TM. Skin Fibroblast
CRL-2072 .TM. Lung Fibroblast CCL-204 .TM. Bone Marow Stromal
CRL-11882 .TM. Endothelial Vascular CRL-1730 .TM. Murine Fibroblast
CRL-2817 .TM. Bovine Pulmonary Artery CCL-209 .TM. Canine Kidney
CRL-2936 .TM. Hampster Ovary CRL-9606 .TM. Monkey Kidney CRL-1586
.TM. Rabbit Corneal Fibroblast CCL-60 .TM. Rat Liver Epithelial
CRL-1439 .TM. Pig Lung Macrophage CRL-2844 .TM. Cat Lymphocyte
CRL-11967 .TM. Chicken Embryonic Fribroblast CRL-12203 .TM.
Zebrafish Fibroblast CRL-2296 .TM. Trout Ovaries & Testies
CCL-55 .TM. Fungi & Saccharomyces 7754 .TM. Yeast
cerevisiae
[0571] Methods of Culturing Cells
[0572] Cells are generally cultured according to methods known in
the art. Culture media contain a mixture of amino acids, sugars,
salts, vitamins, and other nutrients, and are available either as a
powder or as a liquid form from commercial suppliers. Basal medium
for cell culture may also include vitamins, lipids, proteins (e.g.,
rh-insulin and/or rh-transferrin) Amino acid ingredients which may
be included in the media of the present invention include
L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine,
L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine,
L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline,
L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine. These
amino acids may be obtained commercially. Vitamin ingredients which
may be included in the media of the present invention include
biotin, choline chloride, D-Ca++-pantothenate, folic acid,
i-inositol, niacinamide, pyridoxine, riboflavin, thiamine and
vitamin B.sub.12. These vitamins may be obtained commercially.
Inorganic salt ingredients which may be used in the media include
one or more calcium salts (e.g., CaCl.sub.2), Fe(NO.sub.3).sub.3,
KCl, one or more magnesium salts (e.g., MgCl.sub.2 and/or
MgSO.sub.4), one or more manganese salts (e.g., MnCl.sub.2), NaCl,
NaHCO.sub.3, Na.sub.2HPO.sub.4, and ions of the trace elements
selenium, vanadium and zinc. These trace elements may be provided
in a variety of forms, preferably in the form of salts such as
Na.sub.2SeO.sub.3, NH.sub.4VO.sub.3 and ZnSO.sub.4. Methods of
selecting and using culture media for various cells are known and
are described, e.g., in Arora. 2013. Cell Culture Media: A Review.
Mater Methods 3:175.
[0573] In embodiments of the invention, the medium contains no
non-human animal products, e.g., no non-human serum, e.g., no BSA
or FBS. The medium may be a serum-free medium or may be a
chemically defined medium. The medium may be completely
protein-free. If protein is present, it is preferably a human
protein such as a human recombinant protein (e.g., transferring or
insulin). Transferrin may be replaced by ferric citrate chelates at
a concentration of about 10-100 uM (preferably FeCl.sub.3-sodium
citrate chelate at about 60 uM) or ferrous sulfate chelates at a
concentration of about 10-100 uM (preferably FeSO4-EDTA chelate at
about 40 uM). Insulin may be replaced by one or more
zinc-containing compounds such alone or more zinc salts.
Zinc-containing compounds which may be used include but are not
limited to ZnCl, Zn(NO.sub.3).sub.2, ZnBr, and ZnSO.sub.4, any of
which may be present in their anhydrous or hydrated (i.e., ".H2O")
forms.
[0574] In one embodiment, the cultured cells are cultured in a
serum free medium, e.g., a defined medium.
[0575] In some embodiments, the cells may be cultured in 2 or more
"phases", e.g., a growth phase, wherein the cells are cultured
under conditions to multiply and increase biomass of the culture,
and a "production" phase, wherein the cells are cultured under
conditions to increase mitochondrial quality (e.g., to maximize
mitochondrial phenotype, to increase number or size of
mitochondria, to increase oxidative phosphorylation status).
[0576] In embodiments, the cell culture media does not contain
glucose. In embodiments, the cell culture media contains glucose
during the growth phase but does not contain added glucose during
the production phase.
[0577] In some embodiments, the cells may be synchronized, e.g.,
during a growth phase or the production phase. For example, cells
may be synchronized at G1 phase by elimination of serum from the
culture medium (e.g., for about 12-24 hours) or by the use in the
culture media of DNA synthesis inhibitors such as thymidine,
aminopterin, hydroxyurea and cytosine arabinoside. Additional
methods for mammalian cell cycle synchronization are known and
disclosed, e.g., in Rosner et al. 2013. Nature Protocols 8:602-626
(specifically Table 1 in Rosner).
[0578] In some embodiments, the cells can be evaluated and
optionally enriched for a desirable phenotype or genotype for use
as a source of a chondrisome preparation described herein. For
example, cells can be evaluated and optionally enriched, e.g.,
before culturing, during culturing (e.g., during a growth phase or
a production phase) or after culturing but before isolation of
chondrisomes, for example, for one or more of: membrane potential
(e.g., a membrane potential of -5 to -200 mV; cardiolipin content
(e.g., between 1-20% of total lipid); genetic quality >80%,
>85%, >90%.
[0579] In some embodiments, a cell clone is identified, chosen, or
selected for culturing based on a desirable phenotype or genotype
for use as a source of a chondrisome preparation described herein.
For example, a cell clone is identified, chosen, or selected based
on low mitochondrial mutation load, long telomere length,
differentiation state, or a particular genetic signature (e.g., a
genetic signature to match a recipient).
[0580] In some embodiments, cells are cultured in the presence of
an exogenous agent that modulates mitochondrial structure, function
or activity, e.g., a structure, function or activity described
herein. In embodiments, one or a plurality (e.g., 2, 3, 4, 5 or
more) of the agents or strategies described hereinbelow may be used
to treat a cell culture for preparation of a mitochondrial
preparation.
[0581] Mitochondrial Biogenesis (MB) Agents
[0582] The source subject, tissue or cell may be contacted with a
mitochondrial biogenesis (MB) agent in an amount and for a time
sufficient to increase mitochondrial biogenesis in the source
subject tissue or cell (e.g., by at least 10%, 15%, 20%, 30%, 40%,
50%, 60%, 75%, 80%, 90% or more). Such MB agents are described,
e.g., in Cameron et al. 2016. Development of Therapeutics That
Induce Mitochondrial Biogenesis for the Treatment of Acute and
Chronic Degenerative Diseases. DOI:10.1021/acs.jmedchem.6b00669. In
embodiments, the MB agent is added to the cell culture during the
growth phase and/or during the production phase. In embodiments,
the MB agent is added when the cell culture has a predetermined
target density.
[0583] In one embodiment, the MB agent is an agent extracted from a
natural product or its synthetic equivalent, sufficient to increase
mitochondrial biogenesis in the source subject, tissue or cell.
Examples of such agents include resveratrol, epicatechin, curcumin,
a phytoestrogen (e.g., genistein, daidzein, pyrroloquinoline,
quinone, coumestrol and equol).
[0584] In one embodiment, the MB agent is a transcription factor
modulator sufficient to increase mitochondrial biogenesis in the
source subject, tissue or cell. Examples of such transcription
factor modulators include: thiazolidinediones (e.g., rosiglitazone,
pioglitazone, troglitazone and ciglitazone), estrogens (e.g.,
17.beta.-Estradiol, progesterone) and estrogen receptor agonists;
SIRT1 Activators (e.g., SRT1720, SRT1460, SRT2183, SRT2104).
[0585] In one embodiment, the MB agent is a kinase modulator
sufficient to increase mitochondrial biogenesis in the source
subject, tissue or cell. Examples include: AMPK and AMPK activators
such as AICAR, metformin, phenformin, A769662; and ERK1/2
inhibitors, such as U0126, trametinib.
[0586] In one embodiment, the MB agent is a cyclic nucleotide
modulator sufficient to increase mitochondrial biogenesis in the
source subject, tissue or cell. Examples include modulators of the
NO-cGMP-PKG pathway (for example nitric oxide (NO) donors, such as
sodium nitroprusside, (.+-.)S-nitroso-N-acetylpenicillamine (SNAP),
diethylamine NONOate (DEA-NONOate), diethylenetriamine-NONOate
(DETA-NONOate); sGC stimulators and activators, such as cinaciguat,
riociguat, and BAY 41-2272; and phosphodiesterase (PDE) inhibitors,
such as zaprinast, sildenafil, udenafil, tadalafil, and vardenafil)
and modulators of the cAMP-PKA-CREB Axis, such as phosphodiesterase
(PDE) inhibitors such as rolipram.
[0587] In one embodiment, the MB agent is a modulator of a G
protein coupled receptor (GPCR) such as a GPCR ligand sufficient to
increase mitochondrial biogenesis in the source subject, tissue or
cell.
[0588] In one embodiment, the MB agent is a modulator of a
cannabinoid-1 receptor sufficient to increase mitochondrial
biogenesis in the source subject, tissue or cell. Examples include
taranabant and rimonobant.
[0589] In one embodiment, the MB agent is a modulator of a
5-Hydroxytryptamine receptor sufficient to increase mitochondrial
biogenesis in the source subject, tissue or cell. Examples include
alpha-methyl-5-hydroxytryptamine, DOI, CP809101, SB242084,
serotonin reuptake inhibitors such as fluoxetine, alpha-methyl 5HT,
1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, LY334370, and
LY344864.
[0590] In one embodiment, the MB agent is a modulator of a beta
adrenergic receptor sufficient to increase mitochondrial biogenesis
in the source subject, tissue or cell. Examples include
epinephrine, norepinephrine, isoproterenol, metoprolol, formoterol,
fenoterol and procaterol. E.g., BAT cells in culture express UCP1
following .beta.-adrenergic stimulation.
[0591] In one embodiment, the cultured cells are modified, e.g.,
genetically modified, to express a transcriptional activator of
mitochondrial biogenesis, e.g., a transcription factor or
transcriptional coactivator such as PGC1.alpha.. In some
embodiments, the cells express PGC1.alpha.(e.g., over express an
endogenous, or express an exogenous, PGC1.alpha.).
[0592] Modulators of Metabolic Activity
[0593] The source subject, tissue or cell may be contacted with an
agent that is a modulator of metabolic activity in an amount and
for a time sufficient to increase metabolic activity in the source
subject tissue or cell (e.g., by at least 10%, 15%, 20%, 30%, 40%,
50%, 60%, 75%, 80%, 90% or more, compared to without the agent). In
embodiments, the agent is added to the cell culture during the
growth phase and/or during the production phase. In embodiments,
the agent is added when the cell culture has a predetermined target
density.
[0594] In one embodiment, the agent is a respiratory substrate.
Examples include ADP, carbohydrates, lipids and fatty acids,
proteins and amino acids.
[0595] In one embodiment, the agent is a non-glucose carbon source.
Examples include galactose, glutamine, fructose, maltose.
[0596] In some embodiments, the cells are cultured under low oxygen
conditions, e.g., between 0.1-15% oxygen, between 0.5-10% oxygen,
between 0.5-8% oxygen, between 0.5-6% oxygen, between 1-6% oxygen,
between 2-6% oxygen, between 1-5% oxygen, between 2-5% oxygen. In
embodiments, the cells are cultured under low oxygen conditions
during the production phase but not the growth phase.
[0597] In one embodiment, the agent is an antioxidant. In one
embodiment, the agent is an antireductant. In one embodiment, the
agent is an uncoupler.
[0598] In some embodiments, the cells are cultured in combination
with a second cell population (a co-culture), e.g., a feeder
culture that provides the cultured source cells with desirable
agents, e.g., metabolic substrates.
[0599] Bioreactors
[0600] Cell cultures described herein for preparation of
chondrisome preparations may be cultured in a bioreactor.
[0601] For suspension cells, the bioreactor may be any scale or lot
size, e.g., 100 mL, 500 mL, 1 L, 2 L, 5 L, 10 L, 20 L, 40 L, 50 L,
100 L, or greater. For example, the bioreactor is between 1 L-100
L, e.g., between 1 L-50 L, between 1 L-20 L, between 1 L-10 L. In
some embodiments, the bioreactor is between 100 mL-100 L, e.g.,
between 100 mL-5 L, 100 mL-1 L.
[0602] For adherent cells, such as adult primary cell lines and
human multipotent (MSCs) and pluripotent stem cells (hPSCs), a lot
size of cultured cells for production of chondrisome preparations
may be 1-10.times.10.sup.7, 1-10.times.10.sup.8,
1-10.times.10.sup.9, 1-10.times.10.sup.10, 1-10.times.10.sup.11,
1-10.times.10.sup.12. Adherent cells may be cultured in planar
systems, e.g., flasks, or stacked-plate systems, parallel-plate
vessels, or packed-bed bioreactors (PBRs). Honeycomb bioreactors
may also be used, e.g., as described, e.g., in U.S. Pat. No.
6,777,227. Microcarriers can also be used in bioreactors to
increase surface area. Microcarriers can be made, e.g., from
polystyrene or from cross-linked dextran. Although most
microcarriers are spherical and smooth, others have macroporous
surfaces or may have other shapes such as rod-shaped carriers.
Additional techniques include infusion of magnetic particles that
help in cell separation from beads and chip-based microcarriers
that provide a traditional flat surface for cell growth while
maintaining the high SA:V ratio of traditional microcarriers.
Surface chemistry modifications may improve cell adhesion in
adherent systems. Such methods include, e.g., applying positive or
negative charges and coating with extracellular matrix proteins
such as laminin or vitronectin. See, e.g., Rowley et al. 2012.
Meeting Lot-Size Challenges of Manufacturing Adherent Cells for
Therapy. BioProcess International 10(3) Supplement.
[0603] A chondrisome preparation described herein may be comprised
of chondrisomes from one cellular or tissue source, or from a
combination of sources. For example, a chondrisome preparation may
comprise chondrisomes isolated from xenogeneic sources (plants,
animals, tissue culture of the aforementioned species' cells),
allogeneic, autologous, from specific tissues resulting in
different protein concentrations and distributions (liver,
skeletal, neural, adipose, etc.), from cells of different metabolic
states (e.g., glycolytic, respiring). A preparation may also
comprise chondrisomes in different metabolic states, e.g. coupled
or uncoupled.
Isolation Methods
[0604] The basic steps of mitochondria isolation for research are
described in Pallotti & Lenaz. 2007. Isolation and
subfractionation of mitochondria. Methods in Cell Biology Vol 80.
Generally, preparations of mitochondria are isolated from a donor
tissue or cell culture (or combinations of donor tissues or cells)
as follows: Tissue can be obtained by biopsy (solid tissue) or
syringe draw (fluid tissue) and is typically maintained at
0-4.degree. C. throughout the process of isolation. Solid tissue
may be minced into small pieces. The tissue is ground, dissociated
or homogenized in isolation buffer (IB). A typical IB contains one
or more stabilizing agent such as sucrose or albumin (e.g., human
serum albumin), a chelator such as EGTA, and a buffering system
such as Tris. The resulting homogenized material is centrifuged
and/or filtered (e.g., through a 5 .mu.m filter) to remove cells or
large cell debris. The filtrate is recovered and the mitochondria
are washed and concentrated, e.g., by additional centrifugation,
and resuspended in buffer.
[0605] In embodiments of the invention, preparing a chondrisome
preparation includes the following steps (a)-(e):
[0606] (a) A tissue or cellular source of mitochondria (e.g., a
tissue or cellular source described herein) is provided or
obtained. The tissue or cellular source may be fresh or frozen,
from a live or dead donor. In one embodiment, the source is a
sample of frozen cultured cells. In one embodiment, the source is a
blood product (e.g., whole blood, platelets, leucocytes).
[0607] (b) the tissue or cellular source is dissociated to produce
a subcellular composition (e.g., a homogenate) or manipulated
(e.g., activated) to release chondrisomes or
mitochondria-containing vesicles. In embodiments, the dissociating
step is performed in no more than 10 fold (no more than 7-fold,
5-fold, 4-fold, 3-fold, or 2-fold) the volume of buffer relative to
the volume of the tissue or cellular source. The dissociating may
be performed by any cellular dissociation device or method, e.g.,
by douncing (e.g., with a glass dounce, or by a dissociator device
such as a Miltenyl GentleMACS Dissociator). In embodiments, the
dissociating comprises applying a plurality of shear force steps to
the tissue or cellular source, e.g., a first shear force followed
by at least a second, higher shear force. For example, the first
shear force is applied with a dounce device and the second, higher
shear force is applied by passing the homogenate through one or
more needle, e.g., passing the homogenate (e.g., 1-10 times, 1-8
times, 1-6 times, 1-4 times) through a series of needles having a
gauge between 15-45, e.g., a gauge between 18-30. In embodiments,
the dissociation to produce the subcellular homogenate is performed
in the absence of added proteases. In some embodiments, if the
tissue or cells have been previously treated with proteases (e.g.,
to dissociate adherent cultured cells from their substrate or to
dissociate tissue into individual cells), the proteases may be
washed away before this step. In embodiments, the dissociation
technique (e.g., a douncing step) is optimized to produce a
subcellular composition that results in a yield described
herein.
[0608] (c) the subcellular composition is separated into a cellular
debris fraction (e.g., a solid or pelleted fraction) and a
chondrisome enriched fraction (e.g., a fluid fraction). Separation
may be accomplished by known techniques, e.g., centrifugation or
size filtration. The separation may include a plurality of
centrifugation or size filtration steps.
[0609] (d) the chondrisome enriched fraction is separated into a
fraction containing chondrisomes (e.g., a solid or pellet fraction)
and a fraction (e.g., a supernatant) substantially lacking
chondrisomes, e.g., by centrifugation or size filtration. This
separation may include a plurality of centrifugation or size
filtration steps, e.g., including one or more "wash" steps or
repelleting steps.
[0610] (e) the fraction containing chondrisomes is suspended in
solution. In embodiments, the suspension is performed in no more
than 10 fold (no more than 7-fold, 5-fold, 4-fold, 3-fold, or
2-fold) the volume of buffer relative to the volume of the
fraction. The solution may be a buffer, e.g., a storage buffer, or
a pharmaceutically acceptable solution, e.g., suitable for delivery
or administration to a subject.
[0611] In certain embodiments, the yield of the preparation is
>0.05 (e.g., >0.1, >0.2, >0.5, >1, >2, >3,
>5, >6, >7, >8, >8, >10, >20, >30, >40,
>50, >60, >80, >90, >100, >150, >200, >300)
ug protein/10E6 cells. In certain embodiments, the yield of the
preparation is >100 (e.g., >200, >300, >400, >500,
>600, >700, >800, >900, >1,000, >2,000,
>3,000, >5,000, >7000, >10,000) ug protein/g tissue. In
embodiments, the efficiency of chondrisome yield is 1E9 to 9E12
(e.g., >1E9, >5E9, >1E10, >5E10, >1E11, >5E11,
>1E12, >5E12) particles/mg total protein.
Characterization
[0612] Chondrisome preparations can be assayed for structural and
functional parameters e.g., physical, structural, bioenergetics and
functional parameters, e.g., membrane integrity, purity, stability,
morphology, protein content, lipid content, enzymatic activity,
respiration rate, ATP production, concentration, protein content,
fission capabilities and functional activity (or lack thereof),
such as apoptotic modulation, internalization ability, endosomal
escape, metabolic effects, cardiac-protective effects, e.g., as
described in the Examples section herein.
Encapsulation
[0613] In some embodiments of the compositions and methods
described herein, the chondrisomes can be encapsulated, e.g., in
naturally derived or in engineered lipid membranes. Some cells are
known to eject mitochondria in a membrane bound vesicle (Boudreau
et al. 2014. Platelets release mitochondria serving as substrate
for bactericidal group IIA-secreted phospholipase A2 to promote
inflammation. Blood. 124(14):2173-83; Phinney et al. 2015.
Mesenchymal stem cells use extracellular vesicles to outsource
mitophagy and shuttle microRNAs. Nature Communications. 6:8472).
Such vesicles can surprisingly be used in the methods of the
invention. In other instances, this encapsulation takes the form of
an autologous, allogeneic, xenogeneic or engineered cell such as is
described in Ahmad et al. 2014. Miro1 regulates intercellular
mitochondrial transport & enhances mesenchymal stem cell rescue
efficacy. EMBO Journal. 33(9): 994-1010). In another embodiment the
chondrisomes can be encapsulated in engineered substrates such as
described in, e.g. in Orive. et al. 2015. Cell encapsulation:
technical and clinical advances. Trends in Pharmacology Sciences;
36 (8):537-46; and in Mishra. 2016. Handbook of Encapsulation and
Controlled Release. CRC Press. In some embodiments, mitochondria
can be encapsulated in naturally occurring vesicles themselves
(McBride et al. 2012. A Vesicular Transport Pathway Shuttles Cargo
from mitochondria to lysosomes. Current Biology 22:135-141).
[0614] In some embodiments, a composition described herein includes
mitochondria encapsulated in naturally derived vesicles, e.g.,
membrane vesicles prepared from cells or tissues, which vesicles
carry mitochondria. In one embodiment, the vesicle is a platelet
mitoparticle. In one embodiment, the vesicle is a
mitochondria-containing microvesicle from MSCs or astrocytes. In
one embodiment, the vesicle is an exosome.
[0615] In some embodiments, a composition described herein includes
chondrisomes encapsulated in synthetic vesicles, e.g.,
liposomes.
[0616] In some embodiments, a composition described herein includes
chondrisomes encapsulated in nanoparticles or nanogels.
[0617] In some embodiments, a composition described herein includes
mitochondria encapsulated in naturally derived vesicles, e.g.,
membrane vesicles prepared from cells or tissues, which vesicles
carry mitochondria (McBride et al. 2012. A Vesicular Transport
Pathway Shuttles Cargo from mitochondria to lysosomes. Current
Biology 22:135-141). Some cells are known to eject mitochondria in
a membrane bound vesicle (Boudreau et al. 2014. Platelets release
mitochondria serving as substrate for bactericidal group
IIA-secreted phospholipase A2 to promote inflammation. Blood.
124(14):2173-83; Phinney et al. 2015. Mesenchymal stem cells use
extracellular vesicles to outsource mitophagy and shuttle
microRNAs. Nature Communications. 6:8472). In other instances, this
encapsulation takes the form of an autologous, allogeneic,
xenogeneic or engineered cell such as is described in Ahmad et al.
2014. Miro1 regulates intercellular mitochondrial transport &
enhances mesenchymal stem cell rescue efficacy. EMBO Journal.
33(9):994-1010).
[0618] In another embodiment the chondrisomes can be encapsulated
in engineered substrates such as described in, e.g. in Orive. et
al. 2015. Cell encapsulation: technical and clinical advances.
Trends in Pharmacology Sciences; 36 (8):537-46; and in Mishra.
2016. Handbook of Encapsulation and Controlled Release. CRC Press.
In some embodiments, a composition described herein includes
chondrisomes encapsulated in synthetic vesicles, e.g.,
liposomes.
[0619] Liposomes are spherical vesicle structures composed of a
uni- or multilamellar lipid bilayer surrounding internal aqueous
compartments and a relatively impermeable outer lipophilic
phospholipid bilayer. Liposomes may be anionic, neutral or
cationic. Liposomes are biocompatible, nontoxic, can deliver both
hydrophilic and lipophilic drug molecules, protect their cargo from
degradation by plasma enzymes, and transport their load across
biological membranes and the blood brain barrier (BBB) (see, e.g.,
Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID
469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).
[0620] Vesicles can be made from several different types of lipids;
however, phospholipids are most commonly used to generate liposomes
as drug carriers. Vesicles may comprise without limitation DOPE
(dioleoylphosphatidylethanolamine), DOTMA, DOTAP, DOTIM, DDAB,
alone or together with cholesterol to yield DOPE and cholesterol,
DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and
cholesterol, and DDAB and cholesterol. Methods for preparation of
multilamellar vesicle lipids are known in the art (see for example
U.S. Pat. No. 6,693,086, the teachings of which relating to
multilamellar vesicle lipid preparation are incorporated herein by
reference). Although vesicle formation can be spontaneous when a
lipid film is mixed with an aqueous solution, it can also be
expedited by applying force in the form of shaking by using a
homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch
and Navarro, Journal of Drug Delivery, vol. 2011, Article ID
469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).
Extruded lipids can be prepared by extruding through filters of
decreasing size, as described in Templeton et al., Nature Biotech,
15:647-652, 1997, the teachings of which relating to extruded lipid
preparation are incorporated herein by reference.
[0621] As described herein, additives may be added to vesicles to
modify their structure and/or properties. For example, either
cholesterol or sphingomyelin may be added to the mixture in order
to help stabilize the structure and to prevent the leakage of the
inner cargo. Further, vesicles can be prepared from hydrogenated
egg phosphatidylcholine or egg phosphatidylcholine, cholesterol,
and dicetyl phosphate. (see, e.g., Spuch and Navarro, Journal of
Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011.
doi:10.1155/2011/469679 for review). Also vesicles may be surface
modified during or after synthesis to include reactive groups
complementary to the reactive groups on the carrier cells. Such
reactive groups include without limitation maleimide groups. As an
example, vesicles may be synthesized to include maleimide
conjugated phospholipids such as without limitation
DSPE-MaL-PEG2000.
[0622] A vesicle formulation may be mainly comprised of natural
phospholipids and lipids such as
1,2-distearoryl-sn-glycero-3-phosphatidyl choline (DSPC),
sphingomyelin, egg phosphatidylcholines and monosialoganglioside.
Formulations made up of phospholipids only are less stable in
plasma. However, manipulation of the lipid membrane with
cholesterol reduces rapid release of the encapsulated bioactive
compound into the plasma or
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases
stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery,
vol. 2011, Article ID 469679, 12 pages, 2011.
doi:10.1155/2011/469679 for review).
[0623] In another embodiment, lipids may be used to form lipid
microparticles. Lipids include, but are not limited to,
DLin-KC2-DMA4, C12-200 and colipids disteroylphosphatidyl choline,
cholesterol, and PEG-DMG may be formulated (see, e.g.,
Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4;
doi:10.1038/mtna.2011.3) using a spontaneous vesicle formation
procedure. The component molar ratio may be about 50/10/38.5/1.5
(DLin-KC2-DMA or C12-200/disteroylphosphatidyl
choline/cholesterol/PEG-DMG). Tekmira has a portfolio of
approximately 95 patent families, in the U.S. and abroad, that are
directed to various aspects of lipid microparticles and lipid
microparticles formulations (see, e.g., U.S. Pat. Nos. 7,982,027;
7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397;
8,101,741; 8,188,263; 7,915,399; 8,236,943 and 7,838,658 and
European Pat. Nos. 1766035; 1519714; 1781593 and 1664316), all of
which may be used and/or adapted to the present invention.
[0624] Some vesicles and lipid-coated polymer particles are able to
spontaneously adsorb to cell surfaces.
[0625] In some embodiments, a composition described herein includes
chondrisomes encapsulated in microparticles or microgels.
[0626] Microparticles are comprised of one or more solidified
polymer(s) that is arranged in a random manner. The microparticles
may be biodegradable. Biodegradable microparticles may be
synthesized using methods known in the art including without
limitation solvent evaporation, hot melt microencapsulation,
solvent removal, and spray drying. Exemplary methods for
synthesizing microparticles are described by Bershteyn et al., Soft
Matter 4:1787-1787, 2008 and in US 2008/0014144 A1, the specific
teachings of which relating to microparticle synthesis are
incorporated herein by reference.
[0627] As discussed herein, some microparticles are biodegradable
in nature and thus they gradually degrade in an aqueous environment
such as occurs in vivo. Chondrisomes may be released from the
microparticles as the microparticle degrades or chondrisome
products may be released through pores within the microparticles.
Release kinetic studies have been performed and they demonstrate
that protein and small-molecule drugs can be released from such
microparticles over time-courses ranging from 1 day to at least 2
weeks.
[0628] Exemplary synthetic polymers which can be used to form the
biodegradable microparticles include without limitation aliphatic
polyesters, poly (lactic acid) (PLA), poly (glycolic acid) (PGA),
copolymers of lactic acid and glycolic acid (PLGA),
polycarprolactone (PCL), polyanhydrides, poly(ortho)esters,
polyurethanes, poly(butyric acid), poly(valeric acid), and
poly(lactide-co-caprolactone), and natural polymers such as
albumin, alginate and other polysaccharides including dextran and
cellulose, collagen, chemical derivatives thereof, including
substitutions, additions of chemical groups such as for example
alkyl, alkylene, hydroxylations, oxidations, and other
modifications routinely made by those skilled in the art), albumin
and other hydrophilic proteins, zein and other prolamines and
hydrophobic proteins, copolymers and mixtures thereof. In general,
these materials degrade either by enzymatic hydrolysis or exposure
to water in vivo, by surface or bulk erosion.
[0629] The microparticles' diameter ranges from 0.1-1000
micrometers (.mu.m). In some embodiments, their diameter ranges in
size from 1-750 .mu.m, or from 50-500 .mu.m, or from 100-250 .mu.m.
In some embodiments, their diameter ranges in size from 50-1000
.mu.m, from 50-750 .mu.m, from 50-500 .mu.m, or from 50-250 .mu.m.
In some embodiments, their diameter ranges in size from 0.05-1000
.mu.m, from 10-1000 .mu.m, from 100-1000 .mu.m, or from 500-1000
.mu.m. In some embodiments, their diameter is about 0.5 .mu.m,
about 10 .mu.m, about 50 .mu.m, about 100 .mu.m, about 200 .mu.m,
about 300 .mu.m, about 350 .mu.m, about 400 .mu.m, about 450 .mu.m,
about 500 .mu.m, about 550 .mu.m, about 600 .mu.m, about 650 .mu.m,
about 700 .mu.m, about 750 .mu.m, about 800 .mu.m, about 850 .mu.m,
about 900 .mu.m, about 950 .mu.m, or about 1000 .mu.m. As used in
the context of microparticle diameters, the term "about" means+/-5%
of the absolute value stated. Thus, it is to be understood that
although these particles are referred to herein as microparticles,
the invention intends to embrace nanoparticles as well.
[0630] In some embodiments, a ligand is conjugated to the surface
of the microparticle via a functional chemical group (carboxylic
acids, aldehydes, amines, sulfhydryls and hydroxyls) present on the
surface of the particle and present on the ligand to be attached.
Functionality may be introduced into the microparticles by, for
example, during the emulsion preparation of microparticles,
incorporation of stabilizers with functional chemical groups.
[0631] Another example of introducing functional groups to the
microparticle is during post-particle preparation, by direct
crosslinking particles and ligands with homo- or heterobifunctional
crosslinkers. This procedure may use a suitable chemistry and a
class of crosslinkers (CDI, EDAC, glutaraldehydes, etc. as
discussed in more detail below) or any other crosslinker that
couples ligands to the particle surface via chemical modification
of the particle surface after preparation. This also includes a
process whereby amphiphilic molecules such as fatty acids, lipids
or functional stabilizers may be passively adsorbed and adhered to
the particle surface, thereby introducing functional end groups for
tethering to ligands.
[0632] In some embodiments, the microparticles may be synthesized
to comprise one or more targeting groups on their exterior surface
to target a specific cell or tissue type (e.g., cardiomyocytes).
These targeting groups include without limitation receptors,
ligands, antibodies, and the like. These targeting groups bind
their partner on the cells' surface. In some embodiments, the
microparticles will integrate into a lipid bilayer that comprises
the cell surface and the chondrisomes are delivered to the
cell.
[0633] The microparticles may also comprise a lipid bilayer on
their outermost surface. This bilayer may be comprised of one or
more lipids of the same or different type. Examples include without
limitation phospholipids such as phosphocholines and
phosphoinositols. Specific examples include without limitation
DMPC, DOPC, DSPC, and various other lipids such as those described
herein for liposomes.
[0634] In some embodiments, the vesicles or microparticles
described herein are functionalized with a diagnostic agent.
Examples of diagnostic agents include, but are not limited to,
commercially available imaging agents used in positron emissions
tomography (PET), computer assisted tomography (CAT), single photon
emission computerized tomography, x-ray, fluoroscopy, and magnetic
resonance imaging (MRI); and contrast agents. Examples of suitable
materials for use as contrast agents in MRI include gadolinium
chelates, as well as iron, magnesium, manganese, copper, and
chromium.
Modified Preparations
Source Modification
[0635] In one aspect, a modification is made to the source, such as
a subject, tissue or cell, that affects the mitochondria or
chondrisome preparation, such as producing mitochondria with a
heterologous function or a structural change in the mitochondria.
Such modifications can be effective to, e.g., improve mitochondrial
activity, function or structure. Modifications to the source can
include, but are not limited to, changes to the cellular metabolic
state (e.g. through different culture conditions or through
transfected regulatory modulators); changes to the cellular
regulatory state; and changes to the source cells' differentiation
state.
[0636] Stress Treatment
[0637] The source may be treated to modulate mitochondrial
activity, function or structure prior to isolation of chondrisomes.
In some embodiments, chondrisomes are obtained from a source that
has been stressed. A stress condition can include nutritional
stress (reduction in carbon (e.g., sugar) and/or amino acid
source), osmotic stress, hypoxia, temperature stress, injury. Such
stress conditions may enhance mitochondrial biogenesis or function
in the source.
[0638] In one embodiment, chondrisomes can be obtained from a
source exposed to different temperatures: e.g., isolated
chondrisomes from a source below freezing (e.g., at -20.degree. C.
or lower, -4.degree. C. or lower, lower than 0.degree. C.), a cold
or chilled source (e.g., between 0.degree. C.-10.degree. C., e.g.,
0.degree. C., 4.degree. C., 10.degree. C.), a source at or around
room temperature (between 15.degree. C.-25.degree. C., e.g.,
15.degree. C., 20.degree. C., 25.degree. C.) or a warmed source
(between 25.degree. C.-42.degree. C., e.g., 32.degree. C.,
37.degree. C.). In another embodiment, the invention includes a
composition of chondrisomes isolated from a source exposed to a
time and temperature sufficient to modulate mitochondrial activity,
function, structure, or any combination thereof (e.g., by at least
10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more). The
source is exposed to the temperature difference for a time
sufficient to modulate its mitochondrial activity, function,
structure, or any combination thereof (e.g., by at least 10%, 15%,
20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more).
[0639] In another example, chondrisomes are obtained from a source
exposed to a reference oxygen concentrations, e.g., hypoxia (e.g.,
about 0% to about 4%), normoxia (e.g., about 1% to about 14%),
hyperoxia (e.g., about 5% or higher), or any concentration
therebetween. Normal O.sub.2 concentration varies significantly
between tissues. In another embodiment, the invention includes a
composition of chondrisomes isolated from a source exposed to an
O.sub.2 concentration for a time sufficient to modulate its
mitochondrial activity, function, structure, or any combination
thereof (e.g., by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%,
80%, 90% or more). O.sub.2 concentrations in parenchymal organs
(liver, kidneys, heart) varies from about 4% to about 14%; in the
brain, it varies from about 0.5% to about 7%; in the eye (retina,
corpus vitreous), it varies from about 1 to about 5%; and in the
bone marrow, it varies from almost 0% to about 4%. The source is
exposed to an oxygen concentration that is sufficient to modulate
mitochondrial activity, function, structure, or any combination
thereof (e.g., by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%,
80%, 90% or more). The source is exposed to the oxygen
concentration for a time sufficient to modulate its mitochondrial
activity, function, structure, or any combination thereof (e.g., by
at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or
more).
[0640] In some embodiments, chondrisomes are obtained from a source
exposed to starvation conditions, e.g., lack of added glucose or
other sugar substrate, amino acids, or a combination thereof. In
another embodiment, the invention includes a composition of
chondrisomes isolated from a source exposed to starvation
conditions for a time sufficient to modulate its mitochondrial
activity, function, structure, or any combination thereof (e.g., by
at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more).
The source is exposed to starvation conditions that are sufficient
to modulate its mitochondrial activity, function, structure, or any
combination thereof (e.g., by at least 10%, 15%, 20%, 30%, 40%,
50%, 60%, 75%, 80%, 90% or more). The source is exposed to the
starvation conditions for a time sufficient to modulate its
mitochondrial activity, function, structure, or any combination
thereof (e.g., by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%,
80%, 90% or more).
[0641] In another embodiment, chondrisomes are obtained from a
source exposed to specified concentrations of one or more
nutrients, e.g., reduced concentration of glucose or other sugar
substrate, amino acids, or a combination thereof. In another
embodiment, the invention includes a composition of chondrisomes
isolated from a source exposed to one or more nutrient
concentrations for a time sufficient to modulate its mitochondrial
activity, function, structure, or any combination thereof (e.g., by
at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more).
The source is exposed to a nutrient concentration or combination
(e.g., ratio) of nutrients that is sufficient to modulate its
mitochondrial activity, function, structure, or any combination
thereof (e.g., by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%,
80%, 90% or more). The source is exposed to the nutrient
concentration or combination (e.g., ratio) of nutrients for a time
sufficient to modulate its mitochondrial activity, function,
structure, or any combination thereof (e.g., by at least 10%, 15%,
20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more).
[0642] In another embodiment, chondrisomes are obtained from a
source exposed to osmotic stress, e.g., increase or decrease in
solute concentration. In another embodiment, the invention includes
a composition of chondrisomes isolated from a source exposed to
osmotic stress for a time sufficient to modulate its mitochondrial
activity, function, structure, or any combination thereof (e.g., by
at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more).
The source is exposed to a solute concentration that is sufficient
to modulate its mitochondrial activity, function, structure, or any
combination thereof (e.g., by at least 10%, 15%, 20%, 30%, 40%,
50%, 60%, 75%, 80%, 90% or more). The source is exposed to the
solute concentration for a time sufficient to modulate its
mitochondrial activity, function, structure, or any combination
thereof (e.g., by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%,
80%, 90% or more).
[0643] In some embodiments, chondrisomes are obtained from a source
that has been injured or a source undergoing the wound healing
process. At least about 20% of the source may be injured, at least
about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, or up to 100% of the source may be injured.
Chondrisomes may be obtained from a source that has been injured
within about 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day,
20 hours, 18 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12
hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5
hours, 4 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour, 50
mins, 40 mins, 30 mins, 25 mins, 20 mins, 15 mins, 10 mins, 5 mins,
or less. The source may be injured by any physical, chemical, or
other process described herein or known to cause injury to a
source. In another embodiment, the invention includes a composition
of chondrisomes isolated from a source exposed to injury for a time
sufficient to modulate its mitochondrial activity, function,
structure, or any combination thereof (e.g., by at least 10%, 15%,
20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more).
[0644] Toxin Treatment
[0645] The source may be treated with a toxin to modulate its
mitochondrial activity, function or structure. In some embodiments,
chondrisomes are obtained from source that has been treated with or
exposed to one or more toxins (e.g., a mitochondrial toxin), such
as an inhibitor of complex 1 activity, e.g., metformin. Additional
toxins are described in
http://www.mitoaction.org/files/MitoToxins_0. pdf Treating a source
with a toxin or injury inducing chemical agent induces processes
within the source, such as mechanisms to compensate for toxin
injury, that may enhance mitochondrial biogenesis or activity or
function.
[0646] In some embodiments, chondrisomes are obtained from a source
that has been exposed to a toxin described herein. At least about
20% of the source may be exposed to the toxin, at least about 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, or up to 100% of the source is exposed to the toxin.
Chondrisomes may be obtained from a source that has been exposed to
the toxin within about 7 days, 6 days, 5 days, 4 days, 3 days, 2
days, 1 day, 20 hours, 18 hours, 16 hours, 15 hours, 14 hours, 13
hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6
hours, 5 hours, 4 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1
hour, 50 mins, 40 mins, 30 mins, 25 mins, 20 mins, 15 mins, 10
mins, 5 mins, or less. In another embodiment, the invention
includes a composition of chondrisomes isolated from a source
exposed to a toxin for a time sufficient to modulate its
mitochondrial activity, function, structure, or any combination
thereof (e.g., by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%,
80%, 90% or more).
[0647] Infectious Agent Treatment
[0648] In another embodiment, a source may be treated with one or
more infectious agents, such as a virus or bacteria (e.g.,
hepatitis C virus (HCV) and hepatitis B virus (HBV)). A viral
infection causes many physiological alterations in a source and
many of those alterations can directly affect its mitochondrial
dynamics and mitophagy. For example, expression of HCV core and
NS5a proteins perturb complex 1 activity and promote mitochondrial
Ca.sup.2+ uptake, ROS production, and mitochondrial permeability
transition. In another example, source infection with an infectious
agent, such as a virus or bacteria, may induce mitochondrial
dysfunction that activates the innate immune response to fight the
infection. In some embodiments, the source is exposed to an
infectious agent that is sufficient to modulate its mitochondrial
activity, function, structure, or any combination thereof (e.g., by
at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more).
The source is exposed to the infectious agent for a time sufficient
to modulate its mitochondrial activity, function, structure, or any
combination thereof (e.g., by at least 10%, 15%, 20%, 30%, 40%,
50%, 60%, 75%, 80%, 90% or more). In another embodiment, the
invention includes a composition of chondrisomes isolated from a
source exposed to an infectious agent for a time sufficient to
modulate its mitochondrial activity, function, structure, or any
combination thereof (e.g., by at least 10%, 15%, 20%, 30%, 40%,
50%, 60%, 75%, 80%, 90% or more).
[0649] Increasing Bioenergy in Source
[0650] In some embodiments, communication between the mitochondrion
and the cytosol across the outer mitochondrial membrane and the
remarkably high-resistance inner membrane is dependent on numerous
transporters. Thus modulation of these transporters via protein
phosphorylation affects the ability of the cytosol to influence
mitochondrial reaction pathways via the exchange of metabolites and
signaling molecules, as well as proteins. For example,
phosphorylation of mitochondrial pyruvate dehydrogenase (PDH) is
metabolically controlled by enzyme phosphorylation via the PDH
kinase (PDHK) and PDH phosphatase (PDHP) system. In one embodiment,
the source is treated with dephosphorylated pyruvate dehydrogenase
to catabolize glucose and gluconeogenesis precursors. In another
embodiment, the source is treated with phosphorylated pyruvate
dehydrogenase to shift metabolism toward fat utilization. In
another embodiment, the invention includes a composition of
chondrisomes comprising phosphorylated mitochondrial pyruvate
dehydrogenase (e.g., at least 10%, 15%, 20%, 30%, 40%, 50%, 60%,
75%, 80%, 90% or more pyruvate dehydrogenase is phosphorylated). In
another embodiment, the invention includes a composition of
chondrisomes isolated from a source exposed to pyruvate
dehydrogenase kinase for a time sufficient to phosphorylate
mitochondrial pyruvate dehydrogenase (e.g., increase
phosphorylation by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%,
80%, 90% or more).
[0651] Targeting to Mitochondria
[0652] Modifications to the source may also change the distribution
and/or quantity of nuclearly encoded mitochondrial targeted
proteins. In some embodiments, these modifications can involve
targeting proteins or RNA not normally present in the mitochondria
(including both endogenous and exogenous genes) to the mitochondria
by the addition of a targeting sequence to non-mitochondrial
proteins or a mitochondrial import signal appended to RNA. In one
embodiment, the invention includes a composition of chondrisomes
comprising non-mitochondrial proteins (e.g., at least 10%, 15%,
20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more proteins are
non-mitochondrial proteins).
[0653] Of the many proteins involved in mitochondrial function,
only a handful are encoded by the mitochondrial genome and the rest
are expressed by the nuclear genome and transported into the
mitochondria. Mitochondrial proteins may be targeted to the
mitochondrial matrix, inner membrane, outer membrane, or the
intermembrane space. In one embodiment, the invention includes a
composition of chondrisomes comprising non-mitochondrial proteins
in the mitochondrial matrix, inner membrane, outer membrane, or the
intermembrane space (e.g., at least 10%, 15%, 20%, 30%, 40%, 50%,
60%, 75%, 80%, 90% or more non-mitochondrial are located in the
mitochondrial matrix, inner membrane, outer membrane, or the
intermembrane space).
[0654] In addition, mitochondria import a modest number of RNAs
(e.g., small noncoding RNAs, miRNAs, tRNAs, and possibly lncRNAs
and viral RNAs). RNAs are processed within mitochondria and may
have functions different from their cytosolic or nuclear
counterparts. In some tissues, RNA splice-variants are
differentially present in mitochondria or the cytosol. For example,
variants that code for mitochondrial and cytosolic
selenocysteine-containing isoforms possess identical glutaredoxin
(Grx) and thioredoxin reductase (TR) domains but differ exclusively
in their N termini. In one embodiment, the invention includes a
composition of chondrisomes comprising cytosolic RNA or nuclear
RNA. Alternative trans-splicing may create a long or a short
spliced variant of trypanosomal isoleucyl-tRNA synthetase (IleRS).
The protein product of the longer spliced variant possesses an
amino-terminal presequence and is found exclusively in
mitochondria. In contrast, the shorter spliced variant is
translated to a cytosol-specific isoform lacking the presequence.
In some embodiments, a distribution of alternative splice variants,
such as in the cytosol or the mitochondria, is altered by
increasing the presence of one or more forms or decreasing the
presence of one or more forms. In one embodiment, the invention
includes a composition of chondrisomes comprising an altered
distribution of alternative splice variants (e.g., at least 10%,
15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more of the
distribution of the splice variants is altered). In another
embodiment, the invention includes a composition of chondrisomes
comprising an increase of the presence of one or more forms or a
decrease in the presence of one or more forms e.g., at least 10%,
15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more of the
distribution of the splice variants is altered.
[0655] Import into mitochondria can be initiated by N-terminal
targeting sequences (presequences) or internal targeting sequences.
Import into the organelle may be mediated by a translocase in the
outer membrane (TOM) complex, molecular chaperone proteins, and
targeting sequences. A translocase in the inner membrane (TIM)
complex mediates transit from the intermembrane space into the
mitochondrial matrix, as well as embedding proteins into the inner
membrane. A carrier translocase (TIM22) also is capable of
embedding carrier proteins and N-terminal targeted inner membrane
proteins. In one embodiment, the invention includes a composition
of chondrisomes comprising a translocase with modulated activity
e.g., an increase or a decrease in activity of at least 10%, 15%,
20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more as compared to a
non-modulated translocase. In one embodiment, the invention
includes a composition of chondrisomes comprising a protein (or
nucleic acid encoding) a fusion of a translocase and a cargo
protein (e.g., a cargo protein described herein, e.g., an agent
listed in Table 4).
[0656] Proteins destined for import into the mitochondria via the
presequence import pathway may have a leader sequence or an
N-terminal targeting sequence capable of forming an amphipathic
helix. In some embodiments, a protein (e.g., a cargo protein) is
engineered fused to a mitochondrial targeting sequence (MTS)
described herein. Some MTS may be about 10 to about 80 amino acids
in length, and generally able to form amphipathic helices. In one
embodiment, a protein is engineered with a MTS between about 10 to
100 amino acids in length, about 10 to 75 amino acids in length,
about 10 to 50 amino acids in length, about 10 to 40 amino acids in
length, about 10 to about 30 amino acids in length, or any range
therebetween. In one embodiment, the invention includes a
composition of chondrisomes comprising an exogenous protein (e.g.,
a exogenous protein described herein) linked to an MTS. In another
embodiment, the MTS forms one or more amphipathic helices. The
amphipathic helix structure is recognized by the import machinery
and is ultimately cleaved off after the protein enters the
mitochondrial matrix by a mitochondrial processing peptidase.
Protein import into the mitochondria is also described in, for
example, Bolender et al., 2008, EMBO Rep. 9, 42-49; Dolezal et al.,
2006, Science 313, 314-318; Gabriel and Pfanner, 2007, Methods Mol.
Biol. 390, 99-117; Pfanner et al., 2004, Nat. Struct. Mol. Biol.
11, 1044-1048.
[0657] Some mitochondrial proteins do not have an N-terminal
targeting sequence and an internal or C-terminal sequence may be
used for localization. Mitochondrial targeting sequences may be
enriched in positive, hydrophobic, and hydroxylated amino acids,
while acidic residues are rare. While some proteins may lack a
targeting sequence, others are dual targeted, e.g., targeted to the
mitochondrion and to one or more additional subcellular
compartments.
[0658] While multiple mitochondrial targeting sequences are
effective at localizing exogenous protein to mitochondria, sequence
and physiochemical characteristics of the amino acids determine the
precise localization. In some embodiments, the mitochondrial
targeting sequence is a sequence from a 5S rRNA, such as the fly 5S
rRNA variant V. In one embodiment, the invention includes a
composition of chondrisomes comprising an exogenous protein
comprising a MTS from 5S rRNA. In some embodiments, the
mitochondrial targeting sequence is from the RNA component of the
endoribonuclease known as MRP, or the RNA component of the
ribonucleoprotein known as RNAse P. In another embodiment, the
invention includes a composition of chondrisomes comprising an
exogenous protein comprising a MTS from RNAse P. In some
embodiments, a mitochondrial targeting sequence may be designed to
target the mitochondrial matrix by replicating the first 69 amino
acids of the precursor of subunit 9 of the mitochondrial Fo-ATPase.
In one embodiment, the invention includes a composition of
chondrisomes comprising an exogenous protein comprising a MTS from
precursor of subunit 9 of the mitochondrial Fo-ATPase. In some
embodiments, a fusion of a mitochondrial targeting sequences, such
as the first 69 amino acids of the precursor of subunit 9 of the
mitochondrial Fo-ATPase, to dihydrofolate reductase (DHFR) imports
the fusion protein into the mitochondrial matrix.
[0659] See, Kunze, M. & Berger, J. The similarity between
N-terminal targeting signals for protein import into different
organelles and its evolutionary relevance. Frontiers in Physiology,
vol 6. 2015.
[0660] For example, e.g., the DSRed2 fluorescent protein is
targeted to the mitochondrial matrix by appending the mitochondrial
targeting sequence from subunit VIII of human cytochrome c oxidase
(ATGTCCGTCCTGACGCCGCTGCTGCTGCGGGGCTTGACAGGCTCGGCCCGGCGGCTCCCAGT
GCCGCGCGCCAAGATCCATTCGTTG, SEQ ID NO:6) to the N-terminus of the
protein. In one embodiment, the invention includes a composition of
chondrisomes comprising DSRed2 fluorescent protein. In one
embodiment, the invention includes a composition of chondrisomes
comprising DSRed2 fluorescent protein with N-terminus mitochondrial
targeting sequence, e.g., from subunit VIII of human cytochrome c
oxidase. In one embodiment, the invention includes a composition of
chondrisomes comprising an exogenous polypeptide fused to the
mitochondrial targeting sequence from subunit VIII of human
cytochrome c oxidase (SEQ ID NO:6).
[0661] In some embodiments, cytosolic proteins, such as proteases
or enzymes, are modified for targeting to the mitochondria.
Cytosolic proteins may be engineered to include a mitochondrial
targeting sequence, e.g., first 69 amino acids of the precursor of
subunit 9 of the mitochondrial Fo-ATPase. For example, cytosolic
enzymes (e.g., proteases, phosphatases, kinases, demethylases,
methyltransferases, acetylases) may be relocalized to the
mitochondria. In one embodiment, the invention includes a
composition of chondrisomes comprising cytosolic enzymes. In one
embodiment, the invention includes a composition of chondrisomes
comprising a cytosolic enzyme, e.g., a protease, phosphatase,
kinase, demethylase, methyltransferase, acetylase, or any
combination thereof.
[0662] In some embodiments, the source is modified to express
nuclearly encoded proteins typically targeted to a mitochondrial
space without their mitochondrial targeting sequence. For example,
a mitochondrial translocase protein that is nuclearly encoded and
cytosolically expressed is engineered in the source to lack a
mitochondrial targeting sequence, thereby altering the
mitochondrial to lack or have reduced mitochondrial translocase
proteins. In one embodiment, the invention includes a composition
of chondrisomes comprising a decreased level or a lack of a
nuclearly encoded and cytosolically expressed mitochondrial
protein, e.g., mitochondrial translocase protein. Mitochondria or
chondrisome preparations that lack such mitochondrial translocase
proteins have reduced protein translocation capacity.
[0663] Source Engineering
[0664] A source may be genetically modified using recombinant
methods known in the art. A nucleic acid sequence coding for a
desired gene can be obtained using recombinant methods known in the
art, such as, for example by screening libraries from cells
expressing the gene, by deriving the gene from a vector known to
include the same, or by isolating directly from cells and tissues
containing the same, using standard techniques. Alternatively, a
gene of interest can be produced synthetically, rather than
cloned.
[0665] Expression of natural or synthetic nucleic acids is
typically achieved by operably linking a nucleic acid encoding the
gene of interest to a promoter, and incorporating the construct
into an expression vector. The vectors can be suitable for
replication and integration in eukaryotes. Typical cloning vectors
contain transcription and translation terminators, initiation
sequences, and promoters useful for expression of the desired
nucleic acid sequence.
[0666] Additional promoter elements, e.g., enhancers, regulate the
frequency of transcriptional initiation. Typically, these are
located in the region 30-110 bp upstream of the start site,
although a number of promoters have recently been shown to contain
functional elements downstream of the start site as well. The
spacing between promoter elements frequently is flexible, so that
promoter function is preserved when elements are inverted or moved
relative to one another. In the thymidine kinase (tk) promoter, the
spacing between promoter elements can be increased to 50 bp apart
before activity begins to decline. Depending on the promoter, it
appears that individual elements can function either cooperatively
or independently to activate transcription.
[0667] One example of a suitable promoter is the immediate early
cytomegalovirus (CMV) promoter sequence. This promoter sequence is
a strong constitutive promoter sequence capable of driving high
levels of expression of any polynucleotide sequence operatively
linked thereto. Another example of a suitable promoter is
Elongation Growth Factor-1.alpha. (EF-1.alpha.). However, other
constitutive promoter sequences may also be used, including, but
not limited to the simian virus 40 (SV40) early promoter, mouse
mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long
terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia
virus promoter, an Epstein-Barr virus immediate early promoter, a
Rous sarcoma virus promoter, as well as human gene promoters such
as, but not limited to, the actin promoter, the myosin promoter,
the hemoglobin promoter, and the creatine kinase promoter.
[0668] Further, the invention should not be limited to the use of
constitutive promoters. Inducible promoters are also contemplated
as part of the invention. The use of an inducible promoter provides
a molecular switch capable of turning on expression of the
polynucleotide sequence which it is operatively linked when such
expression is desired, or turning off the expression when
expression is not desired. Examples of inducible promoters include,
but are not limited to a metallothionine promoter, a glucocorticoid
promoter, a progesterone promoter, and a tetracycline promoter.
[0669] The expression vector to be introduced into the source can
also contain either a selectable marker gene or a reporter gene or
both to facilitate identification and selection of expressing cells
from the population of cells sought to be transfected or infected
through viral vectors. In other aspects, the selectable marker may
be carried on a separate piece of DNA and used in a co-transfection
procedure. Both selectable markers and reporter genes may be
flanked with appropriate regulatory sequences to enable expression
in the host cells. Useful selectable markers include, for example,
antibiotic-resistance genes, such as neo and the like.
[0670] Reporter genes may be used for identifying potentially
transfected cells and for evaluating the functionality of
regulatory sequences. In general, a reporter gene is a gene that is
not present in or expressed by the recipient source and that
encodes a polypeptide whose expression is manifested by some easily
detectable property, e.g., enzymatic activity. Expression of the
reporter gene is assayed at a suitable time after the DNA has been
introduced into the recipient cells. Suitable reporter genes may
include genes encoding luciferase, beta-galactosidase,
chloramphenicol acetyl transferase, secreted alkaline phosphatase,
or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000
FEBS Letters 479: 79-82). Suitable expression systems are well
known and may be prepared using known techniques or obtained
commercially. In general, the construct with the minimal 5'
flanking region showing the highest level of expression of reporter
gene is identified as the promoter. Such promoter regions may be
linked to a reporter gene and used to evaluate agents for the
ability to modulate promoter-driven transcription.
[0671] In some embodiments, the source may be genetically modified
to alter expression of one or more proteins. Expression of the one
or more proteins may be modified for a specific time, e.g.,
development or differentiation state of the source. In one
embodiment, the invention includes a composition of chondrisomes
isolated from a source genetically modified to alter expression of
one or more proteins, e.g., mitochondrial proteins or
non-mitochondrial proteins that affect mitochondrial activity,
structure or function. Expression of the one or more proteins may
be restricted to a specific location(s) or widespread throughout
the source. Alternative trans-splicing also creates variants that
may be differentially targeted. In some embodiments, the source is
engineered to create a long or a short spliced variant, e.g.,
trypanosomal isoleucyl-tRNA synthetase (IleRS), to differentially
target the protein products, e.g., the longer spliced variant is
found exclusively in mitochondria and the shorter spliced variant
is translated to a cytosol-specific isoform. In some embodiments, a
distribution of alternative splice variants, such as in the cytosol
or the mitochondria, is altered by increasing the presence of one
or more forms or decreasing the presence of one or more forms. In
one embodiment, the invention includes a composition of
chondrisomes isolated from a source genetically modified to alter
expression of alternative splice variants, e.g., distribution of
the splice variants or protein products from the splice variants.
In one embodiment, the invention includes a composition of
chondrisomes comprising modified expression of alternative splice
variants, e.g., RNA or protein products from the splice
variants.
[0672] In some embodiments, the expression of a structural gene is
modified. For example, such structural gene may encode OMP25,
(MNGRVDYLVTEEEINLTRGPSGLGFNIVGGTDQQYVSNDSGIYVSRIKENGAAALDGRLQEGDK
ILSVNGQDLKNLLHQDAVDLFRNAGYAVSLRVQHRLQVQNGPIGHRGEGDPSGIPIFMVLVPVF
ALTMVAAWAFMRYRQQL, SEQ ID NO:10) or a protein at least about 85%,
90%, 95%, 100% identical to SEQ ID NO:10. In one embodiment, the
invention includes a composition of chondrisomes comprising
modified expression of a structural gene, e.g., an increase or a
decrease in expression of OMP25 by at least 10%, 15%, 20%, 30%,
40%, 50%, 60%, 75%, 80%, 90% or more.
[0673] In some embodiments, the expression of a membrane targeted
protein is modified. In some embodiments, such membrane proteins
can be chemical or ion transporters (e.g. MPC1/2 (Mitochondrial
Pyruvate carrier) or UCP1, SEQ ID NO:1). In some embodiments, a
decreased expression results in reduced flux of compounds
transported across the membrane or an alteration of the source's
ability to dynamically control said flux. In one embodiment, the
invention includes a composition of chondrisomes comprising
modified expression of a membrane targeted protein, e.g., an
increase or a decrease in expression of a chemical or ion
transporter by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%,
80%, 90% or more.
[0674] In some embodiments, the expression of a translocase in the
outer membrane (such as TOM22,
MAAAVAAAGAGEPQSPDELLPKGDAEKPEEELEEDDDEELDETLSERLWGLTEMFPERVRSAA
GATFDLSLFVAQKMYRFSRAALWIGTTSFMILVLPVVFETEKLQMEQQQQLQQRQILLGPNTGLS
GGMPGALPSLPGKI, SEQ ID NO: 11) complex, a translocase in the inner
membrane (such as TIM17A,
MEEYAREPCPWRIVDDCGGAFTMGTIGGGIFQAIKGFRNSPVGVNHRLRGSLTAIKTRAPQLGGS
FAVWGGLFSMIDCSMVQVRGKEDPWNSITSGALTGAILAARNGPVAMVGSAAMGGILLALIEG
AGILLTRFASAQFPNGPQFAEDPSQLPSTQLPSSPFGDYRQYQ, SEQ ID NO:12, or
TIM17B,
MEEYAREPCPWRIVDDCGGAFTMGVIGGGVFQAIKGFRNAPVGIRHRLRGSANAVRIRAPQIGG
SFAVWGGLFSTIDCGLVRLRGKEDPWNSITSGALTGAVLAARSGPLAMVGSAMMGGILLALIEG
VGILLTRYTAQQFRNAPPFLEDPSQLPPKDGTPAPGYPSYQQYH, SEQ ID NO:13)
complex, or a carrier translocase (such as TIM22,
MAAAAPNAGGSAPETAGSAEAPLQYSLLLQYLVGDKRQPRLLEPGSLGGIPSPAKSEEQKMIEK
AMESCAFKAALACVGGFVLGGAFGVFTAGIDTNVGFDPKDPYRTPTAKEVLKDMGQRGMSYA
KNFAIVGAMFSCTECLIESYRGTSDWKNSVISGCITGGAIGFRAGLKAGAIGCGGFAAFSAAIDYY
LR, SEQ ID NO:14) is modified. In some embodiments, the source is
engineered to express a protein at least 85%, 90%, 95%, 100%
identical to SEQ ID NOs: 11, 12, 13, or 14. In one embodiment, the
invention includes a composition of chondrisomes comprising
modified expression of a translocase in the outer membrane complex,
a translocase in the inner membrane complex, or a carrier
translocase, e.g., an increase or a decrease in expression of the
translocase by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%,
80%, 90% or more.
[0675] In another embodiment, the expression of one or more
metabolic conversion enzymes is altered to adjust the metabolic
capacity of the chondrisome preparation. In some embodiments, such
enzymes alter the capacity of the chondrisome preparation to alter
a patient's metabolic concentration, e.g. OTC (ornithine
transcarbamylase), such as by altering the ability to address urea
cycle disorders. In some embodiments, such enzymes can be selected
for their ability to adjust redox balancing and cycling, such as
NADH oxidases (e.g. the heterologous LbNOX, see for example, Titov,
D. V., et al., 2016, Science, 352(6282):231-235). In one
embodiment, the invention includes a composition of chondrisomes
comprising modified expression of one or more metabolic conversion
enzymes, e.g., an increase or a decrease in expression of the
metabolic conversion enzyme by at least 10%, 15%, 20%, 30%, 40%,
50%, 60%, 75%, 80%, 90% or more.
[0676] In some embodiments, the source may be engineered to express
a cytosolic enzyme (e.g., proteases, phosphatases, kinases,
demethylases, methyltransferases, acetylases) that targets a
mitochondrial protein. In some embodiments, the source may be
engineered to express one or more enzymes that is relocated to the
mitochondria. In some embodiments, the enzyme affects one or more
mitochondrial proteins (such as membrane transporters, intermediary
metabolism enzymes, and the complexes of oxidative phosphorylation)
by altering post-translational modifications. Post-translational
protein modifications of proteins may affect responsiveness to
nutrient availability and redox conditions, and protein-protein
interactions. In one embodiment, the invention includes a
composition of chondrisomes comprising proteins with altered
post-translational modifications, e.g., an increase or a decrease
in post-translational modifications by at least 10%, 15%, 20%, 30%,
40%, 50%, 60%, 75%, 80%, 90% or more on membrane transporters,
intermediary metabolism enzymes, and the complexes of oxidative
phosphorylation.
[0677] In some embodiments, a source is engineered to up- or
down-regulate expression of an enzyme that controls a
post-translational modification in the mitochondria. For example,
PDH (pyruvate dehydrogenase) can be activated by deacetylation to
alter the metabolic connection between glycolysis and the citric
acid cycle by overexpression of SIRT3 or a protein at least 85%,
90%, 95%, 100% identical to SEQ ID NO:7 in the source. Similarly,
phosphorylation of pyruvate dehydrogenase driven by increased
expression pyruvate dehydrogenase kinase
(MRLARLLRGAALAGPGPGLRAAGFSRSFSSDSGSSPASERGVPGQVDFYARFSPSPLSMKQFLDF
GSVNACEKTSFMFLRQELPVRLANIMKEISLLPDNLLRTPSVQLVQSWYIQSLQELLDFKDKSAE
DAKAIYDFTDTVIRIRNRHNDVIPTMAQGVIEYKESFGVDPVTSQNVQYFLDRFYMSRISIRMLLN
QHSLLFGGKGKGSPSHRKHIGSINPNCNVLEVIKDGYENARRLCDLYYINSPELELEELNAKSPGQ
PIQVVYVPSHLYHMVFELFKNAMRATMEHHANRGVYPPIQVHVTLGNEDLTVKMSDRGGGVPL
RKIDRLFNYMYSTAPRPRVETSRAVPLAGFGYGLPISRLYAQYFQGDLKLYSLEGYGTDAVIYIK
ALSTDSIERLPVYNKAAWKHYNTNHEADDWCVPSREPKDM TTFRSA, SEQ ID NO:8) in
the source to inhibit PDH metabolic flux. In one embodiment, the
invention includes a composition of chondrisomes comprising
proteins with increased or decreased phosphorylation, e.g., an
increase or a decrease in phosphorylation by at least 10%, 15%,
20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more on membrane
transporters, intermediary metabolism enzymes, and/or the complexes
of oxidative phosphorylation. In another example, O-GlcNAc
transferase
(MASSVGNVADSTEPTKRMLSFQGLAELAHREYQAGDFEAAERHCMQLWRQEPDNTGVLLLLS
SIHFQCRRLDRSAHFSTLAIKQNPLLAEAYSNLGNVYKERGQLQEAIEHYRHALRLKPDFIDGYIN
LAAALVAAGDMEGAVQAYVSALQYNPDLYCVRSDLGNLLKALGRLEEAKACYLKAIETQPNF
AVAWSNLGCVFNAQGEIWLAIHHFEKAVTLDPNFLDAYINLGNVLKEARIFDRAVAAYLRALSL
SPNHAVVHGNLACVYYEQGLIDLAIDTYRRAIELQPHFPDAYCNLANALKEKGSVAEAEDCYNT
ALRLCPTHADSLNNLANIKREQGNIEEAVRLYRKALEVFPEFAAAHSNLASVLQQQGKLQEALM
HYKEAIRISPTFADAYSNMGNTLKEMQDVQGALQCYTRAIQINPAFADAHSNLASIHKDSGNIPE
AIASYRTALKLKPDFPDAYCNLAHCLQIVCDWTDYDERMKKLVSIVADQLEKNRLPSVHPHHS
MLYPLSHGFRKAIAERHGNLCLDKINVLHKPPYEHPKDLKLSDGRLRVGYVSSDFGNHPTSHLM
QSIPGMHNPDKFEVFCYALSPDDGTNFRVKVMAEANHFIDLSQIPCNGKAADRIHQDGIHILVNM
NGYTKGARNELFALRPAPIQAMWLGYPGTSGALFMDYIITDQETSPAEVAEQYSEKLAYMPHTF
FIGDHANMFPHLKKKAVIDFKSNGHIYDNRIVLNGIDLKAFLDSLPDVKIVKMKCPDGGDNADSS
NTALNMPVIPMNTIAEAVIEMINRGQIQITINGFSISNGLATTQINNKAATGEEVPRTIIVTTRSQYG
LPEDAIVYCNFNQLYKIDPSTLQMWANILKRVPNSVLWLLRFPAVGEPNIQQYAQNMGLPQNRII
FSPVAPKEEHVRRGQLADVCLDTPLCNGHTTGMDVLWAGTPMVTMPGETLASRVAASQLTCLG
CLELIAKNRQEYEDIAVKLGTDLEYLKKVRGKVWKQRISSPLFNTKQYTMELERLYLQMWEHY
AAGNKPDHMIKPVEVTESA, SEQ ID NO:9 or a sequence at least about 85%,
90%, 95%, 100% identical to SEQ ID NO:9) is overexpressed in the
source to reduce the ETC complex 1 activities by altering
O-GlcNAcylation. In one embodiment, the invention includes a
composition of chondrisomes comprising proteins with increased or
decreased O-GlcNAcylation, e.g., an increase or a decrease in
altering O-GlcNAcylation by at least 10%, 15%, 20%, 30%, 40%, 50%,
60%, 75%, 80%, 90% or more on membrane transporters, intermediary
metabolism enzymes, and/or the complexes of oxidative
phosphorylation.
[0678] In another embodiment, the source is modified to alter
expression of kinases or phosphatases. Such alteration changes
phosphorylation states in the mitochondria or chondrisome
preparation. In some embodiments, one or more enzymes is selected
that alters energy buffering, such as CKs (creatine kinase). By
changing production levels of phosphocreatine, ATP buffering can be
modified. In some embodiments, one or more kinases is selected
based on a distribution of post-translational modifications that
controls signaling and/or metabolic flux control, such as AMPK and
its ability to alter fatty acid oxidation. In one embodiment, the
invention includes a composition of chondrisomes comprising
proteins with increased or decreased levels of phosphocreatine,
e.g., an increase or a decrease in levels of phosphocreatine by at
least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more.
[0679] Mitochondria proteins have a strikingly high percentage of
proteins that are acetylated on one or more lysines. Thousands of
mitochondrial acetylation sites have now been identified and the
mitochondrial protein deacetylase, Sirt3, is one of the enzymes
that controls acetylation in mitochondria. In one embodiment, the
mitochondria of the preparation are modified to express a protein
deacetylase, e.g., SIRT3. In one embodiment, the source is
engineered such that the mitochondria express a protein at least
85%, 90%, 95%, 100% identical to the sequence of human SIRT3 (or
SEQ ID NO:7), wherein the protein has deacetylase activity in the
mitochondria. In another embodiment, the invention includes a
composition of chondrisomes comprising an exogenous protein at
least 85%, 90%, 95%, 100% identical to the sequence of human SIRT3
(or SEQ ID NO:7). In another example, the source is modified to
alter expression of a transcription factor, such as human TFAM
(MAFLRSMWGVLSALGRSGAELCTGCGSRLRSPFSFVYLPRWFSSVLASCPKKPVSSYLRFSKEQ
LPIFKAQNPDAKTTELIRRIAQRWRELPDSKKKIYQDAYRAEWQVYKEEISRFKEQLTPSQIMSLE
KEIMDKHLKRKAMTKKKELTLLGKPKRPRSAYNVYVAERFQEAKGDSPQEKLKTVKENWKNL
SDSEKELYIQHAKEDETRYHNEMKSWEEQMIEVGRKDLLRRTIKKQRKYGAEEC, SEQ ID
NO:15). In one embodiment, the source is engineered such that the
mitochondria express a protein at least 85%, 90%, 95%, 100%
identical to the sequence of human TFAM (or SEQ ID NO:15). In
another embodiment, the invention includes a composition of
chondrisomes comprising an exogenous protein at least 85%, 90%,
95%, 100% identical to the sequence of human TFAM (or SEQ ID
NO:15).
[0680] In some embodiments, one or more transcription factors is
physically associated with the mitochondria. In some embodiments,
one or more transcription factors alters a mitochondrial process,
such as PGC-1 alpha (peroxisome proliferator-activated receptor
gamma coactivator 1-alpha,
MAWDMCNQDSESVWSDIECAALVGEDQPLCPDLPELDLSELDVNDLDTDSFLGGLKWCSDQSE
IISNQYNNEPSNIFEKIDEENEANLLAVLTETLDSLPVDEDGLPSFDALTDGDVTTDNEASPSSMPD
GTPPPQEAEEPSLLKKLLLAPANTQLSYNECSGLSTQNHANHNHRIRTNPAIVKTENSWSNKAKSI
CQQQKPQRRPCSELLKYLTTNDDPPHTKPTENRNSSRDKCTSKKKSHTQSQSQHLQAKPTTLSLP
LTPESPNDPKGSPFENKTIERTLSVELSGTAGLTPPTTPPHKANQDNPFRASPKLKSSCKTVVPPPS
KKPRYSESSGTQGNNSTKKGPEQSELYAQLSKSSVLTGGHEERKTKRPSLRLFGDHDYCQSINSK
TEILINISQELQDSRQLENKDVSSDWQGQICSSTDSDQCYLRETLEASKQVSPCSTRKQLQDQEIR
AELNKHFGHPSQAVFDDEADKTGELRDSDFSNEQFSKLPMFINSGLAMDGLFDDSEDESDKLSY
PWDGTQSYSLFNVSPSCSSFNSPCRDSVSPPKSLFSQRPQRMRSRSRSFSRHRSCSRSPYSRSRSRS
PGSRSSSRSCYYYESSHYRHRTHRNSPLYVRSRSRSPYSRRPRYDSYEEYQHERLKREEYRREYE
KRESERAKQRERQRQKAIEERRVIYVGKIRPDTTRTELRDRFEVFGEIEECTVNLRDDGDSYGFIT
YRYTCDAFAALENGYTLRRSNETDFELYFCGRKQFFKSNYADLDSNSDDFDPASTKSKYDSLDF
DSLLKEAQRSLRR, SEQ ID NO:16) drives mitochondrial biogenesis. In
some embodiments, the source is engineered such that the
mitochondria express human PGC-1alpha or a protein at least 85%,
90%, 95%, 100% identical to SEQ ID NO:16. In one embodiment, the
invention includes a composition of chondrisomes comprising an
exogenous protein at least 85%, 90%, 95%, 100% identical to the
sequence of human PGC-1alpha (or SEQ ID NO:16).
[0681] In another example, the source is modified to express an
engineered affinity domain protein. In some embodiments, such an
affinity domain, e.g., a FLAG tag (DYKDDDDK, SEQ ID NO:17) or
tandem repeat of the FLAG tag, is tethered to a mitochondrial
membrane protein, such as 3XFLAG-EGFP-OMP25 (see, for example,
Chen, W. W., et al., Cell, 2016, 166(5):1324-1337). In some
embodiments, such an affinity domain is cleavable so that it may be
removed prior to therapeutic delivery of the chondrisome
preparation. In one embodiment, the invention includes a
composition of chondrisomes comprising a protein with a FLAG tag,
e.g., a protein fusion with a FLAG tag, e.g., FLAG-OMP25.
[0682] In some embodiments, the source is engineered to lack a
protein that affects a mitochondrial function, such as inhibiting
the source's expression of a mitochondrial protein, or inhibiting a
specific combination of endogenous genes that are targeted to the
mitochondria (e.g. through inducible siRNA, through RNA CRISPR as
described elsewhere herein). In one embodiment, the invention
includes a composition of chondrisomes that lack one or more
mitochondrial proteins, e.g., a membrane protein, a translocase, or
a membrane complex protein.
[0683] Among .about.1,500 mitochondrial proteins participate in
mitochondrial biogenesis including the oxidative phosphorylation.
Around 13 proteins are encoded from the mitochondrial genome and
the rest of the mitochondrial proteins are expressed from the
nuclear genome and actively transported to the mitochondria. In
some embodiments, the source is engineered to express a
mitochondrial protein at a modulated level, e.g., over-expression,
under-expression or loss of nuclearly encoded mitochondrial
proteins. In one embodiment, the source is engineered to express a
protein that is at least 85%, 90%, 95%, 100% identical to a
mitochondrial protein. In another embodiment, the invention
includes a composition of chondrisomes comprising an exogenous
protein that is at least 85%, 90%, 95%, or more identical to a
mitochondrial protein. In another embodiment, the invention
includes a composition of chondrisomes comprising a modulated level
of one or more nuclearly encoded mitochondrial proteins, e.g.,
over-expression, under-expression or loss of a nuclearly encoded
mitochondrial protein, such as a membrane protein, a translocase,
or an enzyme that controls post-translational modification of
mitochondrial proteins.
[0684] In some embodiments, the mitochondria are engineered to
translate a nucleic acid, such as an exogenous nucleic acid, in the
mitochondria. In some embodiments, the mitochondria of the
preparation are modified to express a chemical transporter, e.g.,
UCP1, UCP2, UCP3, UCP4 or UCP5. The expressed transporter may be
endogenous or exogenous to the source mitochondria (e.g., the
transporter may be naturally expressed), or the mitochondria may be
modified (e.g., genetically modified or loaded) to express or
over-express the transporter. In one embodiment, the mitochondria
are engineered to express a protein at least 85%, 90%, 95%, 100%
identical to the sequence of human UCP1
(MGGLTASDVHPTLGVQLFSAGIAACLADVITFPLDTAKVRLQVQGECPTSSVIRYKGVLGTITAV
VKTEGRMKLYSGLPAGLQRQISSASLRIGLYDTVQEFLTAGKETAPSLGSKILAGLTTGGVAVFIG
QPTEVVKVRLQAQSHLHGIKPRYTGTYNAYRIIATTEGLTGLWKGTTPNLMRSVIINCTELVTYD
LMKEAFVKNNILADDVPCHLVSALIAGFCATAMSSPVDVVKTRFINSPPGQYKSVPNCAMKVFT
NEGPTAFFKGLVPSFLRLGSWNVIMFVCFEQLKRELSKSRQTMDCAT, SEQ ID NO:1),
wherein the protein has transporter activity in the mitochondria.
In another embodiment, the invention includes a composition of
chondrisomes comprising an exogenous protein at least 85%, 90%,
95%, 100% identical to the sequence of human UCP1 (or SEQ ID
NO:1).
[0685] In one embodiment, the mitochondria are engineered to
express a protein at least 85%, 90%, 95%, 100% identical to the
sequence of human UCP2
(MVGFKATDVPPTATVKFLGAGTAACIADLITFPLDTAKVRLQIQGESQGPVRATASAQYRGVM
GTILTMVRTEGPRSLYNGLVAGLQRQMSFASVRIGLYDSVKQFYTKGSEHASIGSRLLAGSTTGA
LAVAVAQPTDVVKVRFQAQARAGGGRRYQSTVNAYKTIAREEGFRGLWKGTSPNVARNAIVN
CAELVTYDLIKDALLKANLMTDDLPCHFTSAFGAGFCTTVIASPVDVVKTRYMNSALGQYSSAG
HCALTMLQKEGPRAFYKGFMPSFLRLGSWNVVMFVTYEQLKRALMAACTSREAPF, SEQ ID
NO:2), wherein the protein has transporter activity in the
mitochondria. In another embodiment, the invention includes a
composition of chondrisomes comprising an exogenous protein at
least 85%, 90%, 95%, 100% identical to the sequence of human UCP2
(or SEQ ID NO:2).
[0686] In one embodiment, the mitochondria are engineered to
express a protein at least 85%, 90%, 95%, 100% identical to the
sequence of human UCP3
(MVGLKPSDVPPTMAVKFLGAGTAACFADLVTFPLDTAKVRLQIQGENQAVQTARLVQYRGVL
GTILTMVRTEGPCSPYNGLVAGLQRQMSFASIRIGLYDSVKQVYTPKGADNSSLTTRILAGCTTG
AMAVTCAQPTDVVKVRFQASIHLGPSRSDRKYSGTMDAYRTIAREEGVRGLWKGTLPNIMRNAI
VNCAEVVTYDILKEKLLDYHLLTDNFPCHFVSAFGAGFCATVVASPVDVVKTRYMNSPPGQYFS
PLDCMIKMVAQEGPTAFYKGFTPSFLRLGSWNVVMFVTYEQLKRALMKVQMLRESPF, SEQ ID
NO:3), wherein the protein has transporter activity in the
mitochondria. In another embodiment, the invention includes a
composition of chondrisomes comprising an exogenous protein at
least 85%, 90%, 95%, 100% identical to the sequence of human UCP3
(or SEQ ID NO:3).
[0687] In one embodiment, the mitochondria are engineered to
express a protein at least 85%, 90%, 95%, 100% identical to the
sequence of human UCP4
(MSVPEEEERLLPLTQRWPRASKFLLSGCAATVAELATFPLDLTKTRLQMQGEAALARLGDGAR
ESAPYRGMVRTALGIIEEEGFLKLWQGVTPAIYRHVVYSGGRMVTYEHLREVVFGKSEDEHYPL
WKSVIGGMMAGVIGQFLANPTDLVKVQMQMEGKRKLEGKPLRFRGVHHAFAKILAEGGIRGL
WAGWVPNIQRAALVNMGDLTTYDTVKHYLVLNTPLEDNIMTHGLSSLCSGLVASILGTPADVIK
SRIMNQPRDKQGRGLLYKSSTDCLIQAVQGEGFMSLYKGFLPSWLRMTPWSMVFWLTYEKIRE
MSGVSPF, SEQ ID NO:4), wherein the protein has transporter activity
in the mitochondria. In one embodiment, the invention includes a
composition of chondrisomes comprising an exogenous protein at
least 85%, 90%, 95%, 100% identical to the sequence of human UCP4
(or SEQ ID NO:4).
[0688] In one embodiment, the mitochondria are engineered to
express a protein at least 85%, 90%, 95%, 100% identical to the
sequence of human UCP5
(MGIFPGIILIFLRVKFATAAVIVSGHQKSTTVSHEMSGLNWKPFVYGGLASIVAEFGTFPVDLTKT
RLQVQGQSIDARFKEIKYRGMFHALFRICKEEGVLALYSGIAPALLRQASYGTIKIGIYQSLKRLF
VERLEDETLLINMICGVVSGVISSTIANPTDVLKIRMQAQGSLFQGSMIGSFIDIYQQEGTRGLWR
GVVPTAQRAAIVVGVELPVYDITKKHLILSGMMGDTILTHFVSSFTCGLAGALASNPVDVVRTR
MMNQRAIVGHVDLYKGTVDGILKMWKHEGFFALYKGFWPNWLRLGPWNIIFFITYEQLKRLQI,
SEQ ID NO:5), wherein the protein has transporter activity in the
mitochondria. In another embodiment, the invention includes a
composition of chondrisomes comprising an exogenous protein at
least 85%, 90%, 95%, 100% identical to the sequence of human UCP5
(or SEQ ID NO:5).
[0689] A source may contain many hundreds of mitochondria with
hundreds of copies of mitochondrial DNA. It is common for mutations
to affect only some mitochondria, while leaving others unaffected,
a state known as heteroplasmy. In one embodiment, the invention
includes a composition of chondrisomes comprising heteroplasmic
mtDNA. Detrimental heteroplasmic alleles can shift in percentage
during both mitotic and meiotic cell division, leading to a
potentially continuous array of defects, a process known as
replicative segregation. As the percentage of mutant mtDNAs
increases, the resulting defect becomes increasingly severe.
Heteroplasmic alleles may be eliminated through differential
cleavage (enzymes that recognize the heteroplasmic allele but not
the wildtype or healthy allele). In some embodiments, a source is
engineered to express an enzyme that specifically cleaves the
heteroplasmic allele, thereby leaving the non-heteroplasmic allele
intact. In one embodiment, the invention includes a composition of
chondrisomes comprising heteroplasmic mtDNA, wherein a subset of
the heteroplasmic mtDNA comprises an enzyme recognition sequence
cleavable by an enzyme. In another embodiment, the invention
includes a composition of chondrisomes comprising heteroplasmic
mtDNA, wherein a subset of the heteroplasmic mtDNA is cleaved by an
enzyme at an enzyme recognition sequence in the subset of mtDNA. In
some embodiments, a source is treated with an enzyme that
specifically cleaves the heteroplasmic allele, and leaving the
non-heteroplasmic allele intact.
[0690] Mitochondrial diseases are commonly caused by mutations in
the mitochondrial DNA. Pathogenic mtDNA mutations are
heteroplasmic, and residual wild-type mtDNA can partially
compensate for the mutated mtDNA. The levels of mutated mtDNA in
affected tissues have to reach a high threshold, usually above 80%,
for biochemical and clinical manifestations. In some embodiments, a
source is engineered to express an enzyme that specifically cleaves
the deleterious heteroplasmic allele, while leaving the
non-heteroplasmic or wildtype allele intact. See, for example,
mitoTALEN described in Bacman, et al., Nat. Med., vol.
19(9):1111-1113. In one embodiment, the invention includes a
composition of chondrisomes comprising heteroplasmic mtDNA, wherein
a subset of the heteroplasmic mtDNA comprises a deleterious
mutation that is specifically recognized and cleaved by an enzyme.
In another embodiment, the invention includes a composition of
chondrisomes comprising a subset of mtDNA with a deleterious
mutation, wherein only mtDNA with the deleterious mutation
interacts with and activates an enzyme that cleaves the mtDNA with
the deleterious mutation while leaving the mtDNA without the
deleterious mutation intact. In another embodiment, the invention
includes a composition of chondrisomes comprising mtDNA with about
a 5 Kb deletion, m.8483_13459 del4977. This mutation is known as
the "common deletion" because it is present in approximately 30% of
all patients with mtDNA deletions. The source may be engineered to
express an enzyme, e.g., mitoTALEN, or treated with the enzyme that
specifically cleaves the deleterious heteroplasmic allele, thereby
leaving the non-deleterious heteroplasmic allele intact and capable
of replication.
Mitochondria Modification
[0691] In one aspect, a modification is made to the chondrisome
preparations as described herein to, e.g., produce chondrisomes
with a heterologous function or induce a structural change in the
chondrisomes. Such modifications can be effective to, e.g., improve
chondrisome activity, function or structure. Modifications to the
chondrisomes can include, but are not limited to, changes to the
mitochondrial or chondrisome metabolic state; changes to the
mitochondrial or chondrisome respiratory state, and changes to the
mitochondrial or chondrisome lipid and/or protein content. These
changes can result in changing the distribution and quantity of
chondrisome proteins.
[0692] Engineered Chondrisomes
[0693] In some embodiments, engineered chondrisomes with
heterologous function are produced as a result of genetically
engineering the endogenous mitochondrial genome prior to
therapeutic delivery.
[0694] The human mitochondrial genome is a circular double stranded
DNA molecule with a size of 16,569 bp. The mtDNA has no intron but
retains compactly arranged 37 genes (13 proteins, 22 tRNAs and 2
rRNAs) critical for producing energy through OXPHOS. Major
noncoding regions in the mtDNA genome involve the D-loop sequence
and the origin of L-strand replication (OL), which controls mtDNA
transcription and replication within mitochondria. The 13
protein-coding genes encode subunits of the OXPHOS enzyme
complexes. The genes encoded in the mtDNA can be found in Table
2.
TABLE-US-00002 TABLE 2 Mitochondrial Genes. Start site End site
Gene ID in rCRS in rCRS Description Mitochondrial encoded rRNA
MT-RNR1 648 1601 12S ribosomal RNA MT-RNR2 1671 3229 16S ribosomal
RNA MT-RNR3 3206 3229 5S-like sequence Mitochondrial Encoded
Proteins MT-ATP6 8527 9207 ATP synthase F0 subunit 6 MT-ATP8 8366
8572 ATP synthase F0 subunit 8 MT-CYB 14747 15887 Cytochrome b
MT-CO1 5904 7445 Cytochrome c oxidase subunit I MT-CO2 7586 8269
Cytochrome c oxidase subunit II MT-CO3 9207 9990 Cytochrome c
oxidase subunit III MT-ND1 3307 4262 NADH Dehydrogenase subunit 1
MT-ND2 4470 5511 NADH dehydrogenase subunit 2 MT-ND3 10059 10404
NADH dehydrogenase subunit 3 MT-ND4 10760 12137 NADH dehydrogenase
subunit 4 MT-ND4L 10470 10766 NADH dehydrogenase subunit 4L MT-ND5
12337 14148 NADH dehydrogenase subunit 5 MT-ND6 14149 14673 NADH
dehydrogenase subunit 6 Mitochondrial Encoded Peptides HM 2634 2707
Humanin SHLP1 2561 2490 small humanin-like peptide 1 SHLP2 2170
2092 small humanin-like peptide 2 SHLP3 1821 1707 small
humanin-like peptide 3 SHLP4 2524 2446 small humanin-like peptide 4
SHLP5 2856 2785 small humanin-like peptide 5 SHLP6 2992 3051 small
humanin-like peptide 6 MOTS-c 1343 1392 mitochondrial open reading
frame of the 12S rRNA-c Mitochondrial Encoded tRNA MT-TA 5587 5655
tRNA alanine MT-TR 10405 10469 tRNA arginine MT-TN 5657 5729 tRNA
asparagine MT-TD 7518 7585 tRNA aspartic acid MT-TC 5761 5826 tRNA
cysteine MT-TE 14674 14742 tRNA glutamic acid MT-TQ 4329 4400 tRNA
glutamine MT-TG 9991 10058 tRNA glycine MT-TH 12138 12206 tRNA
histidine MT-TI 4263 4331 tRNA isoleucine MT-TL1 3230 3304 tRNA
leucine 1 MT-TL2 12266 12336 tRNA Ieucine2 MT-TK 8295 8364 tRNA
lysine MT-TM 4402 4469 tRNA methionine MT-TF 577 647 tRNA
phenylalanine MT-TP 15956 16023 tRNA proline MT-TS1 7446 7514 tRNA
serine 1 MT-TS2 12207 12265 tRNA serine2 MT-TT 15888 15953 tRNA
threonine MT-TW 5512 5579 tRNA tryptophan MT-TY 5826 5891 tRNA
tyrosine MT-TV 1602 1670 tRNA valine
[0695] The accepted consensus mitochondrial sequence is the revised
Cambridge Reference Sequence (rCRS) (GenBank Accession
NC_012920.1). However, every individual comprises a degree of
sequence variability, potentially benign/healthy and/or
disease/pathology linked, from this reference sequence. These
changes are catalogued in a number of sequence databases, such as
the Human Mitochondrial DataBase
(http://www.hmtdb.uniba.it:8080/hmdb/; see also, Rubino, F., et
al., Nucleic Acid Res., 2012, 40:D1150-D1159).
[0696] Examples of mitochondrial engineering may include, but are
not limited to, modifying the genome whereby one to all the bases
in the mitochondrial genome are modified to a different nucleobase;
deleting a defined region of the endogenous mitochondrial genome of
any length between 1 base to the entire length of the genome (mtDNA
removal); inserting novel genetic sequence into the genome; and
changing the structure or order of the genetic components by
inversion or rearrangement.
[0697] In some embodiments, the mitochondrial genome is modified
such that between at least one to all the bases in the
mitochondrial genome are modified to a different nucleobase. In one
embodiment, the invention includes a composition of chondrisomes
comprising at least 1 base to the entire length of the genome
(mtDNA) is modified to a different nucleobase. In some embodiments,
the mitochondrial genome is modified such that a defined region of
the endogenous mitochondrial genome is removed. The length may be
any length between at least 1 base to the entire length of the
genome (mtDNA removal). In one embodiment, the invention includes a
composition of mitochondria in a source or chondrisomes comprising
at least 1 base to the entire length of the genome (mtDNA) is
deleted.
[0698] In some embodiments, the mitochondrial genome is modified
such that a novel genetic sequence is inserted into the genome. The
length may be any length between at least 1 base to 100,000 bases.
In one embodiment, the invention includes a composition of
mitochondria in a source or chondrisomes comprising mtDNA with an
insertion of at least 1 base to 100,000 bases.
[0699] In some embodiments, the mitochondrial genome is modified
such that a structure of the genome is modified or the order of the
genetic components is modified, such as by inversion or
rearrangement. In some embodiments, the mitochondrial genome is
modified such that a secondary or tertiary structure of the genome
is modified or a genomic folding structure is modified that allows
or inhibits genomic expression. In one embodiment, the invention
includes a composition of mitochondria in a source or chondrisomes
comprising mtDNA with a modified secondary or tertiary structure or
a modified genomic folding structure.
[0700] In some embodiments, the mitochondria are modified with an
exogenous nucleic acid that comprises a translation initiation
sequence upstream (5') of a translational start codon. The
mitochondrial translation initiation sequence can be any nucleic
acid sequence that mediates the initiation of translation of an RNA
in mitochondria. For example, suitable translation initiation
sequences can be found upstream (5') of a translational start codon
of a mitochondrial gene. In some embodiments, the mitochondrial
translation initiation sequence is at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%
or at least 99% identical to about 10 to about 40 nucleotides found
upstream (5') of a translational start codon of a mitochondrial
gene. In one embodiment, the invention includes a composition of
mitochondria in a source or chondrisomes comprising an exogenous
nucleic acid with a mitochondrial translation initiation sequence,
e.g., at least 80%, at least 85%, at least 90%, at least 95%, at
least 96%, at least 97%, at least 98% or at least 99% identical to
about 10 to about 40 nucleotides found upstream (5') of a
translational start codon of a mitochondrial gene.
[0701] Mitochondrial engineering can be performed either within the
mitochondria by acting on the endogenous mitochondrial genome or in
vitro. In vitro modification is on either through modification of
isolated genetic material, or synthesis of genetic material or some
combination thereof followed by reintroduction of the modified
mitochondrial genome into the mitochondrial matrix. Transgenomic
mitochondria are described, e.g., in US20070128726A1.
[0702] In some embodiments, tRNA sequences in the mitochondria are
modified. For example, mitochondrial tRNA sequences may be modified
to resemble cytosolic tRNA sequences. In another example, tRNA
sequences are modified to alter the wobble position or third base
position. In one embodiment, the invention includes a composition
of mitochondria in a source or chondrisomes comprising one or more
modified tRNA sequences, e.g., a tRNA sequence is modified to a
cytosolic tRNA sequence or a tRNA sequence is modified to alter the
wobble position or third base position.
[0703] In some embodiments, mitochondria are modified to reduce or
eliminate pathogenic mtDNA mutations. In some embodiments,
mitochondria are modified within a source to reduce the levels of
mutated mtDNA in affected sources, e.g., below about 80% to reduce
biochemical and clinical manifestations. In some embodiments,
mitochondria are modified within a source with an enzyme that
specifically cleaves the deleterious heteroplasmic allele, while
leaving the non-heteroplasmic or wildtype allele intact. See, for
example, mitoTALEN described in Bacman, et al., Nat. Med., vol.
19(9):1111-1113. In one embodiment, the invention includes a
composition of mitochondria in a source or chondrisomes isolated
from a source that comprises heteroplasmic mtDNA, wherein a subset
of the heteroplasmic mtDNA comprises a deleterious mutation that is
specifically recognized and cleaved by an enzyme. In another
embodiment, the invention includes a composition of mitochondria in
a source or chondrisomes isolated from a source that comprises a
subset of mtDNA with a deleterious mutation, wherein only mtDNA
with the deleterious mutation interacts with and activates an
enzyme that cleaves the mtDNA with the deleterious mutation while
leaving the mtDNA without the deleterious mutation intact. In
another embodiment, the invention includes a composition of
mitochondria in a source or chondrisomes isolated from a source
that comprises mtDNA with the common deletion, e.g., about a 5 Kb
deletion, m.8483_13459 del4977. The source may be treated with the
enzyme, e.g., mitoTALEN, to specifically cleave the mutant mtDNA
while leaving the non-mutant mtDNA intact.
[0704] Protein Modification in Mitochondria
[0705] In some embodiments, mitochondria or chondrisomes are
modified by loading the mitochondria with modified proteins (e.g.
enable novel functionality, alter post-translational modifications,
bind to the mitochondrial membrane and/or mitochondrial membrane
proteins, form a cleavable protein with a heterologous function,
form a protein destined for proteolytic degradation, assay the
agent's location and levels, or deliver the agent via the
mitochondria as a carrier). In one embodiment, the invention
includes a composition of mitochondria in a source, or
chondrisomes, loaded with modified proteins.
[0706] In some embodiments, an exogenous protein is non-covalently
bound to the mitochondrial outer membrane and/or mitochondrial
outer membrane proteins, loaded into a mitochondrial matrix, within
the intermembrane space, or bound to the outer or inner membrane.
In one embodiment, the invention includes a composition of
mitochondria in a source or chondrisomes comprising an exogenous
protein non-covalently bound to the mitochondrial outer membrane
and/or mitochondrial outer membrane proteins, loaded into a
mitochondrial matrix, within the intermembrane space, or bound to
the outer or inner membrane. The protein may include a cleavable
domain for release into the exterior, the matrix or the
intermembrane space. In one embodiment, the invention includes a
composition of mitochondria in a source or chondrisomes comprising
an exogenous protein with a cleavable domain.
[0707] Mitochondrial peripheral membrane proteins are known to
modulate actin binding. Altered distribution and concentration of
mitochondrial peripheral membrane proteins can, among other
behaviors and effects, alter the efficiency of mitochondrial uptake
as demonstrated by the in vitro uptake assay outlined above.
Candidate proteins include, but are not limited to, nuclear
encoded, engineered, exogenous or xenogeneic proteins, and surface
associating compounds can be used to modulate uptake, and behavior
following delivery, e.g., lymphatic clearance, degradation,
physiological stability intra and intercellularly. See Boldogh, I.
R., Methods in Cell Biology, 2007, 80:683-706. In one embodiment,
the invention includes a composition of mitochondria in a source or
chondrisomes with modulated actin binding, e.g., altered
distribution and/or concentration of mitochondrial peripheral
membrane proteins that bind actin.
[0708] In some embodiments, one or more mitochondrial proteins
(such as membrane transporters, intermediary metabolism enzymes,
and the complexes of oxidative phosphorylation) are altered by
post-translational modifications. Post-translational protein
modifications of proteins located in the mitochondria affect
responsiveness to nutrient availability and redox conditions, and
protein-protein interactions to modify diverse mitochondrial
functions. Examples of post-translational modifications include,
but are not limited to, physiologic redox signaling via reactive
oxygen and nitrogen species, phosphorylation, O-GlcNAcylation,
S-nitrosylation, nitration, glutathionylation, acetylation,
succinylation, and others. Key regulators are known for each of
these pathways, e.g., Bckdha phosphorylation, Hmgcs2 acetylation
and phosphorylation, and Acadl acetylation. In one embodiment, the
invention includes a composition of mitochondria in a source or
chondrisomes comprising one or more exogenous enzymes that regulate
post-translational modifications. Interestingly, Acat1 Lys-265 was
also recently identified as a prominent site of reversible
succinylation, further suggesting that this is an unusually
important site of post-translational regulation. In one embodiment,
mitochondria in a source described herein are loaded with Acat1
(MAVLAALLRSGARSRSPLLRRLVQEIRYVERSYVSKPTLKEVVIVSATRTPIGSFLGSLSLLPATK
LGSIAIQGAIEKAGIPKEEVKEAYMGNVLQGGEGQAPTRQAVLGAGLPISTPCTTINKVCASGMK
AIMMASQSLMCGHQDVMVAGGMESMSNVPYVMNRGSTPYGGVKLEDLIVKDGLTDVYNKIH
MGSCAENTAKKLNIARNEQDAYAINSYTRSKAAWEAGKFGNEVIPVTVTVKGQPDVVVKEDEE
YKRVDFSKVPKLKTVFQKENGTVTAANASTLNDGAAALVLMTADAAKRLNVTPLARIVAFADA
AVEPIDFPIAPVYAASMVLKDVGLKKEDIAMWEVNEAFSLVVLANIKMLEIDPQKVNINGGAVS
LGHPIGMSGARIVGHLTHALKQGEYGLASICNGGGGASAMLIQKL, SEQ ID NO:18)
acetylated at Lys-260 and Lys-265 to inhibit its activity by
disrupting CoA binding. In one embodiment, the invention includes a
composition of mitochondria in a source or chondrisomes comprising
exogenous Acat1 acetylated at Lys-260 and Lys-265.
[0709] In some embodiments, a distribution of posttranslational
modifications is altered in the mitochondria or chondrisomes by up
or downregulating levels of enzymes (e.g., proteases, phosphatases,
kinases, demethylases, methyltransferases, acetylases) that control
the modifications in the mitochondria. For example, PDH (pyruvate
dehydrogenase) can be activated by deacetylation to alter the
metabolic connection between glycolysis and the citric acid cycle
by increases levels of SIRT3. Similarly, increased activity of
pyruvate dehydrogenase kinase results in the phosphorylation of
pyruvate dehydrogenase to drive PDH metabolic flux. In another
example, O-GlcNAcylation can be modified by O-GlcNAc transferase to
control the electron transport chain by reducing ETC complex 1
activities.
[0710] In some embodiments, the mitochondria in the source or the
chondrisomes are loaded with an agent that enables the mitochondria
to have novel functionality within the source. In some embodiments,
the mitochondria are loaded with an agent that binds the
mitochondrial membrane and/or mitochondrial membrane proteins. For
example, the source may be treated with an agent that loads the
mitochondria, such as with a tag or marker, to assay the agent's
location and levels, the mitochondria's location and activities
within the source, e.g., DS-red.
[0711] As described herein, communication between the mitochondrion
and the cytosol is dependent on numerous transporters. These
transporters may be modulated via protein phosphorylation to affect
the exchange of metabolites and signaling molecules, as well as
proteins. In one embodiment, mitochondria in a source are loaded
with dephosphorylated pyruvate dehydrogenase to catabolize glucose
and gluconeogenesis precursors. In another embodiment, mitochondria
in a source are loaded with phosphorylated pyruvate dehydrogenase
to shift metabolism toward fat utilization.
[0712] Cleavable Proteins
[0713] In some embodiments, the mitochondria in a source or
chondrisomes are modified with a cleavable protein. In some cases,
proteins may be engineered to target to the inner membrane or outer
membrane with a fused intermembrane domain that can be released by
intermembrane proteases. The engineered fusion protein may bind any
domain of a transmembrane mitochondrial proteins (e.g. GDP). The
engineered fusion protein may be linked by a cleavage peptide to a
protein domain located within the intermembrane space. The cleavage
peptide may be cleaved by one or a combination of intermembrane
proteases listed in Table 3 (e.g. HTRA2/OMI which requires a
non-polar aliphatic amino acid--valine, isoleucine or methionine
are preferred--at position P1, and hydrophilic residues--arginine
is preferred--at the P2 and P3 positions).
TABLE-US-00003 TABLE 3 Proteases Location Type Protease Class
Enzyme EC Number Clevage Site Cytosol Endopeptidase Serine Protease
Trypsin E.C.3.4.21.4 Arg-|-Xaa and Lys-|-Xaa Thrombin E.C.3.4.21.5
Arg-|-Gly Cysteine Protease Cathepsin B E.C.3.4.22.1 Arg-Arg-|-Xaa
Calpain-1 E.C.3.4.22.52 Met-|-Xaa, Tyr-|-Xaa and Arg-|-Xaa (with
Leu or Val as the P2 residue) Aspartic Acid Protease Pepsin
E.C.3.4.23.1 Preferentially Phe-|-Xaa with Xaa = Phe, Trp, or Tyr
Cathepsin D E.C.3.4.23.5 Preferentially Phe-|-Xaa, Tyr-|-Xaa and
Leu-|-Xaa, ideally with Xaa /= Ala or Val Metalloprotease
Neprilysin E.C.3.4.24.11 Xaa-|-Tyr, Xaa-|-Phe Thimet E.C.3.4.24.15
Xaa-|-Arg, Xaa-|-Ser oligopeptidase Exopeptidase Amino peptidase
Leucyl- E.C.3.4.11.1 Preferentially Leu-|-Xaa, but not Arg-|-Xaa
aminopeptidase and Lys-|-Xaa di/tri peptidyl peptidases Prolyl
tripeptyl- E.C.3.4.14.12 Xaa-Yaa-Pro-|-Zaa if Zaa /= Pro peptidase
Peptidyl-dipeptidases Peptidyl- E.C.3.4.15.1 Xaa-|-Yaa-Zaa, if Yaa
/= Pro, or Zaa /= dipeptidase A Asp or Glu Metallo-
carboxypeptidases Carboxypeptidase U E.C.3.4.17.20 Xaa-|-Arg and
Xaa-|-Lys Mitochondrial Outer Membrane Cysteine Protease EC:
3.4.19.12 USP30 Mitochondrial Intermembrane Metalloprotease EC:
3.4.24 ATP23 Mitochondrial Inner Membrane Metalloprotease EC:
3.4.24 SPG7 Mitochondrial Matrix Metalloprotease EC: 3.4.24
PITRM1
[0714] Proteolytic Degradation
[0715] In some embodiments, mitochondria in a source chondrisomes
are modified with a protein destined for proteolytic degradation.
Mitochondria contain a variety of proteases that recognize specific
protein amino acid sequences and target the proteins for
degradation. These protein degrading enzymes can be used to
specifically degrade mitochondrial proteins having a proteolytic
degradation sequence. In one embodiment, the invention includes a
composition of mitochondria in a source or chondrisomes comprising
modulated levels of one or more protein degrading enzymes, e.g., an
increase or a decrease in protein degrading enzymes by at least
10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more.
[0716] In some embodiments, mitochondrial proteins are engineered
by any methods known in the art or any method described herein to
comprise a proteolytic degradation sequence, e.g., a mitochondrial
or cytosolic degradation sequence. Mitochondrial proteins may be
engineered to include, but is not limited to a proteolytic
degradation sequence, e.g., the preferred Capsase 2 protein
sequence (Val-Asp-Val-Ala-Asp-l-) or other proteolytic sequences
(see, for example, Gasteiger et al., The Proteomics Protocols
Handbook; 2005: 571-607), a modified proteolytic degradation
sequence that has at least 75%, 80%, 85%, 90%, 95% or greater
identity to the wildtype proteolytic degradation sequence, a
cytosolic proteolytic degradation sequence, e.g., ubiquitin, or a
modified cytosolic proteolytic degradation sequence that has at
least 75%, 80%, 85%, 90%, 95% or greater identity to the wildtype
proteolytic degradation sequence. In one embodiment, the invention
includes a composition of mitochondria in a source or chondrisomes
comprising a protein modified with a proteolytic degradation
sequence, e.g., at least 75%, 80%, 85%, 90%, 95% or greater
identity to the wildtype proteolytic degradation sequence, a
cytosolic proteolytic degradation sequence, e.g., ubiquitin, or a
modified cytosolic proteolytic degradation sequence that has at
least 75%, 80%, 85%, 90%, 95% or greater identity to the wildtype
proteolytic degradation sequence.
[0717] In some embodiments, mitochondria may be modified with a
protein comprising a protease domain that recognizes specific
mitochondrial proteins, e.g., over-expression of a mitochondrial
protease, e.g., an engineered fusion protein with mitochondrial
protease activity. For example, a protease or protease domain from
a protease, such as mitochondrial processing peptidase,
mitochondrial intermediate peptidase, inner membrane peptidase. In
one embodiment, the invention includes a composition of
mitochondria in a source or chondrisomes comprising an exogenous
protein with a protease domain that recognizes specific
mitochondrial proteins, e.g., over-expression of a mitochondrial
protease, e.g., an engineered fusion protein with mitochondrial
protease activity.
[0718] See, Alfonzo, J. D. & Soll, D. Mitochondrial tRNA
import--the challenge to understand has just begun. Biological
Chemistry 390: 717-722. 2009; Langer, T. et al. Characterization of
Peptides Released from Mitochondria. THE JOURNAL OF BIOLOGICAL
CHEMISTRY. Vol. 280, No. 4. 2691-2699, 2005; Vliegh, P. et al.
Synthetic therapeutic peptides: science and market. Drug Discovery
Today. 15(1/2). 2010; Quiros P. M. m et al., New roles for
mitochondrial proteases in health, ageing and disease. Nature
Reviews Molecular Cell Biology. V16, 2015; Weber-Lotfi, F. et al.
DNA import competence and mitochondrial genetics. Biopolymers and
Cell. Vol. 30. N 1. 71-73, 2014.
Chondrisome Modification
[0719] Cleavage of Heteroplasmic mtDNA
[0720] Pathogenic mtDNA mutations are heteroplasmic, and residual
wild-type mtDNA can partially compensate for the mutated mtDNA. In
some embodiments, chondrisomes are modified to reduce the levels of
mutated mtDNA, e.g., below about 80% to reduce biochemical and
clinical manifestations. In some embodiments, chondrisomes are
modified with an enzyme that specifically cleaves the deleterious
heteroplasmic allele, while leaving the non-heteroplasmic or
wildtype allele intact. See, for example, mitoTALEN described in
Bacman, et al., Nat. Med., vol. 19(9):1111-1113. In one embodiment,
the invention includes a composition of chondrisomes comprising
heteroplasmic mtDNA, wherein a subset of the heteroplasmic mtDNA
comprises a deleterious mutation that is specifically recognized
and cleaved by an enzyme. In another embodiment, the invention
includes a composition of chondrisomes comprising a subset of mtDNA
with a deleterious mutation, wherein only mtDNA with the
deleterious mutation interacts with and activates an enzyme that
cleaves the mtDNA with the deleterious mutation while leaving the
mtDNA without the deleterious mutation intact. In another
embodiment, the invention includes a composition of chondrisomes
comprising mtDNA with the common deletion, e.g., about a 5 Kb
deletion, m.8483_13459 del4977. The chondrisomes may be treated
with the enzyme, e.g., mitoTALEN, to specifically cleave the mutant
mtDNA while leaving the non-mutant mtDNA intact.
[0721] Chondrisome Targeted Proteins
[0722] The MTS is recognized by the mitochondrial import complexes
(translocases of the outer membrane (TOM) and the inner membrane
(TIM)) and mediates mitochondrial localization, and subsequent
delivery of mitochondrial proteins to the matrix compartment. In
some embodiments, chondrisome preparations described herein are
modified with a modifying agent, e.g., a biologic or drug, targeted
to the chondrisome. The chondrisome preparation may be directly
modified with the modifying agent targeted to the chondrisome by
directly contacting the chondrisome preparation with the modifying
agent. In some embodiments, the chondrisome preparation is modified
with a modifying agent that is targeted to the chondrisome by any
of the methods described herein. In one embodiment, the invention
includes a composition of chondrisomes comprising a modifying
agent, e.g., loaded with a modifying agent described herein.
[0723] Import into the chondrisome can be initiated by N-terminal
targeting sequences (presequences) or internal targeting sequences.
Import into the organelle may be mediated by a translocase in the
outer membrane (TOM) complex, molecular chaperone proteins, and
targeting sequences, a translocase in the inner membrane (TIM)
complex to mediate transit from the intermembrane space into the
mitochondrial matrix, as well as embedding proteins into the inner
membrane, or a carrier translocase (TIM22) for embedding carrier
proteins or N-terminal targeted inner membrane proteins. In some
embodiments, the chondrisome preparation is modified by treating
the preparation with a modifying agent that interferes with
translocase function, e.g., an inhibitor of a translocase or a
protease that directly targets molecular chaperone proteins, such
that the number of proteins imported or the makeup of the proteins
imported is altered. In one embodiment, the invention includes a
composition of chondrisomes comprising decreased translocase
activity or decreased levels of one or more chaperone proteins.
[0724] In some embodiments, the chondrisome preparation is modified
with a protein targeted to, e.g., the DSRed2 fluorescent protein,
the chondrisome matrix by appending the protein with a
mitochondrial targeting sequence, e.g., from subunit VIII of human
cytochrome c oxidase
(ATGTCCGTCCTGACGCCGCTGCTGCTGCGGGGCTTGACAGGCTCGGCCCGGCGGCTCCCAGT
GCCGCGCGCCAAGATCCATTCGTTG, SEQ ID NO:6), to the N-terminus of the
protein.
[0725] In some embodiments, the chondrisome preparation is modified
with a cytosolic protein, such as a protease or enzyme, that is
targeted to the chondrisome. Cytosolic proteins may be produced
with a mitochondrial targeting sequence, e.g., first 69 amino acids
of the precursor of subunit 9 of the mitochondrial Fo-ATPase, then
contacted with the chondrisome preparation to modify the
chondrisomes with the retargeted cytosolic proteins.
[0726] Protein Modification in Chondrisome
[0727] In some embodiments, the chondrisome preparation is modified
by loading with modified proteins to (e.g. enable novel
functionality, alter post-translational modifications, bind to the
chondrisome membrane and/or chondrisome membrane proteins, form a
cleavable protein with a heterologous function, form a protein
destined for proteolytic degradation, assay the agent's location
and levels, or deliver the agent via the chondrisome as a carrier).
In one embodiment, the invention includes a composition of
chondrisomes loaded with modified proteins.
[0728] In some embodiments, the chondrisome preparations described
herein are modified by a non-covalently bound protein to the
chondrisome outer membrane and/or chondrisome outer membrane
proteins. In one embodiment, the invention includes a composition
of chondrisomes comprising an exogenous protein non-covalently
bound to the mitochondrial outer membrane and/or mitochondrial
outer membrane proteins, loaded into a mitochondrial matrix, within
the intermembrane space, or bound to the outer or inner membrane.
Altered distribution and/or concentration of peripheral membrane
proteins can, among other behaviors and effects, alter the
efficiency of protein uptake as demonstrated by the in vitro uptake
assay described herein. Candidate proteins include, but are not
limited to, nuclear encoded, engineered, exogenous or xenogeneic
proteins, and surface associating compounds can be used to modulate
uptake, and behavior following delivery, e.g., lymphatic clearance,
degradation, physiological stability intra and intercellularly. See
Boldogh, I. R. Cell-Free Assays for Mitochondria-Cytoskeleton
Interactions. Methods in Cell Biology Vol 80 2007-b. In one
embodiment, the invention includes a composition of chondrisomes
comprising an altered distribution and/or concentration of one or
more peripheral membrane proteins.
[0729] In some embodiments, the chondrisomes are loaded with a
modifying agent, such as an antibody or transcription factor or
drug, that utilizes the chondrisome as a carrier to deliver the
agent. In one embodiment, the invention includes a composition of
chondrisomes comprising an exogenous antibody or transcription
factor or drug. Examples may include, but are not limited to,
transcription factors such as GPS2:
(MPALLERPKLSNAMARALHRHIMMERERKRQEEEEVDKMMEQKMKEEQERRKKKEMEERMS
LEETKEQILKLEEKLLALQEEKHQLFLQLKKVLHEEEKRRRKEQSDLTTLTSAAYQQSLTVHTGT
HLLSMQGSPGGHNRPGTLMAADRAKQMFGPQVLTTRHYVGSAAAFAGTPEHGQFQGSPGGAY
GTAQPPPHYGPTQPAYSPSQQLRAPSAFPAVQYLSQPQPQPYAVHGHFQPTQTGFLQPGGALSLQ
KQMEHANQQTGFSDSSSLRPMHPQALHPAPGLLASPQLPVQMQPAGKSGFAATSQPGPRLPFIQ
HSQNPRFYHK, SEQ ID NO:19 or a protein at least 85%, 90%, 95%, 98%
identical to SEQ ID NO:19); or YBX1:
(MSSEAETQQPPAAPPAAPALSAADTKPGTTGSGAGSGGPGGLTSAAPAGGDKKVIATKVLGTV
KWFNVRNGYGFINRNDTKEDVFVHQTAIKKNNPRKYLRSVGDGETVEFDVVEGEKGAEAANVT
GPGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNYQNSESGEKNEGSESAPEGQAQQRRPYR
RRRFPPYYMRRPYGRRPQYSNPPVQGEVMEGADNQGAGEQGRPVRQNMYRGYRPRFRRGPPR
QRQPREDGNEEDKENQGDETQGQQPPQRRYRRNFNYRRRRPENPKPQDGKETKAADPPAENSS
APEAEQGGAE, SEQ ID NO:20 or a protein at least 85%, 90%, 95%, 98%
identical to SEQ ID NO:20), or structural control elements such as
actin, OPA1:
(MWRLRRAAVACEVCQSLVKHSSGIKGSLPLQKLHLVSRSIYHSHHPTLKLQRPQLRTSFQQFSSL
TNLPLRKLKFSPIKYGYQPRRNFWPARLATRLLKLRYLILGSAVGGGYTAKKTFDQWKDMIPDL
SEYKWIVPDIVWEIDEYIDFEKIRKALPSSEDLVKLAPDFDKIVESLSLLKDFFTSGSPEETAFRAT
DRGSESDKHFRKVSDKEKIDQLQEELLHTQLKYQRILERLEKENKELRKLVLQKDDKGIHHRKL
KKSLIDMYSEVLDVLSDYDASYNTQDHLPRVVVVGDQSAGKTSVLEMIAQARIFPRGSGEMMT
RSPVKVTLSEGPHHVALFKDSSREFDLTKEEDLAALRHEIELRMRKNVKEGCTVSPETISLNVKG
PGLQRMVLVDLPGVINTVTSGMAPDTKETIFSISKAYMQNPNAIILCIQDGSVDAERSIVTDLVSQ
MDPHGRRTIFVLTKVDLAEKNVASPSRIQQIIEGKLFPMKALGYFAVVTGKGNSSESIEAIREYEE
EFFQNSKLLKTSMLKAHQVTTRNLSLAVSDCFWKMVRESVEQQADSFKATRFNLETEWKNNYP
RLRELDRNELFEKAKNEILDEVISLSQVTPKHWEEILQQSLWERVSTHVIENIYLPAAQTMNSGTF
NTTVDIKLKQWTDKQLPNKAVEVAWETLQEEFSRFMTEPKGKEHDDIFDKLKEAVKEESIKRHK
WNDFAEDSLRVIQHNALEDRSISDKQQWDAAIYFMEEALQARLKDTENAIENMVGPDWKKRW
LYWKNRTQEQCVHNETKNELEKMLKCNEEHPAYLASDEITTVRKNLESRGVEVDPSLIKDTWH
QVYRRHFLKTALNHCNLCRRGFYYYQRHFVDSELECNDVVLFWRIQRMLAITANTLRQQLTNT
EVRRLEKNVKEVLEDFAEDGEKKIKLLTGKRVQLAEDLKKVREIQEKLDAFIEALHQEK, SEQ ID
NO:21 or a protein at least 85%, 90%, 95%, 98% identical to SEQ ID
NO:21), MFN1:
(MAEPVSPLKHFVLAKKAITAIFDQLLEFVTEGSHFVEATYKNPELDRIATEDDLVEMQGYKDKL
SIIGEVLSRRHMKVAFFGRTSSGKSSVINAMLWDKVLPSGIGHITNCFLSVEGTDGDKAYLMTEG
SDEKKSVKTVNQLAHALHMDKDLKAGCLVRVFWPKAKCALLRDDLVLVDSPGTDVTTELDSW
IDKFCLDADVFVLVANSESTLMNTEKHFFHKVNERLSKPNIFILNNRWDASASEPEYMEDVRRQ
HMERCLHFLVEELKVVNALEAQNRIFFVSAKEVLSARKQKAQGMPESGVALAEGFHARLQEFQ
NFEQIFEECISQSAVKTKFEQHTIRAKQILATVKNIMDSVNLAAEDKRHYSVEEREDQIDRLDFIR
NQMNLLTLDVKKKIKEVTEEVANKVSCAMTDEICRLSVLVDEFCSEFHPNPDVLKIYKSELNKHI
EDGMGRNLADRCTDEVNALVLQTQQEIIENLKPLLPAGIQDKLHTLIPCKKFDLSYNLNYHKLCS
DFQEDIVFPFSLGWSSLVHRFLGPRNAQRVLLGLSEPIFQLPRSLASTPTAPTTPATPDNASQEELM
ITLVTGLASVTSRTSMGIIIVGGVIWKTIGWKLLSVSLTMYGALYLYERLSWTTHAKERAFKQQF
VNYATEKLRMIVSSTSANCSHQVKQQIATTFARLCQQVDITQKQLEEEIARLPKEIDQLEKIQNNS
KLLRNKAVQLENELENFTKQFLPSSNEES, SEQ ID NO:22 or a protein at least
85%, 90%, 95%, 98% identical to SEQ ID NO:22) or MFN2:
(MSLLFSRCNSIVTVKKNKRHMAEVNASPLKHFVTAKKKINGIFEQLGAYIQESATFLEDTYRNA
ELDPVTTEEQVLDVKGYLSKVRGISEVLARRHMKVAFFGRTSNGKSTVINAMLWDKVLPSGIGH
TTNCFLRVEGTDGHEAFLLTEGSEEKRSAKTVNQLAHALHQDKQLHAGSLVSVMWPNSKCPLL
KDDLVLMDSPGIDVTTELDSWIDKFCLDADVFVLVANSESTLMQTEKHFFHKVSERLSRPNIFIL
NNRWDASASEPEYMEEVRRQHMERCTSFLVDELGVVDRSQAGDRIFFVSAKEVLNARIQKAQG
MPEGGGALAEGFQVRMFEFQNFERRFEECISQSAVKTKFEQHTVRAKQIAEAVRLIMDSLHMAA
REQQVYCEEMREERQDRLKFIDKQLELLAQDYKLRIKQITEEVERQVSTAMAEEIRRLSVLVDDY
QMDFHPSPVVLKVYKNELHRHIEEGLGRNMSDRCSTAITNSLQTMQQDMIDGLKPLLPVSVRSQI
DMLVPRQCFSLNYDLNCDKLCADFQEDIEFHFSLGWTMLVNRFLGPKNSRRALMGYNDQVQRP
IPLTPANPSMPPLPQGSLTQEEFMVSMVTGLASLTSRTSMGILVVGGVVWKAVGWRLIALSFGLY
GLLYVYERLTWTTKAKERAFKRQFVEHASEKLQLVISYTGSNCSHQVQQELSGTFAHLCQQVD
VTRENLEQEIAAMNKKIEVLDSLQSKAKLLRNKAGWLDSELNMFTHQYLQPSR, SEQ ID NO:23
or a protein at least 85%, 90%, 95%, 98% identical to SEQ ID
NO:23).
[0730] In some embodiments, the chondrisome preparation is modified
by contact with a cytosolic enzyme (e.g., protease, phosphatase,
kinase, demethylase, methyltransferase, acetylase) to alter
post-translational modification of proteins in the preparation. In
some embodiments, one or more chondrisome proteins (such as
membrane transporters, intermediary metabolism enzymes, and the
complexes of oxidative phosphorylation) are altered by
post-translational modifications. Post-translational protein
modifications of proteins may affect responsiveness to nutrient
availability and redox conditions, and protein-protein
interactions. Examples of post-translational modifications include,
but are not limited to, physiologic redox signaling via reactive
oxygen and nitrogen species, phosphorylation, O-GlcNAcylation,
S-nitrosylation, nitration, glutathionylation, acetylation,
succinylation, and others. Key regulators are known for each of
these pathways, e.g., Bckdha phosphorylation, Hmgcs2 acetylation
and phosphorylation, and Acadl acetylation. In one embodiment, the
invention includes a composition of chondrisomes comprising one or
more exogenous enzymes that regulate post-translational
modifications. Interestingly, Acat1 Lys-265 was also recently
identified as a prominent site of reversible succinylation, further
suggesting that this is an unusually important site of
post-translational regulation. In one embodiment, chondrisomes in
preparations described herein are loaded with Acat1 acetylated at
Lys-260 and Lys-265 to inhibit its activity by disrupting CoA
binding. In one embodiment, the invention includes a composition of
chondrisomes comprising exogenous Acat1 acetylated at Lys-260 and
Lys-265.
[0731] In another embodiment, the chondrisome preparation is
modified by treatment with a kinase or phosphatase. Such treatment
changes the phosphorylation state of the chondrisome preparation.
In some embodiments, one or more enzymes is selected that alters
energy buffering, such as CKs (creatine kinase). By changing
production levels of phosphocreatine, ATP buffering can be
modified. In some embodiments, one or more kinases is selected
based on a distribution of post-translational modifications that
controls signaling and/or metabolic flux control, such as AMPK and
its ability to alter fatty acid oxidation. In one embodiment, the
invention includes a composition of chondrisomes comprising one or
more proteins with an altered phosphorylation state, e.g., an
increase or a decrease in protein phosphorylation by at least 10%,
15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more.
[0732] In one embodiment, the chondrisome preparation described
herein is contacted with a dephosphorylated pyruvate dehydrogenase
to catabolize glucose and gluconeogenesis precursors. In one
embodiment, the invention includes a composition of chondrisomes
comprising dephosphorylated pyruvate dehydrogenase. In another
embodiment, the chondrisome preparation described herein is
contacted with phosphorylated pyruvate dehydrogenase to shift
metabolism toward fat utilization. In one embodiment, the invention
includes a composition of chondrisomes comprising phosphorylated
pyruvate dehydrogenase.
[0733] In another embodiment, the chondrisome preparation is
contacted with one or more metabolic conversion enzymes to alter
the metabolic capacity of the chondrisome preparation. In some
embodiments, such enzymes can alter the capacity of the chondrisome
preparation to alter a patient's metabolic concentration, e.g. OTC
(ornithine transcarbamylase), such as by altering the ability to
address urea cycle disorders. In some embodiments, such enzymes can
be selected for their ability to adjust redox balancing and
cycling, such as NADH oxidases (e.g. the heterologous LbNOX, see
for example Titov, D. V., et al., 2016, Science,
352(6282):231-235). In one embodiment, the invention includes a
composition of chondrisomes comprising NADH oxidase.
[0734] Alternative Spliced RNA
[0735] In some embodiments, a chondrisome preparation comprises a
modified distribution of alternative splice variants, such as one
or more variants is increased or decreased in the chondrisome
preparation as compared to the splice variant that was present in
the cytosol prior to preparing the chondrisome for isolation. As
described herein, mitochondria import a certain number of RNAs
(e.g., small noncoding RNAs, miRNAs, tRNAs, and possibly lncRNAs
and viral RNAs). RNAs are processed within mitochondria and may
have functions different from their cytosolic or nuclear
counterparts. In some embodiments, the chondrisome preparation
comprises RNA splice-variants that are differentially present in
mitochondria or the cytosol. For example, in one embodiment, the
short spliced variant of trypanosomal isoleucyl-tRNA synthetase
(IleRS) lacking the presequence found exclusively in the cytosol is
present in the chondrisome preparation. In one embodiment, the
invention includes a composition of chondrisomes comprising RNA
splice-variants that are differentially present in mitochondria or
the cytosol, e.g., an increase or a decrease in RNA splice-variants
by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or
more. In another embodiment, the chondrisome preparation lacks the
protein product of the longer spliced variant that is found
exclusively in mitochondria.
Modifying Agent
[0736] The source, mitochondria in the source, or a chondrisome
preparation may be modified or loaded with an agent, such as a
nucleic acid (e.g., DNA, RNA), protein, or chemical compound. In
some embodiment, the source, mitochondria in the source, or a
chondrisome preparation is modified by two or more of the agents
described herein, including mixtures, fusions, combinations and
conjugates, of atoms, molecules, etc. For example, a nucleic acid
may be combined with a polypeptide; two or more polypeptides may be
conjugated to each other; a protein may be conjugated to a
biologically active molecule (which may be a small molecule such as
a prodrug); and the like.
TABLE-US-00004 TABLE 4 Agents for modifying or loading onto a
source, mitochondria in a source or chondrisome preparation
Compound Small Molecules Class Molecule Electron Transport Chain
Modulator Amiodarone (Complex 1) b-Methoxyacrylate (Complex 3)
Malonate (complex 2) n-Propylgallate (AOX) ATPase Modulators
Aurovertin (Complex V) Oligomycin (Complex V) Uncouplers FCCP
Valinomycin 2,4-Dinitrophenol (DNP) Myxobacterial products
melithiazol Adipose Metabolism Modulating Pioglitazone
Hydrochloride Nucleus/Mitochondrial Decouplers podofilox
cycloheximide thimerosal pararosaniline lycorine Calcium transport
modulation CGP37157 Mito Biogenesis Stimulation Bezafibrate Others
Cyclosporin A (CsA) Dichloroacetate (DCA) Surface associating
compounds Lipids Lysobisphosphatidic acid Sphingomyelin (SM)
Ganglioside GM3 Phosphatidylserine (PS) Phosphatidylinositol (PI)
Phosphatidylcholine (PC) Phosphatidylethanolamine (PE)
Lysophosphatidylcholine (LPC) Polypeptides Enzymes citrate synthase
cytochrome P450 (prenenolone processing) Regulators Nucleases Zinc
Finger Nucleases Kinases ABL2_HUMAN Transporters Type I protein
transporter (HylB, HylD, TolC, HylA etc) Porin (ompF) Aminoacid
exporter (eg yddG) Methylases DNMT1 Surface associating compounds
Nucleic Acids RNA let-7b miR-302a miR-93 miR-125b-1* DNA
Oligonucleotide with homology
[0737] Small Molecules
[0738] The source, mitochondria in the source, or a chondrisome
preparation may be contacted with an exogenous agent, such as a
small molecule or synthetic therapeutic agent, that modulates
mitochondrial activity, function or structure. Examples of suitable
small molecules include those described in, "The Pharmacological
Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York,
N.Y., (1996), Ninth edition, under the sections: Drugs Acting at
Synaptic and Neuroeffector Junctional Sites; Drugs Acting on the
Central Nervous System; Autacoids: Drug Therapy of Inflammation;
Water, Salts and Ions; Drugs Affecting Renal Function and
Electrolyte Metabolism; Cardiovascular Drugs; Drugs Affecting
Gastrointestinal Function; Drugs Affecting Uterine Motility;
Chemotherapy of Parasitic Infections; Chemotherapy of Microbial
Diseases; Chemotherapy of Neoplastic Diseases; Drugs Used for
Immunosuppression; Drugs Acting on Blood-Forming organs; Hormones
and Hormone Antagonists; Vitamins, Dermatology; and Toxicology, all
incorporated herein by reference. In one embodiment, the invention
includes a composition of chondrisomes comprising modulated
mitochondrial activity, function or structure by a small molecule
or synthetic therapeutic agent, e.g., a change in mitochondrial
activity, function or structure by at least 10%, 15%, 20%, 30%,
40%, 50%, 60%, 75%, 80%, 90% or more.
[0739] The source, mitochondria in the source, or a chondrisome
preparation may be loaded with a small molecule, including
inorganic and organic chemicals, to enable novel functionality.
Molecules <5 kDa can passively diffuse through the outer
membrane of mitochondria (Benz 1985). In one embodiment, the
invention includes a composition of mitochondria in the source or
chondrisomes comprising a small molecule, e.g., an inorganic and
organic chemical.
[0740] In some embodiments, the small molecule is a
pharmaceutically active agent. In one embodiment, the small
molecule is an inhibitor of a metabolic activity or component.
Useful classes of pharmaceutically active agents include, but are
not limited to, antibiotics, anti-inflammatory drugs, angiogenic or
vasoactive agents, growth factors and chemotherapeutic
(anti-neoplastic) agents (e.g., tumour suppressers). One or a
combination of molecules from the categories and examples described
herein or from (Orme-Johnson 2007, Methods Cell Biol. 2007;
80:813-26) can be used. In one embodiment, the invention includes a
composition of mitochondria in the source or chondrisomes
comprising an antibiotic, anti-inflammatory drug, angiogenic or
vasoactive agent, growth factor or chemotherapeutic agent.
[0741] For example, small molecule drugs can be used to inhibit
membrane targeted proteins. In some embodiments, such membrane
proteins can be ion transporters (e.g. the sodium calcium
exchanger), where the addition of CGP-37157, a benzothiazepine
analogue of diltiazem, is able to decrease the calcium efflux from
mitochondria (DOI: 10.1054/ceca.2000.0171). In one embodiment, the
invention includes a composition of mitochondria in the source or
chondrisomes comprising an ion transporter inhibitor, e.g.,
benzothiazepine analogue of diltiazem.
[0742] In some embodiments, a small molecule drug is used to
inhibit a protein of the mitochondrial transport chain and/or
reduce oxidative phosphorylation. In one embodiment, NADH
dehydrogenase activity is decreased by the addition of metformin, a
common type 2 diabetes drug, resulting in reduced proton gradient
force in the treated cells. In one embodiment, the invention
includes a composition of mitochondria in the source or
chondrisomes comprising a mitochondrial transport chain inhibitor
or oxidative phosphorylation inhibitor, e.g., metformin.
[0743] Biologics
[0744] The source, mitochondria in the source, or a chondrisome
preparation may be treated with an exogenous agent, such as a
biologic, that modulates mitochondrial activity, function or
structure. In some embodiments, the biologic includes a metabolic
enzyme, a transporter, a transcriptional regulator, a nuclease, a
protein modifying enzyme (e.g., a kinase), and a nucleic acid
modifying enzyme (e.g., a methylase). The biologic may be a
polypeptide with at least 85%, 90%, 95%, 100% identity to an
endogenous protein and retains at least one activity, function or
structure of the endogenous protein. A large molecule biologic can
comprise an amino acid or analogue thereof, which may be modified
or unmodified or a non-peptide (e.g., steroid) hormone; a
proteoglycan; a lipid; or a carbohydrate. If the large molecule
biologic is a polypeptide, it can be loaded directly into a
mitochondrion according to the methods described herein. In one
embodiment, the invention includes a composition of mitochondria in
the source or chondrisomes comprising an exogenous large molecule
biologic, e.g., a hormone; a proteoglycan; a lipid; or a
carbohydrate.
[0745] The source, mitochondria in the source, or a chondrisome
preparation may be treated in vitro with purified protein. Prior to
exogenous protein loading, the mitochondria in the source or in the
preparation should be checked to ensure adequate maintenance of
outer membrane integrity and membrane potential. In one embodiment,
the invention includes a composition of mitochondria in the source
or chondrisomes comprising an exogenous protein described
herein.
[0746] The source, mitochondria in the source, or a chondrisome
preparation may be treated with a protein that non-covalently or
covalently binds to the mitochondrial outer membrane and/or
mitochondrial outer membrane proteins. Mitochondrial peripheral
membrane proteins are known to modulate actin binding. Altered
distribution and concentration of mitochondrial peripheral membrane
proteins can, among other behaviors and effects, alter the
efficiency of mitochondrial uptake as demonstrated by the in vitro
uptake assay outlined above. Candidate proteins include, but are
not limited to, nuclear encoded, engineered, exogenous or
xenogeneic proteins, and surface associating compounds can be used
to modulate uptake, and behavior following delivery, e.g.,
lymphatic clearance, degradation, physiological stability intra and
intercellularly. See Boldogh, I. R. Cell-Free Assays for
Mitochondria-Cytoskeleton Interactions. Methods in Cell Biology Vol
80 2007-b.
[0747] Suitable biologics further include toxins, and biological
and chemical warfare agents, see Somani, S. M. (ed.), Chemical
Warfare Agents, Academic Press, New York (1992)).
[0748] The source, mitochondria in the source, or a chondrisome
preparation may be treated with a cleavable protein that integrates
into the mitochondrial membrane. The engineered fusion protein may
include an anchoring domain selected from any of the transmembrane
mitochondrial proteins (e.g. GDP). In one embodiment, the invention
includes a composition of mitochondria in the source or
chondrisomes comprising an engineered fusion protein described
herein. The C-terminus or N-terminus of the protein may be attached
to a protein domain located within the intermembrane space via a
linker peptide. The linker peptide may be cleaved by one or a
combination of intermembrane proteases listed in Table 3 (e.g.
HTRA2/OMI which requires a non-polar aliphatic amino acid--valine,
isoleucine or methionine are preferred--at position P1, and
hydrophilic residues--arginine is preferred--at the P2 and P3
positions). The attached intermembrane domain can be selected from
a variety of endogenous transmembrane proteins. In some
embodiments, the exogenous protein is an engineered fusion protein,
where the C-terminus or N-terminus of the protein is attached to a
protein domain located within the cytosolic space via a linker
peptide. For example, the linker peptide may be designed for
cleavage by one or a combination of the cytosolic proteases
outlined in Table 3 which requires the accompanying cleavage
sequence also included in Table 3. The attached cytosolic domain
can be selected from a variety of molecules as indicated in Table
4.
[0749] The source, mitochondria in the source, or a chondrisome
preparation may be treated with a protein comprising a proteolytic
degradation sequence. Mitochondria contain multiple proteases that
recognize specific amino acid sequences and target the proteins for
degradation. The source, mitochondria in the source, or a
chondrisome preparation may be engineered to express mitochondrial
proteins comprising a mitochondrial proteolytic degradation
sequence, e.g. the preferred Capsase 2 protein sequence
(Val-Asp-Val-Ala-Asp-l-) or other proteolytic sequences (see
Gasteiger et al., The Proteomics Protocols Handbook; 2005: 571-607)
or a modified mitochondrial proteolytic degradation sequence that
has at least 75%, 80%, 85%, 90%, 95% or greater identity to the
wildtype proteolytic degradation sequence.
[0750] The source, mitochondria in the source, or a chondrisome
preparation may be treated with a mitochondrial protein with a
cytosolic proteolytic degradation sequence, e.g., ubiquitin, and a
modified cytosolic proteolytic degradation sequence that has at
least 75%, 80%, 85%, 90%, 95% or greater identity to the wildtype
proteolytic degradation sequence.
[0751] The source, mitochondria in the source, or a chondrisome
preparation may be treated with a protein comprising a protease
domain that recognizes specific mitochondrial proteins. These
protein degrading enzymes can be used to specifically degrade
mitochondrial proteins. Depending on the sub-organellar location of
the target proteins, these enzymes may be active in the
mitochondrial matrix, the intermembrane space or in the cytoplasm
if they are exported. Any mitochondrial protease, a modified
mitochondrial protease that retains at least 10%, 15%, 20%, 30%,
40%, 50%, 60%, 75%, 80%, 90% or more protease activity, a cytosolic
protease that specifically recognizes a mitochondrial protein
(e.g., a modified mitochondrial protein with a cytosolic protease
degradation sequence), and a cytosolic protease modified to
specifically recognize a mitochondrial protein while retaining at
least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more
protease activity may be useful with the invention described
herein.
[0752] See, for example, Quiros P. M. m et al., New roles for
mitochondrial proteases in health, ageing and disease. Nature
Reviews Molecular Cell Biology. V16, 2015; Langer, T. et al.
Characterization of Peptides Released from Mitochondria. THE
JOURNAL OF BIOLOGICAL CHEMISTRY. Vol. 280, No. 4. 2691-2699, 2005;
and Vliegh, P. et al. Synthetic therapeutic peptides: science and
market. Drug Discovery Today. 15(1/2). 2010.
[0753] In some embodiments, the source, mitochondria in the source,
or a chondrisome preparation may be treated with cytosolic
proteins, such as proteases or enzymes, that are modified for
targeting to the mitochondria. Cytosolic proteins may be engineered
to include a mitochondrial localization sequence, e.g., a 5S rRNA,
such as the fly 5S rRNA variant V, the RNA component of the
endoribonuclease known as MRP, or the RNA component of the
ribonucleoprotein known as RNAse P, or the first 69 amino acids of
the precursor of subunit 9 of the mitochondrial Fo-ATPase.
[0754] Further examples of biologics may include, but are not
limited to, metabolic enzymes, transporters, transcriptional
regulators, nucleases, protein modifying enzymes (e.g., kinases),
and nucleic acid modifying enzymes (e.g., methylases), such as
those described in Table 4.
[0755] Nucleic Acids
[0756] The source, mitochondria in the source, or a chondrisome
preparation may be treated with a nucleic acid, including, but not
limited to, an oligonucleotide or modified oligonucleotide, an
aptamer, a cDNA, genomic DNA, an artificial or natural chromosome
(e.g., a yeast artificial chromosome) or a part thereof, RNA,
including an siRNA, a shRNA, mRNA, tRNA, rRNA or a ribozyme, or a
peptide nucleic acid (PNA); a virus or virus-like particles; a
nucleotide or ribonucleotide or synthetic analogue thereof, which
may be modified or unmodified.
[0757] In some embodiments, the source, mitochondria in the source,
or a chondrisome preparation is treated with an exogenous nucleic
acid, such as RNA. Mitochondria import several types of non-coding
RNA, for example, microRNAs, tRNAs, RNA components of RNase P and
MRP endonuclease, and 5S rRNA. The mitochondria may import RNA from
the cytosol. For example, nucleus-encoded RNAs may be targeted to
the mitochondria by using the 20-ribonucleotide stem-loop sequence
of H1 RNA, the RNA component of the RNase P enzyme that regulates
its import. When appended to a nonimported RNA, the H1 RNA import
sequence, designated RP, enables the fusion transcript to be
imported into mitochondria. See, for example, Wang, et al., (2012),
PNAS, 109(13):4840-4845. In one embodiment, the invention includes
a composition of mitochondria in a source or chondrisomes
comprising an exogenous nucleic acid, such as RNA.
[0758] Mitochondria contain a smaller number of tRNA species than
does the cytoplasm. The mitochondria may import tRNA from the
cytosol for optimal protein synthesis. Precursor tRNAs can be
imported into the mitochondria by, for example the protein import
pathway (e.g., coimport with cytoplasmic aminoacyl-tRNA synthetase
or other chaperone protein) or a pathway independent from protein
import that does not require cytosolic factors. In one embodiment,
the invention includes a composition of mitochondria in a source or
chondrisomes comprising an exogenous tRNA.
[0759] In some embodiments, the source is engineered to express a
DNA. The DNA may encode a polypeptide with at least 85%, 90%, 95%,
100% identity to an endogenous protein and retains at least one
activity, function or structure of the endogenous protein. The DNA
may encode a protein that aids a mitochondrial function or
activity, or provides a new function or activity to the
mitochondria, such as transcription or translation in the
mitochondrial matrix. See, Weber-Lotfi, F. et al. DNA import
competence and mitochondrial genetics. Biopolymers and Cell. Vol.
30. N 1. 71-73, 2014.
[0760] A nucleic acid sequences coding for a desired gene can be
engineered using recombinant methods known in the art, such as, for
example by screening libraries from cells expressing the gene, by
deriving the gene from a vector known to include the same, or by
isolating directly from cells and tissues containing the same,
using standard techniques. Alternatively, a gene of interest can be
produced synthetically, rather than cloned.
[0761] The nucleic acids may be operably linked to a promoter, or
incorporate the nucleic acids into an expression vector. The
vectors can be suitable for replication and integration in
eukaryotes. Typical cloning vectors contain transcription and
translation terminators, initiation sequences, and promoters useful
for expression of the desired nucleic acid sequence.
[0762] Additional promoter elements, e.g., enhancers, may regulate
the frequency of transcriptional initiation. Typically, these are
located in the region 30-110 bp upstream of the start site,
although a number of promoters have recently been shown to contain
functional elements downstream of the start site as well. The
spacing between promoter elements frequently is flexible, so that
promoter function is preserved when elements are inverted or moved
relative to one another. In the thymidine kinase (tk) promoter, the
spacing between promoter elements can be increased to 50 bp apart
before activity begins to decline. Depending on the promoter, it
appears that individual elements can function either cooperatively
or independently to activate transcription.
[0763] One example of a suitable promoter is the immediate early
cytomegalovirus (CMV) promoter sequence. This promoter sequence is
a strong constitutive promoter sequence capable of driving high
levels of expression of any polynucleotide sequence operatively
linked thereto. Another example of a suitable promoter is
Elongation Growth Factor-1.alpha. (EF-1.alpha.). However, other
constitutive promoter sequences may also be used, including, but
not limited to the simian virus 40 (SV40) early promoter, mouse
mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long
terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia
virus promoter, an Epstein-Barr virus immediate early promoter, a
Rous sarcoma virus promoter, as well as human gene promoters such
as, but not limited to, the actin promoter, the myosin promoter,
the hemoglobin promoter, and the creatine kinase promoter.
[0764] Further, the invention should not be limited to the use of
constitutive promoters. Inducible promoters are also contemplated
as part of the invention. The use of an inducible promoter provides
a molecular switch capable of turning on expression of the
polynucleotide sequence which it is operatively linked when such
expression is desired, or turning off the expression when
expression is not desired. Examples of inducible promoters include,
but are not limited to a metallothionine promoter, a glucocorticoid
promoter, a progesterone promoter, and a tetracycline promoter.
[0765] The expression vector to be introduced into the source can
also contain either a selectable marker gene or a reporter gene or
both to facilitate identification and selection of expressing cells
from the population of cells sought to be transfected or infected
through viral vectors. In other aspects, the selectable marker may
be carried on a separate piece of DNA and used in a co-transfection
procedure. Both selectable markers and reporter genes may be
flanked with appropriate regulatory sequences to enable expression
in the host cells. Useful selectable markers include, for example,
antibiotic-resistance genes, such as neo and the like.
[0766] Reporter genes may be used for identifying potentially
transfected cells and for evaluating the functionality of
regulatory sequences. In general, a reporter gene is a gene that is
not present in or expressed by the recipient source and that
encodes a polypeptide whose expression is manifested by some easily
detectable property, e.g., enzymatic activity. Expression of the
reporter gene is assayed at a suitable time after the DNA has been
introduced into the recipient cells. Suitable reporter genes may
include genes encoding luciferase, beta-galactosidase,
chloramphenicol acetyl transferase, secreted alkaline phosphatase,
or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000
FEBS Letters 479: 79-82). Suitable expression systems are well
known and may be prepared using known techniques or obtained
commercially. In general, the construct with the minimal 5'
flanking region showing the highest level of expression of reporter
gene is identified as the promoter. Such promoter regions may be
linked to a reporter gene and used to evaluate agents for the
ability to modulate promoter-driven transcription.
[0767] In some embodiments, the source may be genetically modified
to alter expression of one or more proteins. Expression of the one
or more proteins may be modified for a specific time, e.g.,
development or differentiation state of the source. Expression of
the one or more proteins may be restricted to a specific
location(s) or widespread throughout the source. Alternative
trans-splicing also creates variants that may be differentially
targeted. In some embodiments, the source is engineered to create a
long or a short spliced variant, e.g., trypanosomal isoleucyl-tRNA
synthetase (IleRS), to differentially target the protein products,
e.g., the longer spliced variant is found exclusively in
mitochondria and the shorter spliced variant is translated to a
cytosol-specific isoform. In some embodiments, a distribution of
alternative splice variants, such as in the cytosol or the
mitochondria, is altered by increasing the presence of one or more
forms or decreasing the presence of one or more forms.
[0768] In one embodiment, the source may be modified to
over-express an endogenous nucleic acid or protein, or to express
an exogenous nucleic acid or protein. The nucleic acid may include
one or more mitochondrial genes, such as, a chemical transporter,
e.g., UCP1, UCP2, UCP3, UCP4 or UCP5, or a nucleic acid that
encodes SEQ ID NOs:1, 2, 3, 4, or 5. The nucleic acid may include
any one or more mitochondrial or cytosolic genes, such as, a
protein deacetylase, e.g., Sirt3, or a nucleic acid that encodes
SEQ ID NO:7, or others described herein. The nucleic acid may be a
modified mitochondrial gene that has at least 75%, 80%, 85%, 90%,
95% or greater identity to the wildtype mitochondrial or cytosolic
gene. The nucleic acid may include one or more of the exogenous
genes described herein. The nucleic acid may be a modified
exogenous gene, e.g., comprising a sequence for a mitochondrial
targeting peptide, that has at least 75%, 80%, 85%, 90%, 95% or
greater identity to the exogenous gene.
[0769] The nucleic acid encoding a polypeptide can be obtained
using recombinant methods known in the art, such as, for example by
screening libraries from cells expressing the gene, by deriving the
gene from a vector known to include the same, or by isolating
directly from cells and tissues containing the same, using standard
techniques. Alternatively, a gene of interest can be produced
synthetically, rather than cloned.
[0770] Expression of the nucleic acid may be achieved by direct
introduction of the nucleic acid into the source, mitochondria in
the source, or a chondrisome preparation by one of the methods
described herein or by operably linking the nucleic acid encoding a
polypeptide to a promoter, incorporating the construct into an
expression vector and introducing the vector into the source,
mitochondria in the source, or a chondrisome preparation by one of
the methods described herein. Vectors useful with the invention
should be suitable for replication and integration in eukaryotes.
Typical cloning vectors contain transcription and translation
terminators, initiation sequences, and promoters useful for
expression of the desired nucleic acid sequence.
[0771] Additional promoter elements, e.g., enhancers, may regulate
the frequency of transcriptional initiation. Typically, these are
located in the region 30-110 bp upstream of the start site,
although a number of promoters have recently been shown to contain
functional elements downstream of the start site as well. The
spacing between promoter elements frequently is flexible, so that
promoter function is preserved when elements are inverted or moved
relative to one another. In the thymidine kinase (tk) promoter, the
spacing between promoter elements can be increased to 50 bp apart
before activity begins to decline. Depending on the promoter,
individual elements may function either cooperatively or
independently to activate transcription.
[0772] A constitutive promoter sequence capable of driving high
levels of expression of any polynucleotide sequence operatively
linked thereto may be used, including, but not limited to the
cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early
promoter, mouse mammary tumor virus (MMTV), human immunodeficiency
virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter,
an avian leukemia virus promoter, an Epstein-Barr virus immediate
early promoter, a Rous sarcoma virus promoter, as well as human
gene promoters such as, but not limited to, the actin promoter, the
myosin promoter, the hemoglobin promoter, and the creatine kinase
promoter. Further, the invention should not be limited to the use
of constitutive promoters. Inducible promoters are also
contemplated as part of the invention. The use of an inducible
promoter provides a molecular switch capable of turning on
expression of the polynucleotide sequence which it is operatively
linked when such expression is desired, or turning off the
expression when expression is not desired. Examples of inducible
promoters include, but are not limited to a metallothionine
promoter, a glucocorticoid promoter, a progesterone promoter, and a
tetracycline promoter.
[0773] In one embodiment, the source, mitochondria in the source,
or a chondrisome preparation is treated with a nucleic acid
comprising a gene that encodes a polypeptide, which the gene is
operatively linked to transcriptional and translational regulatory
elements active in a target cell or tissue at a target site.
[0774] Mitochondrial Biogenesis Agent
[0775] The source, mitochondria in the source, or a chondrisome
preparation may be treated with an agent that increases
mitochondrial biogenesis. For example, the source, mitochondria in
the source, or a chondrisome preparation may be contacted with a
mitochondrial biogenesis (MB) agent in an amount and for a time
sufficient to increase mitochondrial biogenesis in the source,
mitochondria in the source, or a chondrisome preparation (e.g., by
at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more).
Such MB agents are described, e.g., in Cameron et al. 2016.
Development of Therapeutics That Induce Mitochondrial Biogenesis
for the Treatment of Acute and Chronic Degenerative Diseases.
DOI:10.1021/acs.jmedchem.6b00669.
[0776] In one embodiment, the MB agent is a an extract of a natural
product or synthetic equivalent sufficient to increase
mitochondrial biogenesis in the source, mitochondria in the source,
or a chondrisome preparation. Examples of such natural products
include resveratrol, epicatechin, curcumin, a phytoestrogen (e.g.,
genistein, daidzein, pyrroloquinoline, quinone, coumestrol and
equol).
[0777] In another embodiment, the MB agent is a metabolite
sufficient to increase mitochondrial biogenesis in the source,
mitochondria in the source, or a chondrisome preparation, e.g., a
primary or secondary metabolite. Such metabolites, e.g., primary
metabolites include alcohols such as ethanol, lactic acid, and
certain amino acids and secondary metabolites include organic
compounds produced through the modification of a primary
metabolite, are described in "Primary and Secondary Metabolites."
Boundless Microbiology. Boundless, 26 May 2016.
[0778] In one embodiment, the MB agent is an energy source
sufficient to increase mitochondrial biogenesis in the source,
mitochondria in the source, or a chondrisome preparation, e.g.,
sugars, ATP, redox cofactors as NADH and FADH2. Such energy source,
e.g., pyruvate or palmitate, are described in Mehlman, M. Energy
Metabolism and the Regulation of Metabolic Processes in
Mitochondria; Academic Press, 1972.
[0779] In one embodiment, the MB agent is a transcription factor
modulator sufficient to increase mitochondrial biogenesis in the
source, mitochondria in the source, or a chondrisome preparation.
Examples of such transcription factor modulators include:
thiazolidinediones (e.g., rosiglitazone, pioglitazone, troglitazone
and ciglitazone), estrogens (e.g., 17.beta.-Estradiol,
progesterone) and estrogen receptor agonists; SIRT1 Activators
(e.g., SRT1720, SRT1460, SRT2183, SRT2104).
[0780] In one embodiment, the MB agent is a kinase modulator
sufficient to increase mitochondrial biogenesis in the source,
mitochondria in the source, or a chondrisome preparation. Examples
include: AMPK and AMPK activators such as AICAR, metformin,
phenformin, A769662; and ERK1/2 inhibitors, such as U0126,
trametinib.
[0781] In one embodiment, the MB agent is a cyclic nucleotide
modulator sufficient to increase mitochondrial biogenesis in the
source, mitochondria in the source, or a chondrisome preparation.
Examples include modulators of the NO-cGMP-PKG pathway (for example
nitric oxide (NO) donors, such as sodium nitroprusside,
(.+-.)S-nitroso-N-acetylpenicillamine (SNAP), diethylamine NONOate
(DEA-NONOate), diethylenetriamine-NONOate (DETA-NONOate); sGC
stimulators and activators, such as cinaciguat, riociguat, and BAY
41-2272; and phosphodiesterase (PDE) inhibitors, such as zaprinast,
sildenafil, udenafil, tadalafil, and vardenafil) and modulators of
the cAMP-PKA-CREB Axis, such as phosphodiesterase (PDE) inhibitors
such as rolipram.
[0782] In one embodiment, the MB agent is a modulator of a G
protein coupled receptor (GPCR), such as a GPCR ligand, sufficient
to increase mitochondrial biogenesis in the source, mitochondria in
the source, or a chondrisome preparation.
[0783] In one embodiment, the MB agent is a modulator of a
cannabinoid-1 receptor sufficient to increase mitochondrial
biogenesis in the source, mitochondria in the source, or a
chondrisome preparation. Examples include taranabant and
rimonobant.
[0784] In one embodiment, the MB agent is a modulator of a
5-Hydroxytryptamine receptor sufficient to increase mitochondrial
biogenesis in the source, mitochondria in the source, or a
chondrisome preparation. Examples include
alpha-methyl-5-hydroxytryptamine, DOI, CP809101, SB242084,
serotonin reuptake inhibitors such as fluoxetine, alpha-methyl 5HT,
1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, LY334370, and
LY344864.
[0785] In one embodiment, the MB agent is a modulator of a beta
adrenergic receptor sufficient to increase mitochondrial biogenesis
in the source, mitochondria in the source, or a chondrisome
preparation. Examples include epinephrine, norepinephrine,
isoproterenol, metoprolol, formoterol, fenoterol and
procaterol.
[0786] RNAi
[0787] In some embodiments, the source, mitochondria in the source,
or a chondrisome preparation is modified with an RNA (of various
sizes to include, but not limited to, siRNA, mRNA, gRNA) targeted
to the mitochondrial intermembrane or matrix. For example, the
source, mitochondria in the source, or a chondrisome preparation
may be modified to under-express an endogenous nucleic acid or
protein.
[0788] Certain RNA can inhibit gene expression through the
biological process of RNA interference (RNAi). RNAi molecules
comprise RNA or RNA-like structures typically containing 15-50 base
pairs (such as about 18-25 base pairs) and having a nucleobase
sequence identical (complementary) or nearly identical
(substantially complementary) to a coding sequence in an expressed
target gene within the cell. RNAi molecules include, but are not
limited to: short interfering RNAs (siRNAs), double-strand RNAs
(dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA),
meroduplexes, and dicer substrates (U.S. Pat. Nos. 8,084,599,
8,349,809 and 8,513,207). In one embodiment, the invention includes
a composition of mitochondria in a source or chondrisomes
comprising an exogenous nucleic acid, such as an RNAi molecule
described herein.
[0789] RNAi molecules comprise a sequence substantially
complementary, or fully complementary, to all or a fragment of a
target gene. RNAi molecules may complement sequences at the
boundary between introns and exons to prevent the maturation of
newly-generated nuclear RNA transcripts of specific genes into mRNA
for transcription. RNAi molecules complementary to specific genes
can hybridize with the mRNA for that gene and prevent its
translation. The antisense molecule can be DNA, RNA, or a
derivative or hybrid thereof. Examples of such derivative molecules
include, but are not limited to, peptide nucleic acid (PNA) and
phosphorothioate-based molecules such as deoxyribonucleic guanidine
(DNG) or ribonucleic guanidine (RNG).
[0790] RNAi molecules can be provided to the cell as "ready-to-use"
RNA synthesized in vitro or as an antisense gene transfected into
cells which will yield RNAi molecules upon transcription.
Hybridization with mRNA results in degradation of the hybridized
molecule by RNAse H and/or inhibition of the formation of
translation complexes. Both result in a failure to produce the
product of the original gene.
[0791] The length of the RNAi molecule that hybridizes to the
transcript of interest should be around 10 nucleotides, between
about 15 or 30 nucleotides, or about 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. The degree
of identity of the antisense sequence to the targeted transcript
should be at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95.
[0792] RNAi molecules may also comprise overhangs, i.e. typically
unpaired, overhanging nucleotides which are not directly involved
in the double helical structure normally formed by the core
sequences of the herein defined pair of sense strand and antisense
strand. RNAi molecules may contain 3' and/or 5' overhangs of about
1-5 bases independently on each of the sense strands and antisense
strands. In one embodiment, both the sense strand and the antisense
strand contain 3' and 5' overhangs. In one embodiment, one or more
of the 3' overhang nucleotides of one strand base pairs with one or
more 5' overhang nucleotides of the other strand. In another
embodiment, the one or more of the 3' overhang nucleotides of one
strand base do not pair with the one or more 5' overhang
nucleotides of the other strand. The sense and antisense strands of
an RNAi molecule may or may not contain the same number of
nucleotide bases. The antisense and sense strands may form a duplex
wherein the 5' end only has a blunt end, the 3' end only has a
blunt end, both the 5' and 3' ends are blunt ended, or neither the
5' end nor the 3' end are blunt ended. In another embodiment, one
or more of the nucleotides in the overhang contains a
thiophosphate, phosphorothioate, deoxynucleotide inverted (3' to 3'
linked) nucleotide or is a modified ribonucleotide or
deoxynucleotide.
[0793] Small interfering RNA (siRNA) molecules comprise a
nucleotide sequence that is identical to about 15 to about 25
contiguous nucleotides of the target mRNA. In some embodiments, the
siRNA sequence commences with the dinucleotide AA, comprises a
GC-content of about 30-70% (about 30-60%, about 40-60%, or about
45%-55%), and does not have a high percentage identity to any
nucleotide sequence other than the target in the genome of the
mammal in which it is to be introduced, for example as determined
by standard BLAST search.
[0794] siRNAs and shRNAs resemble intermediates in the processing
pathway of the endogenous microRNA (miRNA) genes (Bartel, Cell
116:281-297, 2004). In some embodiments, siRNAs can function as
miRNAs and vice versa (Zeng et al., Mol Cell 9:1327-1333, 2002;
Doench et al., Genes Dev 17:438-442, 2003). MicroRNAs, like siRNAs,
use RISC to downregulate target genes, but unlike siRNAs, most
animal miRNAs do not cleave the mRNA. Instead, miRNAs reduce
protein output through translational suppression or polyA removal
and mRNA degradation (Wu et al., Proc Natl Acad Sci USA
103:4034-4039, 2006). Known miRNA binding sites are within mRNA 3'
UTRs; miRNAs seem to target sites with near-perfect complementarity
to nucleotides 2-8 from the miRNA's 5' end (Rajewsky, Nat Genet 38
Suppl:S8-13, 2006; Lim et al., Nature 433:769-773, 2005). This
region is known as the seed region. Because siRNAs and miRNAs are
interchangeable, exogenous siRNAs downregulate mRNAs with seed
complementarity to the siRNA (Birmingham et al., Nat Methods
3:199-204, 2006. Multiple target sites within a 3' UTR give
stronger downregulation (Doench et al., Genes Dev 17:438-442,
2003).
[0795] Lists of known miRNA sequences can be found in databases
maintained by research organizations, such as Wellcome Trust Sanger
Institute, Penn Center for Bioinformatics, Memorial Sloan Kettering
Cancer Center, and European Molecule Biology Laboratory, among
others. Known effective siRNA sequences and cognate binding sites
are also well represented in the relevant literature. RNAi
molecules are readily designed and produced by technologies known
in the art. In addition, there are computational tools that
increase the chance of finding effective and specific sequence
motifs (Pei et al. 2006, Reynolds et al. 2004, Khvorova et al.
2003, Schwarz et al. 2003, Ui-Tei et al. 2004, Heale et al. 2005,
Chalk et al. 2004, Amarzguioui et al. 2004).
[0796] The RNAi molecule modulates expression of RNA encoded by a
gene. Because multiple genes can share some degree of sequence
homology with each other, in some embodiments, the RNAi molecule
can be designed to target a class of genes with sufficient sequence
homology. In some embodiments, the RNAi molecule can contain a
sequence that has complementarity to sequences that are shared
amongst different gene targets or are unique for a specific gene
target. In some embodiments, the RNAi molecule can be designed to
target conserved regions of an RNA sequence having homology between
several genes thereby targeting several genes in a gene family
(e.g., different gene isoforms, splice variants, mutant genes,
etc.). In some embodiments, the RNAi molecule can be designed to
target a sequence that is unique to a specific RNA sequence of a
single gene.
[0797] In some embodiments, the RNAi molecule targets a sequence in
a mitochondrial or cytosol gene, e.g., an enzyme involved in
post-translational modifications including, but are not limited to,
physiologic redox signaling via reactive oxygen and nitrogen
species, kinase, O-GlcNAcylation, S-nitrosylation, nitration,
glutathionylation, acetylation, succinylation, and others. In one
embodiment, the RNAi molecule targets a protein deacetylase, e.g.,
Sirt3. In one embodiment, the invention includes a composition of
mitochondria in a source or chondrisomes comprising an RNAi that
targets a mitochondrial or cytosol gene, e.g., an enzyme involved
in post-translational modifications.
[0798] In some embodiments, the RNAi molecule targets a sequence in
a gene, e.g., a membrane transport protein. In one embodiment, the
RNAi molecule targets a chemical transporter, e.g., UCP1, UCP2,
UCP3, UCP4 or UCP5. In one embodiment, the invention includes a
composition of mitochondria in a source or chondrisomes comprising
an RNAi that targets a chemical transporter gene, e.g., UCP1, UCP2,
UCP3, UCP4 or UCP5.
[0799] Targeted Endonucleases
[0800] Mitochondria-targeted restriction endonucleases (REs) may
also be a useful tool for mitochondrial genome manipulation. The
source, mitochondria in a source, or a chondrisome preparation may
be modified with recombinant REs with mitochondrial localization
signals (MLSs) for import into the mitochondrial matrix where they
can access mtDNA and create site-specific double-strand breaks.
Cleavage of mtDNA in this manner leads primarily to the degradation
of target mtDNA species and if present, expansion of heteroplasmic
species lacking the cleavable sequence. Mitochondria-targeted
endonucleases may recognize sequences only in specific mtDNA.
Recognition and cleavage by the enzyme leads to a reduction in the
relative levels of the target allele through cleavage stimulated
mtDNA degradation. Only the uncleaved mtDNA can replicate and
re-establishment of normal mtDNA levels results in an increased
relative abundance of the mtDNA without the endonuclease
recognition site.
[0801] Some available targeted REs include zinc-finger nucleases
(ZFNs) and transcription activator-like effector nucleases
(TALENs). Both systems share a common basic structure utilizing a
sequencing-independent endonuclease domain from FokI coupled to a
sequence-specific modular DNA-binding domain. As FokI creates
double-strand breaks as a dimer, both enzyme systems require the
design of pairs of monomers that bind the region of interest
tail-tail in close proximity enabling the dimerization of FokI
domains and double-strand cleavage between the monomer-binding
sites. The principal differences between the systems are in the
modularity of DNA sequence recognition. Both systems employ tandem
repeats of modular DNA-binding domains to create sequence-specific
DNA-binding domains. In ZFNs, each individual zincfinger domain
recognizes 3 bp of DNA, and for TALENs, each TALE domain recognizes
1 bp. See, for example, mitoTALEN described in Bacman, et al., Nat.
Med., vol. 19(9):1111-1113. In one embodiment, the invention
includes a composition of mitochondria in a source or chondrisomes
comprising a targeted RE, e.g., a zinc-finger nuclease (ZFN) or
transcription activator-like effector nuclease (TALEN) described
herein.
[0802] CRISPR
[0803] In one embodiment, a modification is made to the source,
mitochondria in a source, or a chondrisome preparation to modulate
one or more proteins targeted to the mitochondria, such as
producing mitochondria with a heterologous function or structural
changing the proteins in the mitochondria. One method for
modulating proteins targeted to the mitochondria uses clustered
regulatory interspaced short palindromic repeat (CRISPR) system for
gene editing. CRISPR systems are adaptive defense systems
originally discovered in bacteria and archaea. CRISPR systems use
RNA-guided nucleases termed CRISPR-associated or "Cas"
endonucleases (e. g., Cas9 or Cpf1) to cleave foreign DNA. In a
typical CRISPR/Cas system, an endonuclease is directed to a target
nucleotide sequence (e. g., a site in the genome that is to be
sequence-edited) by sequence-specific, non-coding "guide RNAs" that
target single- or double-stranded DNA sequences. Three classes
(I-III) of CRISPR systems have been identified. The class II CRISPR
systems use a single Cas endonuclease (rather than multiple Cas
proteins). One class II CRISPR system includes a type II Cas
endonuclease such as Cas9, a CRISPR RNA ("crRNA"), and a
trans-activating crRNA ("tracrRNA"). The crRNA contains a "guide
RNA", typically an about 20-nucleotide RNA sequence that
corresponds to a target DNA sequence. The crRNA also contains a
region that binds to the tracrRNA to form a partially
double-stranded structure which is cleaved by RNase III, resulting
in a crRNA/tracrRNA hybrid. The crRNA/tracrRNA hybrid then directs
the Cas9 endonuclease to recognize and cleave the target DNA
sequence. The target DNA sequence must generally be adjacent to a
"protospacer adjacent motif" ("PAM") that is specific for a given
Cas endonuclease; however, PAM sequences appear throughout a given
genome. CRISPR endonucleases identified from various prokaryotic
species have unique PAM sequence requirements; examples of PAM
sequences include 5'-NGG (Streptococcus pyogenes), 5'-NNAGAA
(Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus
thermophilus CRISPR3), and 5'-NNNGATT (Neisseria meningiditis).
Some endonucleases, e. g., Cas9 endonucleases, are associated with
G-rich PAM sites, e. g., 5'-NGG, and perform blunt-end cleaving of
the target DNA at a location 3 nucleotides upstream from (5' from)
the PAM site. Another class II CRISPR system includes the type V
endonuclease Cpf1, which is smaller than Cas9; examples include
AsCpf1 (from Acidaminococcus sp.) and LbCpf1 (from Lachnospiraceae
sp.). Cpf1-associated CRISPR arrays are processed into mature
crRNAs without the requirement of a tracrRNA; in other words a Cpf1
system requires only the Cpf1 nuclease and a crRNA to cleave the
target DNA sequence. Cpf1 endonucleases, are associated with T-rich
PAM sites, e. g., 5'-TTN. Cpf1 can also recognize a 5'-CTA PAM
motif. Cpf1 cleaves the target DNA by introducing an offset or
staggered double-strand break with a 4- or 5-nucleotide 5'
overhang, for example, cleaving a target DNA with a 5-nucleotide
offset or staggered cut located 18 nucleotides downstream from (3'
from) from the PAM site on the coding strand and 23 nucleotides
downstream from the PAM site on the complimentary strand; the
5-nucleotide overhang that results from such offset cleavage allows
more precise genome editing by DNA insertion by homologous
recombination than by insertion at blunt-end cleaved DNA. See, e.
g., Zetsche et al. (2015) Cell, 163:759-771.
[0804] For the purposes of gene editing, CRISPR arrays can be
designed to contain one or multiple guide RNA sequences
corresponding to a desired target DNA sequence; see, for example,
Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature
Protocols, 8:2281-2308. At least about 16 or 17 nucleotides of gRNA
sequence are required by Cas9 for DNA cleavage to occur; for Cpf1
at least about 16 nucleotides of gRNA sequence is needed to achieve
detectable DNA cleavage. In practice, guide RNA sequences are
generally designed to have a length of between 17-24 nucleotides
(e.g., 19, 20, or 21 nucleotides) and complementarity to the
targeted gene or nucleic acid sequence. Custom gRNA generators and
algorithms are available commercially for use in the design of
effective guide RNAs. Gene editing has also been achieved using a
chimeric "single guide RNA" ("sgRNA"), an engineered (synthetic)
single RNA molecule that mimics a naturally occurring
crRNA-tracrRNA complex and contains both a tracrRNA (for binding
the nuclease) and at least one crRNA (to guide the nuclease to the
sequence targeted for editing). Chemically modified sgRNAs have
also been demonstrated to be effective in genome editing; see, for
example, Hendel et al. (2015) Nature Biotechnol., 985-991. In one
embodiment, the invention includes a composition of mitochondria in
a source or chondrisomes comprising a sgRNA.
[0805] Whereas wild-type Cas9 generates double-strand breaks (DSBs)
at specific DNA sequences targeted by a gRNA, a number of CRISPR
endonucleases having modified functionalities are available, for
example: a "nickase" version of Cas9 generates only a single-strand
break; a catalytically inactive Cas9 ("dCas9") does not cut the
target DNA but interferes with transcription by steric hindrance.
dCas9 can further be fused with an effector to repress (CRISPRi) or
activate (CRISPRa) expression of a target gene. For example, Cas9
can be fused to a transcriptional repressor (e.g., a KRAB domain)
or a transcriptional activator (e.g., a dCas9-VP64 fusion). A
catalytically inactive Cas9 (dCas9) fused to FokI nuclease
("dCas9-FokI") can be used to generate DSBs at target sequences
homologous to two gRNAs. See, e. g., the numerous CRISPR/Cas9
plasmids disclosed in and publicly available from the Addgene
repository (Addgene, 75 Sidney St., Suite 550A, Cambridge, Mass.
02139; addgene.org/crispr/). A "double nickase" Cas9 that
introduces two separate double-strand breaks, each directed by a
separate guide RNA, is described as achieving more accurate genome
editing by Ran et al. (2013) Cell, 154:1380-1389. In one
embodiment, the invention includes a composition of mitochondria in
a source or chondrisomes comprising a CRISPR endonuclease.
[0806] CRISPR technology for editing the genes of eukaryotes is
disclosed in US Patent Application Publications 2016/0138008A1 and
US2015/0344912A1, and in U.S. Pat. Nos. 8,697,359, 8,771,945,
8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418,
8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. Cpf1
endonuclease and corresponding guide RNAs and PAM sites are
disclosed in US Patent Application Publication 2016/0208243 A1.
CRISPR technology for generating mtDNA dysfunction in the
mitochondrial genome with the CRISPR/Cas9 system is disclosed in
Jo, A., et al., BioMed Res. Int'l, vol 2015, article ID 305716, 10
pages, http://dx.doi.org/10.1155/2015/305716.
[0807] In some embodiments, mitochondrial DNA is treated with
mitochondrial targeted restriction endonuclease. Replication in
mitochondria harboring mtDNA that is selectively cleaved by the
restriction endonuclease is inhibited and thereby only non-cleaved
mtDNA is allowed to propagate in the mitochondria.
[0808] In some embodiments, the desired genome modification
involves homologous recombination, wherein one or more
double-stranded DNA breaks in the target nucleotide sequence is
generated by the RNA-guided nuclease and guide RNA(s), followed by
repair of the break(s) using a homologous recombination mechanism
("homology-directed repair"). In such embodiments, a donor template
that encodes the desired nucleotide sequence to be inserted or
knocked-in at the double-stranded break is provided to the cell or
subject; examples of suitable templates include single-stranded DNA
templates and double-stranded DNA templates (e. g., linked to the
polypeptide described herein). In general, a donor template
encoding a nucleotide change over a region of less than about 50
nucleotides is provided in the form of single-stranded DNA; larger
donor templates (e. g., more than 100 nucleotides) are often
provided as double-stranded DNA plasmids. In some embodiments, the
donor template is provided to the cell or subject in a quantity
that is sufficient to achieve the desired homology-directed repair
but that does not persist in the cell or subject after a given
period of time (e. g., after one or more cell division cycles). In
some embodiments, a donor template has a core nucleotide sequence
that differs from the target nucleotide sequence (e. g., a
homologous endogenous genomic region) by at least 1, at least 5, at
least 10, at least 20, at least 30, at least 40, at least 50, or
more nucleotides. This core sequence is flanked by "homology arms"
or regions of high sequence identity with the targeted nucleotide
sequence; in embodiments, the regions of high identity include at
least 10, at least 50, at least 100, at least 150, at least 200, at
least 300, at least 400, at least 500, at least 600, at least 750,
or at least 1000 nucleotides on each side of the core sequence. In
some embodiments where the donor template is in the form of a
single-stranded DNA, the core sequence is flanked by homology arms
including at least 10, at least 20, at least 30, at least 40, at
least 50, at least 60, at least 70, at least 80, or at least 100
nucleotides on each side of the core sequence. In embodiments where
the donor template is in the form of a double-stranded DNA, the
core sequence is flanked by homology arms including at least 500,
at least 600, at least 700, at least 800, at least 900, or at least
1000 nucleotides on each side of the core sequence. In one
embodiment, two separate double-strand breaks are introduced into
the cell or subject's target nucleotide sequence with a "double
nickase" Cas9 (see Ran et al. (2013) Cell, 154:1380-1389), followed
by delivery of the donor template.
[0809] In some embodiments, the composition comprising a gRNA and a
targeted nuclease, e.g., a Cas9, e.g., a wild type Cas9, a nickase
Cas9 (e.g., Cas9 D10A), a dead Cas9 (dCas9), eSpCas9, Cpf1, C2C1,
or C2C3, or a nucleic acid encoding such a nuclease, are used to
modulate mitochondrial gene expression. The choice of nuclease and
gRNA(s) is determined by whether the targeted mutation is a
deletion, substitution, or addition of nucleotides, e.g., a
deletion, substitution, or addition of nucleotides to a targeted
sequence. Fusions of a catalytically inactive endonuclease e.g., a
dead Cas9 (dCas9, e.g., D10A; H840A) tethered with all or a portion
of (e.g., biologically active portion of) an (one or more) effector
domain create chimeric proteins that can be linked to the
polypeptide to guide the composition to specific DNA sites by one
or more RNA sequences (sgRNA) to modulate activity and/or
expression of one or more target nucleic acids sequences (e.g., to
methylate or demethylate a DNA sequence).
[0810] In some embodiments, one or more component of a CRISPR
system described hereinabove. In embodiments, the methods described
herein include a method of delivering one or more CRISPR system
component described hereinabove to a source, e.g., to the nucleus
of the source to modulate a mitochondrial protein, mitochondria in
a source, e.g., to the nucleus of the source to modulate a
mitochondrial protein, or a chondrisome preparation. In one
embodiment, the invention includes a composition of mitochondria in
a source or chondrisomes comprising CRISPR modified mtDNA.
[0811] In some embodiments, a zinc finger protein is engineered to
bind a mitochondrial predetermined DNA sequence. Fusing a zinc
finger protein to a nuclease domain creates a zinc-finger nuclease
(ZFN) that can cleave DNA adjacent to the specific ZFP-binding
site. By designing a single chain quasi-dimeric ZFN with a
predetermined DNA binding domain, the ZFN can recognize a
pathogenic point mutation in the mtDNA, selectively cleave and
eliminate the mutant mtDNA and thereby increase the proportion of
wild type mtDNA. In one embodiment, the invention includes a
composition of mitochondria in a source or chondrisomes comprising
ZFN cleaved mtDNA.
[0812] In some embodiments, the CRISPR components target any
mitochondrial gene as described herein. In some embodiments, the
CRISPR components target any cytosolic gene as described
herein.
[0813] Targeting
[0814] In some embodiments, the modifying agent is designed for
specific trafficking the mitochondria or chondrisome described
herein to a target cell or tissue, e.g., cardiac tissue, or stem
cells. The modifying agent may include a targeting group, e.g., a
cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid
or protein, e.g., an antibody, that binds to a specified cell type
such as a cardiac cell or stem cell. In one embodiment, the
invention includes a composition of mitochondria in a source or
chondrisomes comprising a targeting agent, e.g., a cell or tissue
targeting agent, e.g., a lectin, glycoprotein, lipid or protein,
e.g., an antibody, that binds to a specified cell type such as a
cardiac cell or stem cell. A targeting group may include, but is
not limited to, a thyrotropin, melanotropin, lectin, glycoprotein,
surfactant protein A, Mucin carbohydrate, multivalent lactose,
multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine
multivalent mannose, multivalent fucose, glycosylated
polyaminoacids, multivalent galactose, transferrin, bisphosphonate,
polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile
acid, folate, vitamin B12, biotin, or an RGD peptide, RGD peptide
mimetic, or other commonly used targeting group. In another
embodiment, the invention includes a composition of mitochondria in
a source or chondrisomes comprising the targeting group described
herein.
[0815] In some embodiments, protofection is used to insert and
express mitochondrial genomes into living cells. Protofection uses
recombinant human mitochondrial transcription factor A (TFAM)
engineered with an N-terminal protein transduction domain (PTD)
followed by an MTS to deliver an agent to the mitochondria of
living cells. For protocol, see Keeney P. M., et al., Hum Gene Ther
20: 897-907 (2009). TFAM is a major mtDNA-binding protein with two
high mobility group (HMG) domains. It binds to and organizes mtDNA
into a mitochondrial nucleoid structure, which is necessary for
mtDNA transcription and maintenance. The MTD-TFAM
(MTD=PTD+MTS=mitochondrial transduction domain) recombinant protein
would bind mtDNA by interacting with TFAM and rapidly transporting
it across the plasma membranes into the mitochondria with the
assistance of the MTD. In another embodiment, the invention
includes a composition of mitochondria in a source or chondrisomes
comprising recombinant MTD-TFAM as described herein.
Methods of Modifying Sources or Mitochondria
[0816] Methods of introducing a modifying agent into a source,
mitochondria in the source, or a chondrisome preparation include
physical, biological and chemical methods. See, for example, Geng.
& Lu, Microfluidic electroporation for cellular analysis and
delivery. Lab on a Chip. 13(19):3803-21. 2013; Sharei, A. et al. A
vector-free microfluidic platform for intracellular delivery. PNAS
vol. 110 no. 6. 2013; Yin, H. et al., Non-viral vectors for
gene-based therapy. Nature Reviews Genetics. 15: 541-555. 2014.
Suitable methods for modifying a source, mitochondria in the
source, or a chondrisome preparation described herein with such a
modifying agent include, for example, diffusion, osmosis, osmotic
pulsing, osmotic shock, hypotonic lysis, hypotonic dialysis,
ionophoresis, electroporation, sonication, microinjection, calcium
precipitation, membrane intercalation, lipid mediated transfection,
detergent treatment, viral infection, receptor mediated
endocytosis, use of protein transduction domains, particle firing,
membrane fusion, freeze-thawing, mechanical disruption, and
filtration.
[0817] Regardless of the method used to introduce the modifying
agent into a source, mitochondria in the source, or a chondrisome
preparation or otherwise expose the source, mitochondria in the
source, or a chondrisome preparation to the modifying agent
described herein of the present invention, in order to confirm the
presence of the modifying agent in the source, mitochondria in the
source, or a chondrisome preparation, a variety of assays may be
performed. Such assays include, for example, "molecular biological"
assays well known to those of skill in the art, such as Southern
and Northern blotting, RT-PCR and PCR; "biochemical" assays, such
as detecting the presence or absence of a particular peptide, e.g.,
by immunological means (ELISAs and Western blots) or by assays
described herein to identify agents falling within the scope of the
invention.
Physical Methods
[0818] Some examples of physical methods for introducing a
modifying agent, e.g., protein, small molecule, or a
polynucleotide, into a source, mitochondria in the source, or a
chondrisome preparation include calcium phosphate precipitation,
lipofection, particle bombardment, microinjection, electroporation,
cell squeeze, and the like. The modifying agent can be introduced
into a target source, mitochondria in the source, or a chondrisome
preparation using commercially available methods which include
diffusion, osmosis, osmotic pulsing, osmotic shock, hypotonic
lysis, hypotonic dialysis, ionophoresis, sonication,
microinjection, particle firing, membrane fusion, freeze-thawing,
mechanical disruption, filtration, electroporation (Amaxa
Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830
(BTX) (Harvard Instruments, Boston, Mass.), microfluidic delivery
(CellSqueeze, SQZ Biotech, Watertown, Mass.), Gene Pulser II
(BioRad, Denver, Colo.), or multiporator (Eppendort, Hamburg
Germany). See, for example, Geng. & Lu, Microfluidic
electroporation for cellular analysis and delivery. Lab on a Chip.
13(19):3803-21. 2013; Sharei, A. et al. The modifying agent can
also be introduced into a source, mitochondria in the source, or a
chondrisome preparation using cationic liposome mediated
transfection using lipofection, using polymer encapsulation, using
peptide mediated transfection, or using biolistic particle delivery
systems such as "gene guns" (see, for example, Nishikawa, et al.
Hum Gene Ther., 12(8):861-70 (2001), and A vector-free microfluidic
platform for intracellular delivery. PNAS vol. 110 no. 6. 2013;
Yin, H. et al.
[0819] Biological Methods
[0820] Some examples of biological methods for introducing a
modifying agent, e.g., protein, small molecule, or a
polynucleotide, into a source, mitochondria in the source, or a
chondrisome preparation include the use of DNA and RNA vectors,
viral infection, receptor mediated endocytosis, and use of protein
transduction domains. Viral vectors, and especially retroviral
vectors, have become the most widely used method for inserting
genes into mammalian, e.g., human cells. Vectors derived from
retroviruses, such as the lentivirus, are suitable tools to achieve
long-term gene transfer since they allow long-term, stable
integration of a transgene and its propagation in daughter cells.
Lentiviral vectors have the added advantage over vectors derived
from onco-retroviruses, such as murine leukemia viruses, in that
they can transduce non-proliferating cells. They also have the
added advantage of low immunogenicity. Other viral vectors can be
derived from Sendai virus, poxviruses, herpes simplex virus I,
adenoviruses and adeno-associated viruses, and the like.
[0821] Some examples of biological methods for introducing a
modifying agent, e.g., protein, small molecule, or a
polynucleotide, into a source, mitochondria in the source, or a
chondrisome preparation include physical association/contact. For
example, a modifying agent may attach to the source, mitochondria
in the source, or a chondrisome preparation by interacting with a
protein domain, such as C2 domains (4) or PH domains (5), of the
membrane surface. The interaction may be a covalent bond or a
non-covalent interaction. In some embodiments, the modifying agent
may bind to a surface protein or receptor.
[0822] Cell-penetrating peptides (CPPs) are short peptides that
facilitate cellular intake/uptake of various molecular agents (from
nanosize particles to small chemical molecules and large fragments
of nucleic acids). The agent is associated with the CPP either
through a chemical linkage via covalent bonds or through
non-covalent interactions. The function of the CPPs is to deliver
the agent into the cells via a process that commonly occurs through
endocytosis. CPPs can generally be separated into three classes:
peptides derived from proteins, chimeric peptides that are formed
by the fusion of two natural sequences, and synthetic CPPs which
are rationally designed sequences usually based on
structure-activity studies. Other attempts to classify CPPs, in
spite of their diversity, may be based on the physio-chemical
characteristics of the sequences (e.g., their amphipathicity, or
their hydrophobicity). Examples include, but are not limited to,
TAT, dfTAT, penetratin, pVEC, transportan, MPG, Pep-1,
polyarginines, MAP, and R.sub.6W.sub.3.
[0823] Chemical Methods
[0824] Some examples of chemical means for introducing a
polynucleotide into a source, mitochondria in the source, or a
chondrisome preparation include calcium precipitation, membrane
intercalation, detergent treatment, colloidal dispersion systems,
such as macromolecule complexes, nanocapsules, microspheres, beads,
and lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes. An exemplary colloidal system for
use as a delivery vehicle in vitro and in vivo is a liposome (e.g.,
an artificial membrane vesicle). Non-viral vectors for gene-based
therapy. Nature Reviews Genetics. 15: 541-555. 2014.
[0825] Lipids suitable for use can be obtained from commercial
sources. For example, dimyristyl phosphatidylcholine ("DMPC") can
be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP")
can be obtained from K & K Laboratories (Plainview, N.Y.);
cholesterol ("Choi") can be obtained from Calbiochem-Behring;
dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be
obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock
solutions of lipids in chloroform or chloroform/methanol can be
stored at about -20.degree. C. Chloroform is used as the only
solvent since it is more readily evaporated than methanol.
"Liposome" is a generic term encompassing a variety of single and
multilamellar lipid vehicles formed by the generation of enclosed
lipid bilayers or aggregates. Liposomes can be characterized as
having vesicular structures with a phospholipid bilayer membrane
and an inner aqueous medium. Multilamellar liposomes have multiple
lipid layers separated by aqueous medium. They form spontaneously
when phospholipids are suspended in an excess of aqueous solution.
The lipid components undergo self-rearrangement before the
formation of closed structures and entrap water and dissolved
solutes between the lipid bilayers (Ghosh et al, 1991 Glycobiology
5: 505-10). However, compositions that have different structures in
solution than the normal vesicular structure are also encompassed.
For example, the lipids may assume a micellar structure or merely
exist as nonuniform aggregates of lipid molecules. Also
contemplated are lipofectamine-nucleic acid complexes.
Methods for Assaying Protein Modification
[0826] Modifications to the source, mitochondria in the source or
chondrisome preparation can be assayed for loading levels. In the
case where the modifying agent is a protein, quantification of
modification kinetics is performed by using exogenous protein that
has S35 radioisotope labeled methionine and cysteine amino acids
(Sigma). Protein import is performed as described above with the
addition that 5-mM methionine is included in the import buffer to
prevent nonspecific binding of unincorporated radiolabeled amino
acids and to reduce background noise in subsequent autoradiography.
To determine kinetics of protein uptake, the uptake reaction is
terminated by transferring the reaction to ice, followed by
centrifugation after addition of the exogenous protein, the import
reaction is removed for radiography analysis as previously
described in Stojanovski, D., et al, Methods in Cell Biology,
80:783-806, 2007.
[0827] Modifications can be assayed for localization. Sequential
disruption of mitochondrial membranes and analysis allows the
localization of targeted modifying agents. In the case of exogenous
protein loading, sequential treatment with proteases during
membrane disruption allows determination of a protein's relative
protection from protease between sample and untreated control.
Protocols for these assays are detailed Stojanovski 2007, described
herein, but briefly it is performed as follows. Aliquots of the
isolated chondrisomes are prepared as following: one aliquot is
retained as an untreated control; one aliquot is treated with
protease directly to degrade any unincorporated of surface bound
protein; one aliquot is incubated in hypotonic swelling buffer
(10-mM MOPS-KOH, pH 7.2) to induce swelling, outer membrane
rupture, and mitoplast formation. (This process should be monitored
by immunodecoration of proteins that only become accessible to
protease after swelling (e.g., cytochrome c heme lyase) and the
integrity of the inner membrane should be assessed by comparing the
protease resistance of the canonical citrate synthase matrix
proteins in both whole chondrisomes and mitoplasts by Western blot
analysis); finally, one aliquot is solubilized with Triton X-100 to
assess matrix targeted proteins. Aliquots of the chondrisome
samples are split and are subjected to proteinase K to degrade
unprotected protein or a control buffer. The protease is then
inhibited by phenylmethylsulfonyl fluoride treatment. Chondrisomes
are then isolated by centrifugation, washed, TCA precipitated if
required, solubilized in SDS-PAGE-loading dye, and subjected to
SDS-PAGE and Western blots to determine the quantity of the target
protein delivered to the various subcellular locations.
[0828] Alternatively, immunolabeling and microscopy is a classical
approach that can be used to study the sub cellular localization of
loaded. Methods to employ this technique in determining the
sub-organelle localization of a delivered protein are described in
the literature (Sambrook 2012) and should be performed whenever new
cell types or protein targets are employed. Here, the approach to
determine the localization of delivered protein is briefly
outlined. First, the isolated mitochondria are added to
poly(L-lysine)-coated (0.1%) coverslips where they are incubated in
a wet-chamber at room temperature with protein target specific
monoclonal antibody (Molecular Probes, Eugene, Oreg.) in phosphate
buffer saline (PBS) containing bovine serum albumin (BSA). To
quantify background, control sections are incubated without the
primary antibodies. Samples are rinsed with PBS. The samples are
then incubated in a wet chamber with anti-rabbit IgG-fluorescein
(or anti-mouse IgG-Texas red). The samples are once again rinsed
three times with PBS and then mounted on slides for visualization.
In the cases where primary antibodies for the protein of interest
don't exist, an epitope tag (9-aa-long HA1 hemagglutinin) is added
to the coding region of the loaded protein (Pinton 2007). This
allows immunolocalization of most delivered proteins. Stojanovski,
D., et al., Methods in Cell Biology, 80:783-806, 2007; Sambrook, J.
& Green, M. R. Molecular Cloning: A LABORATORY MANUAL. COLD
SPRING HARBOR LABORATORY PRESS. 2012; and Pinton, P., et al.,
Methods in Cell Biology, 80:297-325, 2007.
Methods of Use
[0829] The chondrisome preparations and compositions described
herein are useful in therapeutic (human) applications, or
veterinary applications, in-vivo or ex-vivo.
In-Vivo Applications
[0830] A chondrisome composition or preparation described herein
may be delivered to a subject in an amount and for a time
sufficient to enhance a cell or tissue function in a mammalian
subject, e.g., a human. For example, the chondrisome composition or
preparation is administered to the subject in an amount and for a
time sufficient to improve function in the subject of a cell or
tissue that is in an injured state (e.g., from trauma, disease or
other damage).
[0831] A chondrisome composition or preparation described herein
may be delivered to a subject in an amount and for a time
sufficient to increase target tissue ATP levels, In some
embodiments, target tissue ATP levels are increased by at least 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more.
[0832] A chondrisome composition or preparation described herein
may be delivered to a subject in an amount and for a time
sufficient to reduce ROS in a target tissue (e.g., cardiovascular
tissue or neural tissue). In some embodiments, ROS levels are
decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or
more in the target tissue.
[0833] In some embodiments, a chondrisome composition or
preparation described herein may be delivered (in vivo or ex-vivo)
to increase mitochondrial content and activity in a target cell or
tissue. In a model system, citrate synthase activity can be used to
assess mitochondrial quantity and activity to determine the
increase in mitochondrial content as a result of delivered
exogenous chondrisomes. Briefly, mitochondria extracted from cells
are subjected to three rounds of freeze/thaw using a dry
ice/ethanol slurry. 65 ul of assay reagent (100-mM Tris-HCl, pH
8.0; 100-uM DTNB; 50-uM acetyl coenzyme A; 0.1% (w/v) Triton X-100)
is added to a cuvette and brought up to 0.5 ml with water.
Processed mitochondria (15 ug of total protein) are added and the
reaction is started by adding oxaloacetate to 50-uM and the
reaction is followed for three minutes by monitoring absorbance at
412 nm. In some tissues (e.g. liver) there is significant
background citrate synthase activity and a control reaction without
the addition of oxaloacetate must be performed (Kirby et al.,
1999). Relative or percent change in citrate synthase activity
between an untreated control and a sample that has been treated
with a chondrisome preparation described herein is used to
determine the modulation of mitochondrial activity. Kirby et. Al.
2007. Biochemical Assays of Respiratory Chain Complex Activity.
Methods in Cell Biology. Vol 80. In some embodiments, mitochondrial
content and/or activity is increased in a target tissue by at least
5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more.
[0834] In some embodiments, a chondrisome composition or
preparation described herein is used to increase thermogenesis,
modify adipocyte size and function, and/or modulate serum
composition by delivering such preparations to a subject, e.g., to
adipocytes (e.g., white adipocytes of a subject). In some
embodiments, chondrisome preparations isolated from brown
adipocytes are administered to white adipocytes of a subject. Such
increase in thermogenesis and adipocyte function can result in
increased fat burning ability and/or improved serum composition
and/or weight loss in the subject.
[0835] In some embodiments, delivery of a chondrisome composition
or preparation described herein may induce or block cellular
differentiation, de-differentiation, or trans-differentiation. The
target mammalian cell may be a precursor cell. Alternatively, the
target mammalian cell may be a differentiated cell, and the cell
fate alteration includes driving de-differentiation into a
pluripotent precursor cell, or blocking such de-differentiation,
such as the dedifferentiation of cancer cells into cancer stem
cells. In situations where a change in cell fate is desired,
effective amounts of a chondrisome preparation described herein
encoding a cell fate inductive molecule or signal is introduced
into a target cell under conditions such that an alteration in cell
fate is induced. In some embodiments, a chondrisome preparation
described herein is useful to reprogram a subpopulation of cells
from a first phenotype to a second phenotype. Such a reprogramming
may be temporary or permanent. Optionally, the reprogramming
induces a target cell to adopt an intermediate phenotype.
[0836] Also provided are methods of reducing cellular
differentiation in a target cell population. For example, a target
cell population containing one or more precursor cell types is
contacted with a a chondrisome composition or preparation described
herein, under conditions such that the preparation reduces the
differentiation of the precursor cell. In certain embodiments, the
target cell population contains injured tissue in a mammalian
subject or tissue affected by a surgical procedure. The precursor
cell is, e.g., a stromal precursor cell, a neural precursor cell,
or a mesenchymal precursor cell.
[0837] A chondrisome composition or preparation described herein,
comprising a cargo or payload, may be used to deliver such payload
(e.g., an agent listed in Table 4) to a cell tissue or subject.
Delivery of a payload by administration of a chondrisome
composition or preparation described herein may modify cellular
protein expression levels. In certain embodiments, the administered
preparation directs up-regulation of (via expression in the cell,
delivery in the cell, or induction within the cell) of one or more
payload (e.g., a polypeptide) that provide a functional activity
which is substantially absent or reduced in the cell in which the
polypeptide is delivered. For example, the missing functional
activity may be enzymatic, structural, or regulatory in nature. In
related embodiments, the administered chondrisome preparation
directs up-regulation of one or more polypeptides that increases
(e.g., synergistically) a functional activity which is present but
substantially deficient in the cell in which the polypeptide is
up-regulated.
[0838] The subject may have a disease or condition described
herein.
Ex-Vivo Applications
[0839] In embodiments, a chondrisome composition or preparation
described herein is delivered ex-vivo to a cell or tissue, e.g., a
human cell or tissue. In embodiments, the composition or
preparation improves function of a cell or tissue ex-vivo, e.g.,
improves cell viability, respiration, or other function (e.g.,
another function described herein).
[0840] In some embodiments, the composition or preparation is
delivered to an ex vivo tissue that is in an injured state (e.g.,
from trauma, disease, hypoxia, ischemia or other damage).
[0841] In some embodiments, the composition or preparation is
delivered to an ex-vivo transplant (e.g., a tissue explant or
tissue for transplantation, e.g., a human vein, a musculoskeletal
graft such as bone or tendon, cornea, skin, heart valves, nerves;
or an isolated or cultured organ, e.g., an organ to be transplanted
into a human, e.g., a human heart, liver, lung, kidney, pancreas,
intestine, thymus, eye). The preparation improves viability,
respiration, or other function of the transplant. The composition
or preparation can be delivered to the tissue or organ before,
during and/or after transplantation.
[0842] In some embodiments, the composition or preparation is
delivered, administered or contacted with a cell, e.g., a cell
preparation. The cell preparation may be a cell therapy preparation
(a cell preparation intended for administration to a human
subject). In embodiments, the cell preparation is comprised of
cells expressing a chimeric antigen receptor (CAR), e.g.,
expressing a recombinant CAR. The cells expressing the CAR may be,
e.g., T cells, Natural Killer (NK) cells, cytotoxic T lymphocytes
(CTL), regulatory T cells. In embodiments, the cell preparation is
a neural stem cell preparation. In embodiments, the cell
preparation is a mesenchymal stem cell (MSC) preparation. In
embodiments, the cell preparation is a hemapoietic stem cell (HSC)
preparation. In embodiments, the cell preparation is an islet cell
preparation.
Therapeutic Use
[0843] The preparations of chondrisomes described herein can be
used to treat a subject, e.g., a human, in need thereof. In such
embodiments, the subject may be at risk, may have a symptom of, or
may be diagnosed with or identified as having, a particular disease
or condition (e.g., a disease or condition described herein).
[0844] In some embodiments, the source mitochondria are from the
same subject that is treated with a chondrisome preparation or
composition. In other embodiments they are different. For example,
the source of mitochondria and recipient tissue may be autologous
(from the same subject) or heterologous (from different subjects).
In either case, the donor tissue for chondrisome compositions or
preparations described herein may be a different tissue type than
the recipient tissue. For example, the donor tissue may be muscular
tissue and the recipient tissue may be connective tissue (e.g.,
adipose tissue). In other embodiments, the donor tissue and
recipient tissue may be of the same or different type, but from
different organ systems.
[0845] Diseases, disorders and conditions that may be treated or
prevented by administering a chondrisome preparation described
herein include those associated with but not limited to targets in
the circulatory system, hepatic system, renal system,
cardio-pulmonary system, central nervous system, musculoskeletal
system, lymphatic system, immune system, sensory nervous systems
(sight, hearing, smell, touch, taste), digestive system, endocrine
systems (including adipose tissue metabolic regulation).
[0846] Mitochondrial Disease
[0847] Diseases, disorders and conditions that may be treated or
prevented by administering a chondrisome preparation described
herein include but are not limited to those associated with
mutations of mitochondrial genes: 2-ketoglutarate dehydrogenase
complex deficiency; Aminoglycoside-Induced Deafness; Ataxia,
Friedreich Ataxia, progressive seizures, mental deterioration, and
hearing loss; Autosomal Recessive Cardiomyopathy; Ophthalmoplegia;
Autosomal recessive peripheral neuropathy (CMT4A); Beta-oxidation
defects; Cerebellar ataxia, cataract and diabetes mellitus; Complex
III deficiency; Complex V deficiency; Chronic progressive external
ophthalmoplegia (CPEO); creatine deficiency syndromes; diabetes
mellitus and deafness (DAD); Exercise intolerance; Hypertrophic
cardiomyopathy; Kearns-Sayre Syndrome; LBSL--leukodystrophy; LCHAD;
Leber's hereditary optic neuropathy; Leigh Disease or Syndrome;
Luft Disease; MCAD; Maternally Inherited Diabetes and Deafness
(MIDD); maternally inherited Leigh's syndrome (MILS); mitochondrial
recessive ataxia syndrome (MIRAS); Mitochondrial encephalomyopathy,
lactic acidosis and stroke-like episodes (MELAS); Myoclonic
Epilepsy with Ragged Red Fibers (MERRF); Mitochondrial
neurogastrointestinal encephalopathy disease (MNGIE); Myopathy and
Diabetes; neuropathy, ataxia, retinitis, pigmentosa, and ptosis
(NARP); Optic Atrophy; Pearson Syndrome; Progressive Myoclonus
Epilepsy; Sensory Ataxia Neuropathy Dysarthia Ophthalmoplegia
(SANDO); Short-chain acyl-CoA dehydrogenase deficiency (SCAD);
Short Chain Hydroxy Acyl-CoA Dehydrogenase Deficiency (SCHAD);
Nonsyndromic Hearing Loss and Deafness; SIDS; Hereditary spastic
paraplegia; VLCAD etc.
[0848] Diseases, disorders and conditions that may be treated or
prevented by administering preparation described herein include but
are not limited to those associated with mutations of nuclear genes
whose products are located or associated with the mitochondria:
alpers Disease; carnitine-acyl-carnitine deficiency;
Charcot-Marie-Tooth Disease (Type 2A)/Autosomal dominant peripheral
neuropathy; Complex I deficiency; Complex II deficiency; Complex IV
deficiency; CPT I Deficiency; CPT II Deficiency; Friedreich's
ataxia; Fumarase Deficiency; MADD/Glutaric Aciduria Type II; Maple
Syrup Urine Disease; Ornithine transcarbamylase deficiency; Rett
Syndrome; Barth Syndrome; Hemochromatosis; Batten Disease;
Lesch-Nyhan Syndrome; Hurler Syndrome; Niemann-Pick Disease;
Gaucher Disease; Glycogen Storage Disease; Zellweger Syndrome;
Wilson's Disease; Menkes Disease; methylmalonic Acidemia;
Huntington Disease.
[0849] Ischemia
[0850] The compositions and methods described herein may reduce the
incidence, extent, and/or severity of ischemia (e.g., ischemic
injury) in a subject who has one or more condition or disorder
described herein.
[0851] Ischemia is the condition of an inadequate oxygen or blood
supply to an organ or tissue. Tissue injury and/or death can occur
as a result of an ischemic insult, and/or subsequent damage may be
induced by reperfusion. During prolonged ischemia, ATP levels and
intracellular pH decrease as a result of anaerobic metabolism and
lactate accumulation. As a consequence, ATPase-dependent ion
transport mechanisms become dysfunctional, contributing to
increased intracellular and mitochondrial calcium levels (calcium
overload), cell swelling and rupture, and cell death by necrotic,
necroptotic, apoptotic, and autophagic mechanisms. Although oxygen
levels are restored upon reperfusion, a surge in the generation of
reactive oxygen species occurs and proinflammatory neutrophils
infiltrate ischemic tissues to exacerbate ischemic injury. The
pathologic events induced by ischemia or reperfusion may
orchestrate the opening of the mitochondrial permeability
transition pore (MPTP). (Kalogeris et al. 2012. Cell Biology of
Ischmia/Reperfusion Injury. Int Rev Cell Mol Biol. 298:229-317).
Ischemia/reperfusion (I/R) occurs, e.g., during hypovolemic shock,
thrombolytic therapy, organ transplantation, coronary angioplasty,
aortic cross-clamping, or cardiopulmonary bypass.
[0852] Ischemia may be caused by any mechanism including a partial
or complete blockage (an obstruction), a narrowing (a
constriction), a leak, a rupture or trauma of one or more blood
vessels that supply blood to an organ or tissue. A subject may
suffer from ischemia of the, e.g., brain, kidney, liver, arteries,
heart, intestines, mesentery, skin, ovary, penis, mesenatry, bile
ducts, extremities/limbs, or eye (e.g., the optic nerve).
[0853] The subject may have myocardial ischemia, cerebral ischemia
(e.g., a transient ischemic attack), intestinal ischemia, hepatic
ischemia, critical limb ischemia, testicular ischemia. A subject
who has ischemia may have a blood clot (e.g., a thrombus or an
embolus), vasculitis, atherosclerosis, coronary artery disease,
peripheral artery disease. In some instances, ischemia is caused by
a myocardial infarction, stroke, peripheral vascular disease. A
subject may have acute injury ischemia, e.g., caused by aortic
dissection, acute kidney injury, acute liver injury, acute lung
injury, myocardial infarction, stroke, spinal cord injury,
traumatic brain injury. Ischemia-induced injury (i.e., disease
and/or damage) includes ischemic myelopathy, ischemic optic
neuropathy, ischemic colitis, coronary heart disease, and/or
cardiac heart disease (e.g., angina, heart attack, etc.), among
others. A subject may have a developmental ischemia, such as Marfan
Syndrome, Mitral Valve Stenosis, Tetralogy of Fallot, Ventricular
Septal Defect.
[0854] Other disorders or conditions that may cause, may be caused
by, or may be associated with, ischemia or I/R include: ischemic
cholangiopathy, ischemic stroke, traumatic brain injury (TBI),
subarachnoid hemorrhage, intracerebral hemorrhage, compartment
syndrome, acute peripheral arterial occlusion, peripheral arterial
disease, acute limb ischemia, frostbite, sixth cranial nerve palsy,
diabetic or hypertensive retinopathy, ischemic optic neuropathy,
retinal artery occlusion, acute coronary syndromes (acs), Takayasu
Arteritis, intussusception, intestinal obstruction, renal
atheroembolism, renal vein thrombosis, renal artery stenosis and
occlusion, acute kidney injury, hepatic artery occlusion,
postoperative hepatitis, ischemic hepatitis, pulmonary embolism,
acute respiratory distress syndrome, pulmonary edema, acute
mesenteric ischemia, adnexal torsion, priapism, preeclampsia and
eclampsia, pressure ulcers, diabetic foot ulcers, burns, arterial
gas embolism, shock, transplants. Methods of the invention can be
performed with subjects having such disorders or conditions.
[0855] The compositions and methods described herein are useful to
effect, in a subject in need thereof, one or more (e.g., 2 or more,
3 or more, 4 or more, 5 or more) of: (a) decreased reactive oxygen
species (ROS) in a target tissue; (b) increased ATP levels in a
target tissue; (c) increased intracellular pH in a target tissue;
(d) decreased intracellular and/or mitochondrial calcium levels in
a target tissue; (e) decreased cell death (e.g., decreased cell
death by necrotic, necroptotic, apoptotic, ferroptotic or
autophagic mechanisms) in a target tissue; (f) blocked or reduced
mitochondrial permeability transition (MPT) in a target tissue; (g)
reduction or clearance of pro-inflammatory neutrophil infiltrate in
a target tissue. In some embodiments, a subject in need thereof is
administered a preparation or composition described herein in an
amount and for a time sufficient such that one or more of (a)-(g)
are modulated in a target tissue of subject by at least 5%, 10%,
15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or more, relative
to a control tissue or subject that has not been treated, or
relative to the same subject prior to administration.
[0856] In some embodiments, a subject having cardiac ischemia can
be administered a chondrisome composition described herein in an
amount and for a time sufficient such that: (a) blood flow is
increased to an ischemic tissue of the subject, (b) infarct size is
reduced in an ischemic tissue of the subject, (c) improved ejection
fraction in the heart. In some embodiments, a subject who has
ischemia is administered a chondrisome composition described herein
in an amount and for a time sufficient such that one or more of
(a)-(c) are modulated in an ischemic tissue of subject by at least
5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or more,
relative to a control tissue or subject that has not been treated,
or relative to the same subject prior to administration.
[0857] A subject who has ischemia may be any person (a human
subject) or animal (an animal subject) that has ischemia, I/R
injury, an ischemia-related condition, a history of ischemia,
and/or a significant chance of developing ischemia after a
treatment begins and during a time period in which the treatment is
still effective. An ischemic subject may be selected for treatment
by any suitable criteria. Exemplary criteria include any detectable
symptom of ischemia, a history of ischemia, an event that increases
the risk of (or induces) ischemia (such as a surgical procedure,
trauma, administration of a medication, etc.). A history of
ischemia may involve one or more prior ischemic episodes. In some
examples, a subject selected for treatment may have had an onset of
ischemia that occurred at least about one, two, or three hours
before treatment begins, or a plurality of ischemic episodes (such
as transient ischemic attacks) that occurred less than about one
day, twelve hours, or six hours prior to initiation of
treatment.
[0858] An ischemic subject may be a human, such as a human patient,
or a non-human animal (such as an agricultural animal, e.g., a cow,
a pig, a sheep, a chicken, a goat; or a companion animal, e.g., a
dog or cat).
[0859] Combination Therapy for Ischemia
[0860] In some embodiments, a preparation described herein may be
administered in combination with a second agent to treat ischemia.
For example, the second agent may be aspirin; a nitrate; a beta
blocker; a calcium channel blocker; a cholesterol-lowering agent; a
steroid; an angiotensin-converting enzyme (ACE) inhibitor;
ranolazine (Ranexa); a fibrinolytic agent, e.g., tissue plasminogen
activator (tPA), streptokinase (SK), or urokinase. In some
embodiments relating to I/R, a preparation described herein may be
administered in combination with ischemic preconditioning, an
antioxidant agent (e.g., superoxide dismutase, catalase, mannitol,
allopurinol, vitamin E, N-acetylcysteine, iron chelating compounds
(e.g., desferrioxamine), angiotensin-converting enzyme inhibitors,
or calcium channel antagonists), an anticomplement agent (e.g., C3
convertase inhibitor, anti-C5 agent such as h5G1.1-scFv), an
antileukocyte agent (e.g., leukocyte depletion/filtration, soluble
interleukin-1 receptor antagonists, anti-TNF antibody, or platelet
activation factor-leukotriene B4 antagonists).
[0861] In some embodiments, a preparation described herein may be
administered in combination with a surgical or other procedure,
e.g., angioplasty, thrombectomy, stenting, embolectomy, coronary
artery bypass surgery or enhanced external counterpulsation.
Metabolic Conditions
[0862] Methods of the invention are useful in subjects in need of
increasing thermogenesis, modulating serum composition (such as
reducing serum cholesterol, or reducing serum triglycerides),
and/or reducing adipocyte or fat volume. Subjects may have a
disease or condition associated with undesirably high fat
composition, such as overweight or obesity (e.g., a subject who has
a BMI >25, >26, >27, >28, >29, >30, >31,
>32, >33, >34, >35; a female subject who has a waist
size greater than 35 in, a male subject who has a waist size
greater than 40 in). A suitable subject may have a high cholesterol
level (e.g., total cholesterol level >150 mg/dL, >160, 170,
180, 190, 200, 220, 240 mg/dL); high low-density-lipoprotein (LDL)
levels (e.g., >80 mg/dL, >90, 100, 120, 140, 160, 180, 190
mg/dL), high triglyceride levels (e.g., >150 mg/dL, >200,
300, 400, 500, 600, 700, 800, 900 mg/dL); high blood pressure
(systolic greater than 135, 140, 145, 150; diastolic greater than
85, 90, 95, 100), or has (or is at risk for) cancer, diabetes, or
heart disease. The subject may have familial
hypercholesterolemia.
[0863] In embodiments, a chondrisome preparation or composition
useful in these methods is one wherein the source mitochondria or
chondrisomes express an (endogenous or exogenous) uncoupling
protein (e.g., UCP-1, UCP-2, UCP-3, UCP-4 or UCP-5).
[0864] The methods of the invention may include treating the
subject with a combination of a preparation or composition
described herein and one or more of: an agent for reducing
cholesterol (e.g., a statin, a nicotinic acid, a fibric acid
derivative, a bile acid sequestrant), an agent for reducing high
blood pressure (e.g., a beta blocker, an ACE inhibitor, an
angiotensin II receptor blocker; a calcium channel blocker, an
alpha blocker, an alpha-2 receptor agonist, a central agonist, a
peripheral adrenergic receptor inhibitor, a vasodilator); an
obesity drug (e.g., a lipase inhibitor, a CNS stimulant, an
anorexiant, a GLP-1 agonist, an antidepressant, a dopamine reuptake
inhibitors, an opioid antagonist).
Other Conditions
[0865] Methods of the invention are useful in subjects who have a
disease, disorder or condition such as: neurodegenerative disorders
(e.g. Alzheimer's disease, Duchenne muscular dystrophy, Parkinson's
Disease, amyotrophic lateral sclerosis (ALS), Huntington's
disease); a disease associated with infectious agents or pathogens
(e.g., bacterial, fungal, viral, parasitic infections); diseases
associated with apoptosis, ferroptosis, necrosis; neoplasms, e.g.,
aberrant growths or cancer; metabolic diseases such as an acquired
metabolic disease (e.g., diabetes, diabetic or hypertensive
retinopathy, NASH/NAFLD, obesity, type 2 diabetes) or a rare
metabolic disease (e.g., carnitine palmitoyltransferase deficiency,
citrullinemia, ornithine transcarbomylase deficiency); disease
associated with toxic proteins; diseases associated with the
accumulation of lipids; clotting and anti-clotting diseases; one or
more of the following: autoimmune disorders (e.g. diabetes, lupus,
multiple sclerosis, psoriasis, rheumatoid arthritis); inflammatory
disorders (e.g. arthritis, pelvic inflammatory disease); infectious
diseases (e.g. viral infections (e.g., HIV, HCV, RSV), bacterial
infections, fungal infections, sepsis); autism; cardiovascular
disorders (e.g. atherosclerosis, hypercholesterolemia, thrombosis,
clotting disorders, angiogenic disorders such as macular
degeneration); proliferative disorders (e.g. cancer, benign
neoplasms); respiratory disorders (e.g. chronic obstructive
pulmonary disease); digestive disorders (e.g. inflammatory bowel
disease, ulcers); musculoskeletal disorders (e.g. fibromyalgia,
arthritis); endocrine, metabolic, and nutritional disorders (e.g.
diabetes, osteoporosis); urological disorders (e.g. renal disease);
psychological disorders (e.g. depression, schizophrenia); skin
disorders (e.g. wounds, eczema); blood and lymphatic disorders
(e.g. anemia, hemophilia); and degenerative diseases (e.g.,
osteoarthritis, sarcopenia, progeria, muscular dystrophy).
Non-Human Applications
[0866] Compositions described herein may also be used to similarly
modulate the cell or tissue function or physiology of a variety of
other organisms including but not limited to: farm or working
animals (horses, cows, pigs, chickens etc.), pet or zoo animals
(cats, dogs, lizards, birds, lions, tigers and bears etc.),
aquaculture animals (fish, crabs, shrimp, oysters etc.), plants
species (trees, crops, ornamentals flowers etc), fermentation
species (saccharomyces etc.). Chondrisome preparations described
herein can be made from such non-human sources and administered to
a non-human target cell or tissue or subject. Chondrisome
preparations can be autologous, allogeneic or xenogeneic to the
target.
Formulation and Methods of Delivery
[0867] A preparation or composition described herein can be
delivered via various routes. Parenteral routes include
intramuscular (IM), subcutaneous (SC) and intravenous (IV),
intramyocardial, intracoronary, intrathecal, epidural,
intraarticular, intradermal, intravitreal, and intranasal routes or
direct injection to fat tissue or the bone marrow. Enteral
administration includes but is not limited to sublingual, buccal,
oral and rectal routes. A preparation or composition described
herein can also be delivered via transdermal delivery or topical
application (applied to surface of any epidermis, skin, mouth, or
GI tract). These routes of administration can be used as a method
of either systemic delivery (e.g. systemic venous/arterial
injection or profusion) or for tissue specific delivery of the
payload via carefully selected points of administration (e.g.
intravitreal or injection or perfusion to tissue with well-defined
and isolated venous, arterial or lymphatic vasculature).
[0868] The compositions may be administered once to the subject or,
alternatively, multiple administrations may be performed over a
period of time. For example, two, three, four, five, or more
administrations may be given to the subject during one treatment or
over a period of time. In some embodiments, six, eight, ten, 12, 15
or 20 or more administrations may be given to the subject during
one treatment or over a period of time as a treatment regimen.
[0869] In some embodiments, administrations may be given as needed,
e.g., for as long as symptoms associated with the disease, disorder
or condition persist. In some embodiments, repeated administrations
may be indicated for the remainder of the subject's life. Treatment
periods may vary and could be, e.g., one day, two days, three days,
one week, two weeks, one month, two months, three months, six
months, a year, or longer.
[0870] In some embodiments, the pharmaceutical composition is
administered by a regimen sufficient to alleviate a symptom of the
disease, disorder or condition.
[0871] In embodiments, a pharmaceutical composition or chondrisome
preparation described herein is formulated for administration to a
human subject. The chondrisome preparation may be formulated in a
physiologically acceptable buffer for both storage and
administration. In some embodiments, the chondrisome preparation is
formulated for storage in a first formulation and formulated for
administration with a second formulation (e.g., just before use).
In some embodiments, the storage formulation may be frozen, and
subsequently thawed and reformulated for administration to a
subject. For example, a storage formulation may contain one or more
of: an osmotic regulator, a sugar, a pH buffer, a salt. A
formulation for administration to a subject may contain one or more
of: an osmotic regulator, a sugar, a pH buffer, a salt, autologous
serum (e.g., 5-50% autologous serum).
[0872] In embodiments of the methods described herein, the
chondrisome composition or preparation is treated with an agent,
and/or administered in combination with an agent, to modulate
subcellular targeting of the administered preparation. In
embodiments, the agent enables endosomal/lysosomal escape and/or
enhances cytosolic or non-lysosomal delivery of the preparation. In
embodiments, the agent is a peptide or protein that enhances
cytosolic or non-lysosomal delivery of the preparation, e.g.,
haemagglutinin, diINF-7, penton base, gp41, gp41/polyethylenimine,
TAT, L2 from Papillomavirus, envelope protein (E) of West Nile
virus, listeriolysin O (LLO), Pneumococcal pneumolysin (PLO),
Streptococcal streptolysin O (SLO), Diphtheria toxin (DT),
Pseudomonas aeruginosa exotoxin A (ETA), Shiga toxin, cholera
toxin, ricin, saporin, gelonin, human calcitonin derived peptide,
fibroblast growth factors receptor (FGFR3), melittin, (R-Ahx-R)(4)
AhxB, glycoprotein H (gpH) from herpes simplex, KALA, GALA, a
synthetic surfactant, penetratin (pAntp), R6-Penetratin with
arginine-residues, EB1, bovine prion protein (bPrPp), Poly
(L-histidine), Sweet Arrow Peptide (SAP). In other embodiments, the
agent is a chemical that enhances cytosolic or non-lysosomal
delivery of the preparation, e.g., polyethylenimine (PEI),
Poly(amidoamine)s (PAAs), poly(propylacrylic acid) (PPAA), ammonium
chloride, chloroquine, methylamine. Other such agents are
described, e.g., in Varkouhi et al. 2011. Endosomal escape pathways
for delivery of biologicals. Journal of Controlled Release 151:
220-228.
[0873] Effective doses of compositions and preparations described
herein will vary depending on the mode of administration and the
nature of the subject to be treated. In some instances,
conventional methods of extrapolating human dosage based on doses
administered to an animal model can be carried out. A unit dose
useful in the methods described herein is between 2 ug/kg to 10
mg/kg, e.g., 2 ug/kg to 200 ug/kg, e.g., 2 ug/kg to 2 mg/kg, e.g.,
2 ug/kg to 5 mg/kg, e.g., 2 ug/kg to 1 mg/kg, e.g., 5 ug/kg to 10
mg/kg, e.g., 5 ug/kg to 1 mg/kg, e.g., 5 ug/kg to 5 mg/kg, e.g., 5
ug/kg to 500 ug/kg, e.g., 5 ug/kg to 50 ug/kg, e.g., 5 ug/kg to 100
ug/kg, e.g., 5 ug/kg to 250 ug/kg.
DEFINITIONS
[0874] A used herein, a "mitochondrion" is an organelle capable of
producing ATP through oxidative phosphorylation as it exists in a
living cell or in its natural state in an organism.
[0875] As used herein, a "chondrisome" is a subcellular apparatus
derived and isolated or purified from the mitochondrial network of
a natural cell or tissue source. A "chondrisome preparation" has
bioactivity (can interact with, or have an effect on, a cell or
tissue) and/or pharmaceutical activity.
[0876] As used herein, "douncing" refers to a method of
mechanically grinding tissue between two surfaces (typically
between a container and a tightly fitting pestle) to obtain
subcellular fractions.
[0877] As used herein, a chondrisome preparation described herein
is "stable" when it maintains a predefined threshold level of its
activity and structure over a defined period of time. In some
embodiments, one or more (2 or more, 3 or more, 4 or more, 5 or
more) structural and/or functional characteristics of a chondrisome
preparation described can be used as defining metrics of stability
for chondrisome preparations described herein. These metrics, whose
assay protocols are outlined herein, are determined subsequent to
preparation and prior to storage (e.g., at 4 C, 0 C, -4 C, -20 C,
-80 C) and following removal from storage. The characteristic of
the preparation should not change by more than 95%, 90%, 85%, 80%,
75%, 60%, 50% (e.g., no more than 40%, 35%, 30%, 25%, 20%, 15%,
10%, 5%) over the course of 1, 2, 5, 8, 12, 24, 36, or 48 hours, 3
days, 7 days, 14 days, 21 days, 30 days, 60 days, 90 days, 4
months, 6 months, 9 months, a year or more of storage. In some
embodiments, the characteristic of the chondrisome preparation
described herein should not have changed by more than 50% (e.g., no
more than 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%) over the course of
1, 2, 5, 8, 12, 24, 36, or 48 hours of storage. In some
embodiments, the characteristic of the chondrisome preparation
described herein should not change by more than 50% (e.g., no more
than 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%) over the course of 1,
2, 5, 8, 12, 24, 36, or 48 hours, 3 days, 7 days, 14 days, 21 days,
30 days, 60 days, 90 days, 4 months, 6 months, 9 months, a year or
more of storage.
[0878] As used herein, a "heterologous function" of a chondrisome
preparation described herein is one or more biological activity
different than the biological activity of the source mitochondria
in its native parental cell. For example, a chondrisome preparation
described herein may have an activity not present in the
mitochondria in their native state, may be in a different metabolic
state than the mitochondria in their native state, or a biological
activity may be present at a higher or lower level than in the
native state.
[0879] As used herein, "locally" or "local administration" means
administration at a particular site of the body intended for a
local effect. Examples of local administration include
epicutaneous, inhalational, intra-articular, intrathecal,
intravaginal, intravitreal, intrauterine, intra-lesional
administration, lymph node administration, intratumoral
administration, administration to a fat tissue or mucous membrane
of the subject, wherein the administration is intended to have a
local effect. Local administration may also include perfusion of
the preparation into a target tissue. For example, a preparation
described herein may be delivered locally to the cardiac tissue
(i.e., myocardium, pericardium, or endocardium) by direct
intracoronary injection, or by standard percutaneous catheter based
methods or by perfusion into the cardiac tissue. In another
example, the preparation is infused into the brain or cerebrospinal
fluid using standard methods. In another example, the preparation
is directly injected into adipose tissue of a subject.
[0880] As used herein, a chondrisome preparation is "pure" or
"purified" when separated from its original cellular source and
substantially free (>50%) of other cellular components. In some
embodiments, the weight of the purified chondrisomes constitutes
>50%, >60%, >70%, >80%, >90%, >95%, >98% of
the combined weight of the chondrisomes and other sub-cellular
fractions (see Hartwig et al., Proteomics, 2009, (9) 13209-3214)).
In some embodiments, the weight of the purified chondrisomes
constitutes between 50%-90%, between 50%-80%, between 60-90%,
between 60-%-80%, between 80-95% of the combined weight of the
chondrisomes and other sub-cellular fractions.
[0881] As used herein, "encapsulated" means surrounded by a
protective structure. For example, a preparation of chondrisomes
described herein may be encapsulated in a synthetic or natural
membrane (e.g., an exosome, a vesicle, a host cell, a platelet) or
another natural or synthetic biocompatible material.
[0882] As used herein, a "combination therapy" or "administered in
combination" means that two (or more) different agents or
treatments are administered to a subject as part of a defined
treatment regimen for a particular disease or condition. The
treatment regimen defines the doses and periodicity of
administration of each agent such that the effects of the separate
agents on the subject overlap. In some embodiments, the delivery of
the two or more agents is simultaneous or concurrent and the agents
may be co-formulated. In other embodiments, the two or more agents
are not co-formulated and are administered in a sequential manner
as part of a prescribed regimen. In some embodiments,
administration of two or more agents or treatments in combination
is such that the reduction in a symptom, or other parameter related
to the disorder is greater than what would be observed with one
agent or treatment delivered alone or in the absence of the other.
The effect of the two treatments can be partially additive, wholly
additive, or greater than additive (e.g., synergistic). Sequential
or substantially simultaneous administration of each therapeutic
agent can be effected by any appropriate route including, but not
limited to, oral routes, intravenous routes, intramuscular routes,
and direct absorption through mucous membrane tissues. The
therapeutic agents can be administered by the same route or by
different routes. For example, a first therapeutic agent of the
combination may be administered by intravenous injection while a
second therapeutic agent of the combination may be administered
orally.
[0883] As used herein, the term "pharmaceutical composition" refers
to a medicinal or pharmaceutical formulation for human therapeutic
use that contains one or more active ingredient as well as one or
more excipients and diluents to enable the active ingredient(s)
suitable for the method of administration. The pharmaceutical
composition of the invention includes pharmaceutically acceptable
components that are compatible with the chondrisomes described
herein. The pharmaceutical composition is typically in aqueous form
for intravenous or subcutaneous administration. In embodiments, a
pharmaceutical composition or pharmaceutical preparation is a
composition or preparation produced under good manufacturing
practices (GMP) conditions, having pharmacological activity or
other direct effect in the mitigation, treatment, or prevention of
disease, and/or a finished dosage form or formulation thereof and
is for human use.
[0884] As used herein, the terms "increasing" and "decreasing"
refer to modulating resulting in, respectively, greater or lesser
amounts, function or activity of a metric relative to a reference.
For example, subsequent to administration of a composition
described herein, a functional output may be increased or decreased
in a subject by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or
more relative to prior to administration or relative to an
untreated subject. Generally, the metric is measured subsequent to
administration at a time that the administration has had the
recited effect, e.g., at least one hour, one week, one month, 3
months, 6 months, after a treatment regimen (e.g., a therapy
described herein) has begun.
[0885] As used herein, "percent identity" between two sequences can
be determined by the BLAST 2.0 algorithm, which is described in
Altschul et al., (1990) J. Mol. Biol. 215:403-410. Software for
performing BLAST analyses is publicly available through the
National Center for Biotechnology Information.
[0886] As used herein, "genetic quality" of a chondrisome
preparation means, for all the loci described in Table 5, the
percent of sequencing reads mapping to the wild type allele.
[0887] "Treatment" and "treating," as used herein, refer to the
medical management of a subject with the intent to improve,
ameliorate, stabilize, prevent or cure a disease, pathological
condition, or disorder. This term includes active treatment
(treatment directed to improve the disease, pathological condition,
or disorder), causal treatment (treatment directed to the cause of
the associated disease, pathological condition, or disorder),
palliative treatment (treatment designed for the relief of
symptoms), preventative treatment (treatment directed to minimizing
or partially or completely inhibiting the development of the
associated disease, pathological condition, or disorder); and
supportive treatment (treatment employed to supplement another
therapy).
[0888] As used herein, the term "autologous" refers to a
preparation derived from the same individual to which the
preparation is administered. "Allogeneic" refers to a preparation
derived from a different animal of the same species. "Xenogeneic"
refers to a preparation derived from an animal of a different
species.
[0889] All references cited herein are hereby incorporated by
reference in their entirety.
[0890] The following examples are provided to further illustrate
some embodiments of the present invention, but are not intended to
limit the scope of the invention; it will be understood by their
exemplary nature that other procedures, methodologies, or
techniques known to those skilled in the art may alternatively be
used.
Examples
[0891] 1 Production of Chondrisome preparation 136 [0892] Example
1-1: production of chondrisome preparations from tissue culture
cells 136 [0893] Example 1-2a: production of chondrisome
preparation from skeletal muscle tissue 136 [0894] Example 1-2b:
production of chondrisome preparations from skeletal muscle tissue
for in vivo delivery 137 [0895] Example 1-3a: production of
chondrisome preparations from blood cells 137 [0896] Example 1-3b:
production of chondrisome preparations from platelet cells 138
[0897] Example 1-4: production of chondrisome preparation from
brown adipose tissue 138 A Structural Characteristics of
chondrisome preparations 139 [0898] Example A-1: average size 139
[0899] Example A-2: polydipersity 139 [0900] Example A-3: outer
membrane integrity 140 [0901] Example A-5: protein content 141
[0902] Example A-6: OXPHOS complex levels 141 [0903] Example A-7:
genomic concentration 143 [0904] Example A-8: yield per cell 145
[0905] Example A-9: yield per unit mass of tissue 146 [0906]
Example A-10: chondrisome count per protein mass 146 [0907] Example
A-11: membrane potential state 147 B Bioenergetic Characteristics
of chondrisome preparations 148 [0908] Example B-1: respiratory
control ratio 148 [0909] Example B-2a: individual respiratory
complex activities (I-IV) 149 [0910] Example B-2b: complex V
respiratory activities 150 [0911] Example B-3: reactive oxygen
species production 151 [0912] Example B-4: enzymatic activity 152
[0913] Example B-5: fatty acid oxidation level 154 [0914] Example
B-6: electron transport chain efficiency 155
C Quality Characteristics 155
[0914] [0915] Example C-1: protein carbonyl level 155 [0916]
Example C-2: lipid content 156 [0917] Example C-3: contaminating
and non-contaminating protein levels 160 [0918] Example C-4:
genetic quality 162 [0919] Example C-5: contaminating nuclear DNA
levels 166 [0920] Example C-6: endotoxin levels 167 E Blood derived
preps 168 [0921] Example E-1: preparation of chondrisome containing
mitoparticles 168 [0922] Example E-2: concentration of chondrisome
containing mitoparticles from platelets 168 [0923] Example E-3:
membrane potential of chondrisome containing mitoparticles 169
[0924] Example E-4: quantification of platelet derived chondrisome
mitoparticle delivery to specific subcellular locations 169
F Biological/Functional Characteristics 172
[0924] [0925] Example F-1: apoptosis induction level 172 [0926]
Example F-2: enhancement of cellular respiration 173 [0927] Example
F-3: subcellular targeting levels 174 [0928] Example F-4: delivery
of a loaded cargo 176 [0929] Example F-5: delivery of an engineered
cargo 177 [0930] Example F-6: chemical modulation of subcellular
chondrisome targeting. 178 [0931] Example F-7: proportion of
delivered chondrisomes maintain an active membrane potential 179
[0932] Example F-8: persistence of delivered chondrisomes 180
[0933] Example F-9: quantification of lipid utilization 181 [0934]
Example F-10: quantification of exogenous protein delivery 182
[0935] Example F-11: increase in uncoupled respiration 183 [0936]
Example F-12: inhibition of MPTP opening following delivery of the
chondrisome preparation 184 [0937] Example F-13: increased Akt
activation 185 [0938] Example F-14: modulation of cellular
nicotinamide adenine dinucleotide pools 186 [0939] Example F-15:
improved functional cardiac metrics 187 [0940] Example F-16:
improved functional cardiac metrics 190 [0941] Example F-17: no
acute immune effect 192 [0942] Example F-18: metabolic stimulation
193 [0943] Example F-19: no adaptive immune effect 194
1 Production of Chondrisome Preparation
Example 1-1: Production of Chondrisome Preparations from Tissue
Culture Cells
[0944] Cell culture (primary or cell lines) were trypsinized with
trypsin-EDTA 0.25%, followed by diluting the cells once
trypsinized. Cells were pelleted by centrifugation at 200 g for 5
min at room temperature. Cells were then resuspended in
phosphate-buffered saline to dilute remaining trypsin and
re-centrifuged to obtain the cell pellet. The cells were then
resuspended in 4-6 mL of MSHE (200 mM mannitol, 70 mM sucrose, 10
mM HEPES, 1 mM EDTA, adjust the pH to 7.4 with KOH)+0.5% BSA buffer
and moved to a Potter Elvehjem homogenizer. Cell samples were
homogenized with a glass Potter Elvehjem homogenizer using a Teflon
pestle operated at 1600 rpm for 30-35 strokes, followed by further
membrane disruption via syringe/needle transfer (sample drawn with
18-gauge needle and expelled with 30-gauge needle one time,
followed by drawing with 18-gauge needle and expelling with
23-gauge needle four times).
[0945] The material was centrifuged at 600 g for 10 min at
4.degree. C. The supernatant was centrifuged again at 600 g for 10
min at 4.degree. C. The supernatant was then distributed into 2 mL
microcentrifuge tubes and centrifuged at 10,000 g for 10 min at
4.degree. C. The resulting chondrisome pellets were resuspended in
2 mL of MSHE+0.5% BSA buffer and re-centrifuged at 10,000 g for 10
min at 4.degree. C. The final chondrisome pellet was resuspended in
100-500 uL MSHE buffer. Tissue and chondrisome solutions, including
buffers, were kept on ice at all times. Final chondrisome
suspensions were kept on ice and used within 3 hours of obtaining
final pellet.
Example 1-2a: Production of Chondrisome Preparation from Skeletal
Muscle Tissue
[0946] Tissue (human or mouse) was obtained by dissection or biopsy
and washed in phosphate-buffered saline twice. Tissue and
chondrisome samples, including buffers, were maintained at 4 C
throughout the process of subcellular apparatus isolation. Solid
tissue was minced into small pieces in 2 ml MSHE (200 mM mannitol,
70 mM sucrose, 10 mM HEPES, 1 mM EDTA, adjust the pH to 7.4 with
KOH)+0.5% BSA buffer (0.2-1 g tissue per 8 mL of MSHE+0.5% BSA
buffer) using scissors if necessary and then homogenized with a
glass Potter Elvehjem homogenizer using Teflon pestle operated at
1600 rpm for 9-12 strokes.
[0947] The material was centrifuged at 600 g for 10 min at
4.degree. C. The supernatant was centrifuged again at 600 g for 10
min at 4.degree. C. The supernatant was then distributed into 2 mL
microcentrifuge tubes and centrifuged at 10,000 g for 10 min at
4.degree. C. The resulting chondrisome pellets were resuspended in
2 mL of MSHE+0.5% BSA buffer and re-centrifuged at 10,000 g for 10
min at 4.degree. C. The final chondrisome pellet was resuspended in
100-500 uL MSHE buffer. Tissue and chondrisome solutions, including
buffers, were kept on ice at all times. Final chondrisome
suspensions were kept on ice and used within 3 hours of obtaining
final pellet.
Example 1-2b: Production of Chondrisome Preparations from Skeletal
Muscle Tissue for in Vivo Delivery
[0948] Tissue was obtained by dissection or biopsy and washed in
phosphate-buffered saline twice. Tissue and chondrisome samples,
including buffers, were maintained at 4 C throughout the process of
subcellular apparatus isolation. Solid tissue was minced into small
pieces in 2 ml MSHE (200 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1
mM EDTA, adjust the pH to 7.4 with KOH)+0.5% BSA buffer using
scissors (0.2-1 g tissue per 8 mL of MSHE+0.5% BSA buffer) if
necessary and then homogenized using source-specific homogenization
protocol. Primary solid skeletal muscle tissue (human or mouse)
samples were homogenized with a glass Potter Elvehjem homogenizer
using Teflon pestle operated at 1600 rpm for 15 strokes.
[0949] The material was centrifuged at 1000 g for 10 min at
4.degree. C. The supernatant was then distributed into 6 2 mL
microcentrifuge tubes and centrifuged at 10,000 g for 10 min at
4.degree. C. The resulting chondrisome pellets were resuspended in
2 mL of MSHE+0.5% BSA buffer and re-centrifuged at 10,000 g for 10
min at 4.degree. C. The final chondrisome pellet was resuspended in
1 ml of delivery buffer (250 mM sucrose, 2 mM KH2PO4, 10 mM MgCl2,
20 mM K-HEPES, 0.5 mM K-EGTA, pH 7.4) and then diluted 10.times. in
the same buffer. Tissue and chondrisome solutions, including
buffers, were kept on ice at all times. Final chondrisome
suspensions were kept on ice and used within 1 hour of obtaining
final pellet.
Example 1-3a: Production of Chondrisome Preparations from Blood
Cells
[0950] Human blood was obtained commercially from ZenBio Inc. Blood
cells were obtained by syringe draw (fluid tissue) and maintained
at 4 C throughout the process of chondrisome preparation. Samples
were centrifuged at 2,500.times.g to pellet cells and the cell
pellet was resuspended in 4-8 mL of MSHE (200 mM mannitol, 70 mM
sucrose, 10 mM HEPES, 1 mM EDTA, adjust the pH to 7.4 with
KOH)+0.5% BSA buffer and then homogenized using source-specific
homogenization protocol. Primary human blood samples were
homogenized with a glass Potter Elvehjem homogenizer using a Teflon
pestle operated at 1600 rpm for 100 strokes, followed by further
membrane disruption via syringe/needle transfer (draw sample with
18-gauge needle and expel with 30-gauge needle one time, followed
by drawing with 18-gauge needle and expelling with 23-gauge needle
four times).
[0951] The material was centrifuged at 600 g for 10 min at
4.degree. C. The supernatant was centrifuged again at 600 g for 10
min at 4.degree. C. The supernatant was then distributed into 2 mL
microcentrifuge tubes and centrifuged at 10,000 g for 10 min at
4.degree. C. The resulting chondrisome pellets were resuspended in
2 mL of MSHE+0.5% BSA buffer and re-centrifuged at 10,000 g for 10
min at 4.degree. C. The final chondrisome pellet was resuspended in
100-500 uL MSHE buffer. Tissue and chondrisome solutions, including
buffers, were kept on ice at all times. Final chondrisome
preparations were kept on ice and used within 3 hours of obtaining
final pellet.
Example 1-3b: Production of Chondrisome Preparations from Platelet
Cells
[0952] Platelets were obtained commercially from ZenBio. Platelets
were obtained by syringe draw (fluid tissue) and maintained at 4 C
throughout the process of subcellular apparatus isolation. Samples
were centrifuged at 2,500.times.g to pellet cells and the cell
pellet was resuspended in 4-8 mL of MSHE (200 mM mannitol, 70 mM
sucrose, 10 mM HEPES, 1 mM EDTA, adjust the pH to 7.4 with
KOH)+0.5% BSA buffer and then homogenized with a glass Potter
Elvehjem homogenizer using a Teflon pestle operated at 1600 rpm for
100 strokes, followed by further membrane disruption via
syringe/needle transfer (draw sample with 18-gauge needle and expel
with 30-gauge needle one time, followed by drawing with 18-gauge
needle and expelling with 23-gauge needle four times).
[0953] The material was centrifuged at 600 g for 10 min at
4.degree. C. The supernatant was centrifuged again at 600 g for 10
min at 4.degree. C. The supernatant was then distributed into 2 mL
microcentrifuge tubes and centrifuged at 10,000 g for 10 min at
4.degree. C. The resulting chondrisome pellets were resuspended in
2 mL of MSHE (200 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM
EDTA, adjust the pH to 7.4 with KOH)+0.5% BSA buffer and
re-centrifuged at 10,000 g for 10 min at 4.degree. C. The final
chondrisome pellet was resuspended in 100-500 uL MSHE buffer.
Tissue and chondrisome solutions, including buffers, were kept on
ice at all times. Final chondrisome preparations were kept on ice
and used within 3 hours of obtaining final pellet.
Example 1-4: Production of Chondrisome Preparation from Brown
Adipose Tissue
[0954] Mice were anesthetized and sacrificed using isoflurane, and
then exsanguination was done by cardiac puncture prior to tissue
dissection. Tissue was obtained by dissection or biopsy and washed
in phosphate-buffered saline twice. Tissue and chondrisome samples,
including buffers, were maintained at 4 C throughout the process of
isolation. Solid tissue was minced into small pieces in 2 ml SHE
(Sucrose (250 mM), HEPES (5 mM), EGTA (2 mM), BSA 2%, PH=7.2 with
KOH)+2% BSA buffer (0.8-1.6 g tissue per 8 mL of SHE+2% BSA buffer)
using scissors if necessary and then homogenized with a glass
Potter Elvehjem homogenizer using Teflon pestle operated manually
(by hand) for 9-12 strokes.
[0955] The material was centrifuged at 2100 rpm for 10 min at
4.degree. C. The supernatant was centrifuged again at 2100 g for 10
min at 4.degree. C. The supernatant was then distributed into 2 mL
microcentrifuge tubes and centrifuged at 9,000 g for 10 min at
4.degree. C. The resulting chondrisome pellets were resuspended in
2 mL of SHE+2% BSA buffer and re-centrifuged at 9,000 g for 10 min
at 4.degree. C. This step was repeated in 2 ml SHE buffer with no
BSA. The final chondrisome pellet was resuspended in 100-500 uL SHE
buffer. Tissue and chondrisome solutions, including buffers, were
kept on ice at all times. Final chondrisome suspensions were kept
on ice and used within 3 hours of obtaining final pellet.
A Structural Characteristics of Chondrisome Preparations
Example A-1: Average Size
[0956] The chondrisome preparations were tested to determine the
average size of particles using the commercially available qNANO
GOLD system. The qNANO GOLD with software version 3.3.2.194 was
used according to manufacturer's instructions with the NP300
nanopore, which is designed to analyze particles within the 115 to
1150 nm size range. Chondrisome samples were diluted in
phosphate-buffered saline (PBS) to a final concentration range of
0.01-0.1 ug protein/mL as outlined in Example A-5. Other instrument
settings were adjusted as indicated in the following table:
TABLE-US-00005 Measurement Parameter Setting Pressure 6 Nanopore
type NP300 Calibration sample CPC400_6P Gold standard analysis no
Capture assistant none
[0957] All chondrisome preparations were analyzed within 2 hours of
isolation. The average size of the chondrisomes in the preparations
tested was 175-950 nm. The minimum size range was 50-360 nm; the
maximum size range was 1500-2300 nm.
Example A-2: Polydipersity
[0958] The chondrisome preparations were tested to determine the
average size of particles using the commercially available qNANO
GOLD system, using the same instrument settings as in Example
A-1.
TABLE-US-00006 D50 D90 (diameter (diameter D10 (diameter in nm in
nm in nm that 10% that 50% of the that 90% of the Source of the
particles particles were particles D90/D10 tissue/cells were below)
below) were below) range All tested 150-375 225-550 575-1100
1.6-4.8 Human 150-350 225-425 575-775 1.6-3.6 platelets Human
175-375 350-550 900-1100 2.8-4.8 fibroblasts
Example A-3: Outer Membrane Integrity
[0959] The chondrisome preparation was tested to verify the extent
of outer membrane intactness following isolation. The integrity of
the outer membrane can be evaluated to the degree by which
respiration increases following provision of reduced cytochrome c,
a 12-kDa protein that traverses compromised outer chondrisome
membranes and donate electrons to cytochrome oxidase of the
electron transport chain, leading to increased oxygen consumption
(i.e., respiration).
[0960] Reduced cytochrome c was prepared using the method described
in Spinazzi et al., Nature Protocols 7(6): 1235-1246, 2012.
Briefly, purified cytochrome c from bovine heart was acquired from
Sigma-Aldrich (C3131) and suspended in 1.2 mL of 10 mM phosphate
buffer. Then, 110 mg of ascorbic acid was dissolved in 1 mL of 10
mM phosphate buffer and adjusted to pH 6.5 using Tris base. Three
hundred microliters of this solution was then added to the
cytochrome c, followed by incubation at 4.degree. C. for one hour.
To remove excess ascorbic acid, the solution was passed through a
PD10 disposable desalting column that had been equilibrated with 50
mL of 10 mM phosphate buffer. After elution with the phosphate
buffer, the cytochrome c redox state was examined using a plate
reader set to monitor absorbance at 550 nm. The presence of a peak
at 550 nm that decayed following addition of a few granules of
potassium ferricyanide (an oxidizing agent) indicated successful
reduction of the purified cytochrome.
[0961] Chondrisomes were isolated from cultured human fibroblasts
(see example 1-1) and protein content was assessed by BCA (example
A-5) and the preparation remains on ice until the following
quantification protocol was initiated (within 20 minutes from
isolation). Isolated chondrisomes (0.125 to 1 mg/mL) were suspended
in 1 mL respiration medium (0.3 mM mannitol, 10 mM KH2PO4, 5 mM
MgCl2, and 10 mM KCl (pH 7.2) in a Clark oxygen electrode chamber
(Hansatech Instruments, Norfolk, United Kingdom) maintained at
37.degree. C. Chondrisomes were allowed to equilibrate in the
respiration buffer prior to addition of respiratory substrates.
[0962] First, the respiratory substrates (e.g. glutamate (5 mM) and
malate (1 mM); succinate (5 mM) rotenone (2 .mu.M)) were added to
stimulate the production of NADH from the tricarboxylic acid cycle
and deliver electrons to the electron transport chain. The
resulting oxygen consumption rate was denoted as State 2
respiration, which was caused by a leak of electrons across the
inner chondrisome membrane and the compensatory increased flux of
electrons down the electron transport chain to maintain membrane
potential equilibrium. Next, adenosine diphosphate (ADP) was added
to a final concentration of 100 .mu.M. In the presence of inorganic
phosphate (10 mM) and the NADH-linked substrates, ADP causes a
burst in respiration (termed State 3) as protons were utilized by
the FoF1-ATPase to generate ATP. Upon consumption of the exogenous
ADP, the chondrisomes returned to basal respiration (also termed
State 4). After reaching this state, the reduced cytochrome c was
added to the chondrisome suspension at a working concentration of
10 .mu.M. Following addition of reduced cytochrome c, there was
<5% increase in oxygen consumption rate over state 4 rate,
indicative of an intact outer chondrisome membrane.
Example A-5: Protein Content
[0963] The chondrisome preparation was tested to determine the
protein concentration using a standard BCA. Here a commercially
available Pierce.TM. BCA Protein Assay Kit (Thermo Fischer
product#23225) was used. As per the manufacturer's instructions, a
standard curve was generated using the supplied BSA, from 0 to 20
ug of BSA per well (in triplicate). The chondrisome preparation was
diluted such that the quantity measured was within the range of the
standards. The chondrisome preparation was analyzed in triplicate
and the mean value was used.
Example A-6: OXPHOS Complex Levels
[0964] An ELISA analysis approach was used to determine the
concentration of the chondrisome oxidative phosphorylation
complexes. Complex I levels were determined using a commercially
available kit (Abcam, ab124539).
[0965] Immediately following generation of the chondrisome
preparation the sample was split to quantify total protein levels
(as outlined in example A-5) and to quantify specific complexes. As
per the manufacturer's instructions all buffers were warmed to room
temperature prior to the initiation of the analysis. Within 2 hours
of isolation, 200 ug of the chondrisome preparation was pelleted
and resuspended in 200 uL of supplied Extraction buffer with
protease inhibitors added (Millipore, 539137). The chondrisome
sample was then serially diluted 1/2 in 1.times. Incubation buffer.
50 ul of the samples dilution or 1.times. Incubation buffer (2
replicates of each) were added to the respective wells of the ELISA
plate. The plate was sealed and incubated for 2 hour at room
temperature on a plate shaker set to 300 rpm. The wells were washed
twice by completely removing the fluid by aspiration and then
dispensing 300 ul of 1.times. Wash buffer into each well. After the
final wash the plate was inverted and blotted with paper to remove
all excess liquid. Solutions of 1.times. Detector antibody provided
with the kit were prepared and 50 ul of the Detector antibody
solution was then added to each well used. The sealed plate was
then incubated for 1 hour at room temperature on a plate shaker set
to 300 rpm. The wells were washed 3 times by aspirating and adding
300 uL 1.times. Wash buffer as performed previously. Solutions of
1.times.HRP label provided with the kit were prepared and 50 ul of
the 1.times.HRP label solution was then added to each well used.
The sealed plate was then incubated for 1 hour at room temperature
on a plate shaker set to 300 rpm. The wells were washed 3 times by
aspirating and adding 300 uL 1.times. Wash buffer as performed
previously. 100 ul of the Tetramethyl benzidine substrate solution
(TMB buffer) was added to each well and the plate was immediately
analyzed on a microplate reader by measuring absorbance at 600 nm
every 1 minute for 30 minutes, with shaking between readings.
[0966] To calculate the complex levels, the OD reading for the
1.times. Incubation buffer only wells was subtracted from all the
readings and then all duplicate readings were averaged for the
30-minute time-point. The chondrisome preparation was analyzed in
duplicate and the mean value appropriately adjusted by the dilution
factor used to determine the chondrisome preparation mOD level of
each complex. Lastly the complex level was normalized to the total
protein in the chondrisome preparation as determined by BCA. With
this assay the chondrisome preparation was shown to have a complex
I level of 36.4 mOD/ug total protein. The approach to determine the
levels of the other complexes was analogous to the protocol
described. Appropriate & specific commercially available kits
and the complex levels obtained from them are outlined in the table
below.
TABLE-US-00007 Human Platelet derived chondrisome preparations
(produced via example 1-3b) Complex Level OXPHOS Determined Complex
(mOD/ug Measured Commercial Kit Type Kit ID total protein) Complex
I Human Complex I abcam, ab178011 5.74 .+-. 1.52 ELISA kit Complex
II Human Succinate abcam, ab124536 1.58 .+-. 0.65 Dehydrogenase
ELISA kit Complex III Human Complex III abcam, ab124537 22.3 .+-.
2.0 ELISA kit Complex V Human Complex V abcam, ab124593 36.4 .+-.
3.5 ELISA kit
TABLE-US-00008 Human Fibroblast derived chondrisome preparations
(produced via example 1-1) Complex Level OXPHOS Determined Complex
(mOD/ug Measured Commercial Kit Type Kit ID total protein) Complex
I Human Complex I abcam, ab178011 2.44 .+-. 0.37 ELISA kit Complex
II Human Succinate abcam, ab124536 0.12 .+-. 0.06 Dehydrogenase
ELISA kit Complex III Human Complex III abcam, ab124537 2.43 .+-.
1.0 ELISA kit Complex V Human Complex V abcam, ab124593 6.61 .+-.
2.4 ELISA kit
Example A-7: Genomic Concentration
[0967] This example describes the determination of the mtDNA
concentration of the chondrisome preparation relative to the total
protein content of the preparation. Chondrisomes are isolated from
human skeletal muscle punch biopsies, platelets, or cultured
fibroblasts using the procedures explained in Examples 1-2a and
1-3b and 1-1 respectively. From these preparations, total DNA is
isolated using a Qiagen DNeasy blood and tissue kit (catalog number
69504), followed by determination of DNA concentration using a
Thermo Scientific NanoDrop. After completion of the DNA isolation
procedure, a DNA standard is generated by producing an amplicon
using the human mtDNA specific primers listed below and same the
reaction protocol and machine as used for the semi-quantitative PCR
described below. The PCR reaction is run on a resolving gel to
confirm mtDNA specific amplification (single band and correct
amplicon size). The PCR product is then cleaned to remove primers
using a standard commercially available kit following the
manufacturers recommendations (Zymo Research: DNA Clean &
Concentrator.TM.-5). The DNA concentration of this cleaned amplicon
is determined using a Thermo Scientific NanoDrop and diluted to
generate a 6-point standard curve over 4 orders of magnitude that
is run during the semi-quantitative qPCR analysis. The standard
curve is converted to total mitochondrial DNA by multiplying the
NanoDrop measured quantity by 60.25 (amplicon is 60.25.times.
smaller than mtDNA genome). A portion of the chondrisome
preparation is devoted to the determination of total protein
content of the chondrisome preparation quantified via BCA (example
A-5).
[0968] RT-PCR is carried out using Applied Biosystems PCR Master
Mix (catalog number 4309155) in a 20 .mu.L total reaction volume
using the following reaction template: [0969] SYBR Green Master
Mix: 10 .mu.L [0970] 0.45 .mu.M Forward Primer: 1 .mu.L [0971] 0.45
.mu.M Reverse Primer: 1 .mu.L [0972] DNA Template: 10 ng [0973]
PCR-Grade Water: Variable
[0974] Forward and reverse primers are produced and acquired by
Integrated DNA Technologies. The table below details the primer
pairs and their associated sequences:
TABLE-US-00009 Target Forward Primer Reverse Primer Sequence
(5'.fwdarw. 3') Sequence (5' 3') Human mtDNA CAC CCA AGA ACA GGG
TTT GT TGG CCA TGG GTA TGT TGT TA Human nDNA TGC TGT CTC CAT GTT
TGA TGT TCT CTG CTC CCC ACC TCT ATC T AAG T
[0975] An Applied Biosystems 7900HT Real-Time PCR system is used to
perform the amplification and detection with the following
protocol:
[0976] Denaturation, 94.degree. C. 2 min
[0977] 40 Cycles of the following sequence: [0978] Denaturation,
94.degree. C. 15 sec [0979] Annealing, Extension, 60.degree. C. 1
min
[0980] The Ct number denotes the cycle threshold for chondrisome
mtDNA and the standard curve determined at a fluorescence level of
0.014179736. The Ct value is used to interpolate the DNA content of
the chondrisome prep. Using this assay the expected ratio of mtDNA
mass to protein mass of the prep is determined and outlined in the
table below:
TABLE-US-00010 Source: mtDNA ug/mg protein Human Fibroblast
0.01-0.1 Human Platelet 0.01-0.05 Human Muscle 0.1-0.2
Example A-8: Yield Per Cell
[0981] The yield of chondrisomes derived from cell suspension
starting material (tissue culture) was determined. Example A-5 has
described the quantification of chondrisome protein content in the
chondrisome preparation. This example defines the ratio between the
starting cell amount and the resulting chondrisome protein yield
(total ug protein) which can be used to characterize the
preparation. Tissue culture produced cells were trypsinized from
the culture flasks, and a 10 uL aliquot of cell solution was
assayed on a Hemacytometer to count cell number with a minimum of
10e6 cells counted total. Using this assay the ranges of
chondrisome yield per cell were are shown in the table below for
each particular source material:
TABLE-US-00011 Yield Preparation production range (ug protein per
10.sup.6 Source cells example cells) Human fibroblasts Example 1-1
10-70
[0982] The yield of chondrisomes derived from certain blood
products was also determined. Example A-5 has described the
quantification of chondrisome protein content in the chondrisome
preparation. This example defines the ratio between the starting
cell amount and the resulting chondrisome protein yield (total ug
protein) which can be used to characterize the preparation. Total
cellular content of human whole blood and human platelet
concentrate was determined using a Drew Scientific Inc. Hemavet
950FS hematology analyzer. Total leukocyte, erythrocyte, and
thrombocyte count was included in whole blood, while only total
thrombocyte count was applicable to the platelet concentrate. Using
this assay the ranges of chondrisome yield per cell were are shown
in the table below for each particular source material:
TABLE-US-00012 Yield Preparation production range (ug protein per
10.sup.6 Source cells example cells) Human whole blood Example 1-3a
0.05-0.25 Human platelets Example 1-3b 0.05-0.25
Example A-9: Yield Per Unit Mass of Tissue
[0983] This assay was used to determined yield of chondrisomes
derived from weight of tissue starting material. Example A-5 has
described the quantification of the protein content of the
chondrisome preparation. Here this example defines the ratio
between the starting dry tissue weight (grams) and the resulting
chondrisome yield (total ug chondrisome protein) which can be used
to characterize the preparation. Performing this assay on the
chondrisome preparation produced from human skeletal muscle tissue
(example 1-2a) the chondrisome yield per g tissue was 745 ug
protein/g of tissue. (Range was 400-1200 ug/g of tissue).
Example A-10: Chondrisome Count Per Protein Mass
[0984] The chondrisome preparation was tested to determine the
concentration of chondrisome particles using the commercially
available qNANO GOLD system. The qNANO GOLD with software version
3.3.2.194 was used according to manufacturer's instructions with
the NP300 nanopore, which is designed to analyze particles within
the 115 to 1150 nm size range. Chondrisome samples were diluted in
phosphate-buffered saline (PBS) to a final concentration range of
0.01-0.1 ug/mL of protein determined by Example A-5. Other
instrument settings were adjusted as indicated in the following
table:
TABLE-US-00013 Measurement Parameter Setting Pressure 6 Nanopore
type NP300 Calibration sample CPC400_6P Gold standard analysis no
Capture assistant none
[0985] All chondrisome preparations were analyzed within 2 hours of
isolation. The determined particle concentration (particles/mL) was
normalized to the total protein content (mg/mL) as assessed by
Example A-5. Particle concentrations are shown in the following
table:
TABLE-US-00014 Particle concentration Production (particles/mg
Source tissue/cells Method total protein) Human platelets Example
1-3b 2.51 .times. 10.sup.10 Human fibroblasts Example 1-1 2.03
.times. 10.sup.10
TABLE-US-00015 Particle concentration range (particles/mg total
Source tissue/cells protein) Human platelets 0.5-20 .times.
10.sup.10 Human fibroblasts 0.5-20 .times. 10.sup.10
Example A-11: Membrane Potential State
[0986] The membrane potential of the chondrisome preparation was
quantified using a commercially available dye TMRE for assessing
chondrisome membrane potential (TMRE tetramethyl rhodamine, ethyl
ester, perchlorate, Abcam, Cat# T669). Chondrisomes were isolated
from mouse skeletal muscle (see example 1-2a) and the preparation
remained on ice until the membrane assessment protocol was
initiated (within 2 hours from isolation). While on ice, total
protein content of the chondrisome preparation was quantified via
BCA (see example A-5). The preparation was diluted in respiration
buffer (250 mM sucrose, 2 mM KH2PO4, 10 mM MgCl2, 20 mM K-HEPES and
0.5 mM K-EGTA, pH 7.4) to a final concentration of 10-100 ug
protein/ml in 6.times.200 .mu.L aliquots (untreated and
FCCP-treated triplicates). Chondrisome respiratory substrates 5 mM
glutamate and 1 mM malate were added to the samples, followed by 30
nM TMRE. For each sample, an unstained (no TMRE) sample was also
prepared in parallel. Chondrisome samples were incubated at room
temperature for 15 minutes. The samples were then analyzed on a BD
FACScan flow cytometer with 488 nm argon laser excitation and
emission was collected at 530+/-30 nm. For FCCP-treated samples, 2
uM FCCP was added to the samples and incubated for 5 minutes prior
to analysis.
[0987] Membrane potential values (in millivolts, mV) were
calculated based on the intensity of TMRE. All events were captured
in the forward and side scatter channels (alternatively, a gate can
be applied to select only the chondrisome population). The
fluorescence intensity (FI) value for both the untreated and
FCCP-treated samples, was normalized by subtracting the geometric
mean of the fluorescence intensity of the unstained sample from the
geometric mean of the untreated and FCCP-treated sample. The
membrane potential state for each preparation was calculated using
the normalized fluorescent intensity values with a modified Nernst
equation (see below) that can be used to determine chondrisome
membrane potential based on TMRE fluorescence (as TMRE accumulates
in chondrisomes in a Nernstian fashion).
[0988] Chondrisome membrane potential
(mV)=-61.5*log(FI.sub.untreated-normalized/FI.sub.FCCP-treated-normalized-
). The membrane potential state of the chondrisome preparation was
determined to be -65 mV (Range was -20 to -150 mV).
B Bioenergetic Characteristics of Chondrisome Preparations
Example B-1: Respiratory Control Ratio
[0989] This example describes the physiological respiration of the
chondrisome preparation. Key functional capabilities of isolated
chondrisomes were analyzed by the classical respiratory control
experiments (Chance and Hollunger. The interaction of energy and
electron transfer reactions in chondrisomes. VI. The efficiency of
the reaction. J Biol Chem 236: 1577-1584, 1961; Chance B and
Williams G R. A simple and rapid assay of oxidative
phosphorylation. Nature 175(4469): 1120-1121, 1955). Chondrisomes
were isolated (see appropriate example for each source material)
and protein content was assessed by BCA (example A-5) and the
preparation remained on ice until the following quantification
protocol was initiated (within 20 minutes from isolation). Isolated
chondrisomes (0.125 to 1 mg protein/mL) were suspended in 1 mL
respiration medium (0.3 mM mannitol, 10 mM KH.sub.2PO.sub.4, 5 mM
MgCl.sub.2, and 10 mM KCl (pH 7.2) in a Clark oxygen electrode
chamber (Hansatech Instruments, Norfolk, United Kingdom) maintained
at 37.degree. C. Chondrisomes were allowed to equilibrate in the
respiration buffer prior to the addition of respiratory
substrates.
[0990] First, the respiratory substrates (e.g. glutamate (5 mM) and
malate (1 mM); succinate (5 mM) rotenone (2 .mu.M)) were added to
stimulate the production of NADH from the tricarboxylic acid cycle
and deliver electrons to the electron transport chain. The
resulting oxygen consumption rate was denoted as State 2
respiration, which was caused by a leak of electrons across the
inner chondrisome membrane and the compensatory increased flux of
electrons down the electron transport chain to maintain membrane
potential equilibrium. Next, adenosine diphosphate (ADP) was added
to a final concentration of 100 .mu.M. In the presence of inorganic
phosphate (10 mM) and the NADH-linked substrates, ADP causes a
burst in respiration (termed State 3) as protons were utilized by
the F.sub.oF.sub.1-ATPase to generate ATP. After ADP-stimulated
respiration rate has plateaued, 5 uM oligomycin was added to
inhibit ATPase and determine levels of respiration not coupled to
ATP synthesis (state 4o). Finally, maximally-stimulated respiration
was induced via addition of a chemical uncoupler, such as carbonyl
cyanide-p-trichloromethoxyphenylhydrazone (FCCP) at a concentration
of 4 .mu.M. The chemical uncoupler bypasses the
F.sub.oF.sub.1-ATPase resistance and provides an indication of
maximal electron flux through the electron transport chain.
Respiratory control ratio 3/2 (RCR 3/2) was calculated by taking
the ratio of State 3 to State 2 oxygen consumption rates.
Respiratory control ratio 3/4o (RCR 3/4o) was calculated by taking
the ratio of State 3 to State 4o oxygen consumption rates. The RCRs
obtained with this assay are indicated in the table below.
TABLE-US-00016 Human Fibroblast derived chondrisome preparations
(produced via example 1-1) Substrate Glutamate/Malate
Succinate/Rotenone RCR RCR 3/2 RCR 3/4o RCR 3/2 RCR 3/4o Ranges 1-4
4-16 1.5-5 5-20
TABLE-US-00017 Human Platelet derived chondrisome preparations
(produced via example 1-3b) Substrate Glutamate/Malate
Succinate/Rotenone RCR RCR 3/2 RCR 3/4o RCR 3/2 RCR 3/4o Ranges 1-4
4-13 1-4 1.5-5
Example B-2a: Individual Respiratory Complex Activities (I-IV)
[0991] This example describes electron transport chain complex
activity of chondrisome preparations (here exemplified for Complex
I) via spectrophotometry. Analogous methods can be used to assess
the other Complex activities using immunocapture procedures and
kinetic absorbance measurements.
[0992] Complex I activity can be measured in chondrisomes isolated
from human tissue, whole blood, or cultured fibroblasts via UV-Vis
spectrophotometry as described in Wibom R et al., Analytical
Biochem 311: 139-151, 2002. Chondrisomes are isolated and protein
content is assessed by BCA (example A-5) and the preparation
remains on ice until the following quantification protocol is
initiated (within 20 minutes from isolation). Briefly, chondrisomes
are solubilized with detergent and probed with a species-specific
antibody to Complex I. The immunocaptured Complex in solution is
then incubated in a reaction mixture containing 50 mM
KH.sub.2PO.sub.4, 5 mM MgCl.sub.2, 5 g/L bovine serum albumin, 0.20
mM KCN, 1.2 mg/L antimycin A, and 0.12 mM coenzyme Q.sub.1. NADH is
added to a final concentration of 0.15 mM, the oxidation of which
can be followed at 340 nm before and after the addition of rotenone
(2 mg/mL). The rotenone-sensitive activity can be calculated using
an extinction coefficient of 6.81 L/mmol/cm. Complex I activity is
then expressed as nmol NADH oxidized/min/mg chondrisome protein.
Expected activity ranges for Complex I and for the other
chondrisome respiratory chain complexes are indicated in the table
below. Product numbers for the corresponding abcam MitoTox.TM.
microplate assays have also been included.
TABLE-US-00018 Human chondrisomes derived from tissue, whole blood,
blood-derived products, or cultured cells. Activity abcam MitoTox
.TM. Microplate (nmol/min/mg Chondrisome Complex Assay total
protein) Complex I ab109903 0.5-10 Complex II ab109904 0.5-5
Complex III ab109905 0.5-10 Complex IV ab109906 0.5-10
Example B-2b: Complex V Respiratory Activities
[0993] ATP Synthase (or Complex V) activity can be assessed in
chondrisomes isolated from whole tissue, blood, or blood-derived
products using the abcam MitoTox.TM. Complex V OXPHOS Activity
Microplate Assay. In the absence of an inner chondrisome membrane
potential, the ATP Synthase operates in the reverse direction,
hydrolyzing ATP to generate ADP in organic phosphate. The ADP
produced can be coupled to the oxidation of NADH via the pyruvate
kinase (PK), lactate dehydrogenase (LDH) enzymatic reactions.
[0994] Briefly, chondrisomes isolated from human tissue, whole
blood, or blood derived products are solubilized in detergent and
placed on ice for 30 min. The chondrisome suspension is centrifuged
at 20,000.times.g for 20 min at 4.degree. C. The resulting
supernatant is used to perform a BCA protein assay, after which it
is pipetted into the microplate coated with monoclonal antibodies
against Complex V. The plate is allowed to incubate for two hours,
consistent with the ELISA protocols outlined in Example A-6. To
quantify the Complex V activity, the 96-well microplate is placed
inside a plate reader set to monitor absorbance at 340 nm at
30.degree. C. every 60 seconds. As mentioned, the activity of ATP
Synthase is coupled to the oxidation of NADH over time (i.e., the
conversion of NADH to NAD.sup.+). The most linear oxidation rate of
NADH occurs between 12-50 minutes of absorbance measurements. Using
the rate of decrease in absorbance at 340 nm and the NADH
extinction coefficient (6.22/mM/cm) and the amount of protein
loaded into each well of the plate, the ATP Synthase activity is
expressed as nmol/min/mg protein.
TABLE-US-00019 Chondrisome preparations derived from human tissue,
whole blood, blood-derived products, or cultured cells. Activity
Chondrisome Complex Antibody Capture Kit (nmol/min/mg protein)
Complex V ab109907 10-200
Example B-3: Reactive Oxygen Species Production
[0995] This example describes the measurement of reactive oxygen
species (ROS) production in the chondrisome preparation.
Chondrisome H.sub.2O.sub.2 production was quantified using a system
containing horseradish peroxidase (HRP) in conjunction with Amplex
Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) (ThermoFisher,
Cat# A22188).
[0996] Chondrisomes generate superoxide radicals at Complexes I,
III, and IV of the electron transport chain. This superoxide
quickly undergoes dismutation by MnSOD to form hydrogen peroxide
(H.sub.2O.sub.2), which diffuses through the outer chondrisome
membrane. In the assay, H.sub.2O.sub.2 was used as a substrate by
HRP, leading to the oxidation of Amplex Red and the generation of a
fluorescent product known as resorufin (Starkov 2010; Gram 2015).
The assay was performed per the manufacturer's instructions,
following a modified version of the protocol for Measuring H2O2
Released from Cells.
[0997] First, 100 uL of reaction mixture of 50 uM Amplex Red
reagent and 0.1 U/mL HRP in MAS buffer (70 mM Sucrose, 220 mM
Mannitol, 5 mM KH2PO4, 5 mM MgCl2, 1 mM EGTA, 0.1% BSA fatty
acid-free, 2 mM HEPES, pH 7.4) with 5 mM glutamate and 5 mM malate
as respiratory substrates was added into the appropriate number of
wells of a 96-well plate for H2O2 standard curve and unknown
samples. An H2O2 standard curve was prepared by adding H2O2 stock
to final concentrations of 0, 1, 5, 10, and 50 uM in the
appropriate wells. Chondrisome samples (in MSHE buffer) were added
at 1, 5, 10, and 50 ug amounts to wells with reaction mixture for
the unknown samples. The plate was then read on a fluorescence
microplate reader with excitation of 545 nm and emission recorded
at 590 nm with 20 nm bandwidths. The plate was initially measured 5
minutes after adding H2O2 standards and unknown samples to record
baseline fluorescence. The plate was then incubated at 37 C for 12
hours and fluorescence was read again. At 12 hours, the nmol of
H2O2 for the unknown chondrisome samples was calculated by
correlating the measured fluorescence values with a linear
trendline fitted to the H2O2 standard curve. The moles of H2O2
production was normalized per ug protein of chondrisomes (example
A-5) and per hour of reaction time.
[0998] With this assay the chondrisome preparation (produced as
described in example 1-1) derived from human fibroblasts was shown
to have a glutamate-malate H2O2 production in the range of 2-12
pmol H2O2/ug chondrisome protein/hr. In this specific experiment a
H2O2 production level of 6.03 pmol H2O2/ug chondrisome protein/hr
was measured.
[0999] With this assay the chondrisome preparation (produced as
described in example 1-3b) derived from human platelets was shown
to have a ROS production level in the range of 0.05-4 pmol H2O2/ug
chondrisome protein/hr. In this specific experiment a H2O2
production level from of 0.40 pmol H2O2/ug chondrisome protein was
measured. [1000] Starkov A A. Measurement of mitochondrial ROS
production. Methods Mol Biol 648: 245-255, 2010. [1001] Gram M et
al. Skeletal muscle mitochondrial H.sub.2O.sub.2 emission increases
with immobilization and decreases after aerobic training in young
and older men. J Physiol 593(17): 4011-4027, 2015.
Example B-4: Enzymatic Activity
[1002] This example describes the measurement of citrate synthase
activity in the chondrisome preparation. Citrate synthase is an
enzyme within the tricarboxylic acid (TCA) cycle that catalyzes the
reaction between oxaloacetate (OAA) and acetyl-CoA to generate
citrate. Upon hydrolysis of acetyl-CoA, there was a release of CoA
with a thiol group (CoA-SH). The thiol group reacts with a chemical
reagent, 5,5-Dithiobis-(2-nitrobenzoic acid) (DTNB), to form
5-thio-2-nitrobenzoic acid (TNB), which was a yellow product that
can be measured spectrophotometrically at 412 nm (Green 2008).
Commercially-available kits, such as the Abcam Human Citrate
Synthase Activity Assay Kit (Product #ab119692) provide all the
necessary reagents to perform this measurement.
[1003] The assay was performed as per the manufacturer's
recommendations. Chondrisome preparation was prepared (see example
1-1 and 1-3b) and protein content was assessed by BCA (example A-5)
and the preparation remains on ice until the following
quantification protocol was initiated (within 120 minutes from
isolation). Briefly, 2-20 ug of chondrisome samples were diluted in
1.times. Incubation buffer (final volume=100 uL) in the provided
microplate wells, with one set of wells receiving only 1.times.
Incubation buffer. The plate was sealed and incubated for 4 hours
at room temperature with shaking at 300 rpm. The buffer was then
aspirated from the wells and 300 uL of 1.times. Wash buffer was
added. This washing step was repeated once more. Then, 100 uL of
1.times. Activity solution was added to each well, and the plate
was analyzed on a microplate reader by measuring absorbance at 412
nm every 20 seconds for 30 minutes, with shaking between readings.
Background values (wells with only 1.times. Incubation buffer) were
subtracted from all wells, and the Citrate Synthase activity is
expressed as the change in absorbance per minute per ug of
chondrisomes sample loaded. Only the linear portion from 100-400
seconds of the kinetic measurement is used to calculate the
activity. The output of this and analogous enzymatic activity
assays are outlined in the table below. [1004] Green H J et al.
Metabolic, enzymatic, and transporter response in human muscle
during three consecutive days of exercise and recovery. Am J
Physiol Regul Integr Comp Physiol 295: R1238-R1250, 2008.
TABLE-US-00020 [1004] Human Fibroblast derived chondrisome
preparations (produced via example 1-1) Enzymatic activity measured
Kit ID # Enzymatic Activity Level Citrate Synthase Abcam, ab119692
1.96 .+-. 0.70 mOD/min/ug total protein Alpha ketoglutarate abcam,
ab185440 1.95 .+-. 0.89 mOD/min/ug total protein dehydrogenase
Pyruvate Sigma-Aldrich 2.55 .+-. 0.15 mOD/min/ug total protein
dehydrogenase Pyruvate Dehydrognease Assay Kit, product #MAK183
Aconitase abcam, ab83459 20.01 .+-. 3.00 mOD/min/ug total protein
Creatine Kinase abcam, ab155901 10.00 .+-. 5.00 mOD/min/ug total
protein (expected value)
TABLE-US-00021 Human Platelet derived chondrisome preparations
(produced via example 1-3b) Enzymatic activity measured Kit ID #
Enzymatic Activity Level Citrate Synthase Abcam, ab119692 0.74 .+-.
0.30 mOD/min/ug total protein Alpha ketoglutarate abcam, ab185440
3.19 .+-. 1.16 mOD/min/ug total protein dehydrogenase Pyruvate
Sigma-Aldrich 4.54 .+-. 0.26 mOD/min/ug total protein dehydrogenase
Pyruvate Dehydrognease Assay Kit, product #MAK183 Aconitase abcam,
ab83459 1.07 .+-. 0.14 mOD/min/ug total protein Creatine Kinase
would abcam, ab155901 10.00 .+-. 5.00 mOD/min/ug total protein
similarly be expected to be:
Example B-5: Fatty Acid Oxidation Level
[1005] This example describes the measurement of fatty oxidation
acid in the chondrisome preparation. Chondrisome preparation was
tested to determine fatty acid oxidation level using respiratory
assay with Palmitoyl carnitine and malate as a substrate. Isolated
chondrisomes (0.125 to 1 mg/mL) were suspended in 0.5 mL
respiration medium (115 mM KCL, 10 mM KH2PO4, 2 mM MgCl2, 5 mM
HEPES, 1 mM EGTA, BSA 0.1% (pH 7.2) plus substrate--Palmitoyl
carnitine (25 uM) plus Malate (1 mM)-. Chondrisomes then will be
loaded as 10 ug and 5 ug per well in a seahorse plate for human
platelet and fibroblast isolated chondrisomes respectively.
Respiration was measured by seahorse instrument maintained at
37.degree. C. Chondrisomes were allowed to equilibrate in the
respiration buffer prior to the addition of respiratory
substrates.
[1006] Palmytolyl carnitine and Malate stimulate the production of
FADH2 and NADH from the Beta oxidation pathway inside the
chondrisomes and deliver electrons to the electron transport chain.
The resulting oxygen consumption rate was denoted as State 2
respiration, which is caused by a leak of electrons across the
inner chondrisome membrane and the compensatory increased flux of
electrons down the electron transport chain to maintain membrane
potential equilibrium. Next, adenosine diphosphate (ADP) was added
to a final concentration of 4 mM. ADP causes a burst in respiration
(termed State 3) as protons were utilized by the FoF1-ATPase to
generate ATP. The increased respiration continues until all
exogenous ADP has been consumed, after which the chondrisome
respiration returns to a basal rate (defined as State 4). State 4
can also be artificially induced by adding Oligomycin (ATP synthase
inhibitor). Finally, maximal fatty acid oxidation level of
respiration was induced via addition of a chemical uncoupler, here
using carbonyl cyanide-p-trichloromethoxyphenylhydrazone (FCCP) at
a concentration of 4 .mu.M. The chemical uncoupler bypasses the
FoF1-ATPase resistance and provides an indication of maximal
electron flux through the electron transport chain. The rate of
respiration with the chondrisome preparation under palmitoyl
carnitine as a substrate indicates the capacity of chondrisomes to
oxidize fatty acid.
[1007] With this assay the chondrisome preparations derived from
human platelet and fibroblast (produced as described in examples
1-3b and 1-1 respectively) were shown to have a maximal fatty acid
oxidation level of 2.6+/-0.4 and 22.7+/-2.7 (pmolO2/min/ug
chondrisome protein) respectively. The state 3/state 2 respiratory
control ratio (RCR 3/2) calculated values were as 3.4+/-2.6 and
4.8+/-0.9 for human platelet and fibroblast isolated chondrisomes
respectively.
Example B-6: Electron Transport Chain Efficiency
[1008] This example describes the measurement of chondrisome
electron transport chain (ETC) efficiency, also referred to as ETC
conductance. Isolated chondrisomes (0.125 to 1 mg/mL) are suspended
in 1 mL respiration medium (0.3 mM mannitol, 10 mM KH2PO4, 5 mM
MgCl2, and 10 mM KCl (pH 7.2) in a Clark oxygen electrode chamber
(Hansatech Instruments, Norfolk, United Kingdom) maintained at
37.degree. C. Chondrisomes are allowed to equilibrate in the
respiration buffer prior to the addition of glutamate (10 mM) and
malate (1 mM).
[1009] Steady-state, intermediate oxygen consumption rates are
attained using a creatine kinase energetic clamp as described by
Glancy et al. Effect of Calcium on the Oxidative Phosphorylation
Cascade in Skeletal Muscle Mitochondria. Biochemistry 52(16):
2793-2809, 2013. Briefly, in the presence of a larger creatine
pool, excess creatine kinase (CK), a known concentration of ATP,
and the CK equilibrium constant, the free energy of ATP hydrolysis
(i.e., the reverse driving force of chondrisome respiration) can be
calculated after adding incremental amounts of phosphocreatine:
.DELTA.G.sub.ATP=.DELTA.G.degree..sub.ATP-2.3*RT
log([PCr]*K.sub.CK/([Cr]*[P.sub.i])),
where .DELTA.G.degree..sub.ATP is the standard .DELTA.G.sub.ATP
(-7.592 kcal/mol), R is the gas constant (1.987 calK-1mol-1), and T
is temperature (310 K). Chondrisomes in the presence of 10 mM
glutamate and 1 mM malate are provided 2.5 mM phosphocreatine
(PCr), 5 mM creatine (Cr), 5 mM ATP, and 75 U/mL CK. Subsequent
additions of PCr (to 3.75, 5, 7.5, and 10 mM) are made to slow
chondrisome respiration. Upon completion of the respiration, the
respiratory rates are plotted as a function of the reverse driving
force, .DELTA.G.sub.ATP. The resulting scatter plot is then fitted
with a linear regression, the slope of the line indicating the
conductance (L) of the chondrisome preparation, having a slope of
.DELTA.J.sub.o (in nmol O.sub.2/min/mg protein) divided by
.DELTA.G.sub.ATP (in kcal/mol). The chondrisome preparations are
expected to have 10-400 nmol O.sub.2/min/mg
protein/.DELTA.G.sub.ATP (in kcal/mol).
C Quality Characteristics
Example C-1: Protein Carbonyl Level
[1010] This example describes the quantification of chondrisome
protein carbonyls, which are formed via reactive oxygen
species-(ROS) induced protein damage. The commercially-available
kit from Abcam, Protein Carbonyl Content Assay Kit (Product
#ab126287) provides all the necessary reagents to perform this
measurement. During the assay, the carbonyl groups are chemically
derivatized with 2,4-dinitrophenylhydrazine (DNPH), resulting in
stable hydrazine adducts that are detected via UV-Vis
spectrophotometry at 375 nm.
[1011] Isolated chondrisomes were suspended in distilled H.sub.2O
buffer at a concentration of 51, 2, or 4 .mu.g/.mu.L in a total
volume of 100 .mu.L per microcentrifuge tube. Since nucleic acids
interfere with the assay, 1% streptozocin was added to each sample
and incubated at room temperature for 15 minutes to degrade nucleic
acids. Then, 100 .mu.L of DNPH was added to each sample and was
incubated at room temperature for 10 minutes. Next, 30 .mu.L of
100% trichloroacetic acid (TCA) was added to each well and the
plate was incubated on ice for 5 min. The samples were centrifuged
at 13,000.times.g for 2 minutes, after which the supernatant was
carefully removed. Ice-cold acetone (500 .mu.L) was added to the
pellets and the samples were placed in a sonicating bath for 2
minutes. The samples were then incubated for 5 min at -20.degree.
C. and centrifuged at 13,000.times.g. The acetone was removed from
the pellet, followed by addition of 200 .mu.L of 6 M guanidine to
resolubilize the proteins. Then, 100 .mu.L of each sample was
transferred to a well (in duplicates) of 96-well plate. Lastly, the
plate was read in a spectrophotometer with the detection wavelength
set to 375 nm.
[1012] With this assay the chondrisome preparation (produced as
described in example 1-3b) derived from human platelets was shown
to have a protein carbonyl level in the range of 5-40 nmol
carbonyl/mg chondrisome protein. In this specific experiment 19.7
nmol carbonyl/mg chondrisome protein was measured. Chondrisome
preparations described herein are generally expected to have a
protein carbonyl level less than 100 nmol carbonyl/mg chondrisome
protein.
Example C-2: Lipid Content
[1013] Lipid Extraction:
[1014] Mass spectrometry-based lipid analysis was performed at
Lipotype GmbH (Dresden, Germany) as described (1). Lipids were
extracted using a two-step chloroform/methanol procedure (2).
Samples were spiked with internal lipid standard mixture
containing: cardiolipin 16:1/15:0/15:0/15:0 (CL), ceramide 18:1;
2/17:0 (Cer), diacylglycerol 17:0/17:0 (DAG), hexosylceramide 18:1;
2/12:0 (HexCer), lysophosphatidate 17:0 (LPA),
lyso-phosphatidylcholine 12:0 (LPC), lyso-phosphatidylethanolamine
17:1 (LPE), lyso-phosphatidylglycerol 17:1 (LPG),
lyso-phosphatidylinositol 17:1 (LPI), lyso-phosphatidylserine 17:1
(LPS), phosphatidate 17:0/17:0 (PA), phosphatidylcholine 17:0/17:0
(PC), phosphatidylethanolamine 17:0/17:0 (PE), phosphatidylglycerol
17:0/17:0 (PG), phosphatidylinositol 16:0/16:0 (PI),
phosphatidylserine 17:0/17:0 (PS), cholesterol ester 20:0 (CE),
sphingomyelin 18:1; 2/12:0; 0 (SM) and triacylglycerol
17:0/17:0/17:0 (TAG). After extraction, the organic phase was
transferred to an infusion plate and dried in a speed vacuum
concentrator. 1st step dry extract was re-suspended in 7.5 mM
ammonium acetate in chloroform/methanol/propanol (1:2:4, V:V:V) and
2nd step dry extract in 33% ethanol solution of methylamine in
chloroform/methanol (0.003:5:1; V:V:V). All liquid handling steps
were performed using Hamilton Robotics STARlet robotic platform
with the Anti Droplet Control feature for organic solvents
pipetting.
[1015] MS Data Acquisition:
[1016] Samples were analyzed by direct infusion on a QExactive mass
spectrometer (Thermo Scientific) equipped with a TriVersa NanoMate
ion source (Advion Biosciences). Samples were analyzed in both
positive and negative ion modes with a resolution of
Rm/z=200=280000 for MS and Rm/z=200=17500 for MSMS experiments, in
a single acquisition. MSMS was triggered by an inclusion list
encompassing corresponding MS mass ranges scanned in 1 Da
increments (3). Both MS and MSMS data were combined to monitor CE,
DAG and TAG ions as ammonium adducts; PC, PC O-, as acetate
adducts; and CL, PA, PE, PE O-, PG, PI and PS as deprotonated
anions. MS only was used to monitor LPA, LPE, LPE O-, LPI and LPS
as deprotonated anions; Cer, HexCer, SM, LPC and LPC O- as
acetate.
[1017] Data Analysis and Post-Processing:
[1018] Data were analyzed with in-house developed lipid
identification software based on LipidXplorer as described in the
following references (4,5). Only lipid identifications with a
signal-to-noise ratio >5, and a signal intensity 5-fold higher
than in corresponding blank samples were considered for further
data analysis. [1019] Sampaio J L, Gerl M J, Klose C, Ejsing C S,
Beug H, Simons K, et al. Membrane lipidome of an epithelial cell
line. Proc Natl Acad Sci USA. 2011 Feb. 1; 108(5):1903-7. [1020]
Ejsing C S, Sampaio J L, Surendranath V, Duchoslav E, Ekroos K,
Klemm R W, et al. Global analysis of the yeast lipidome by
quantitative shotgun mass spectrometry. Proc Natl Acad Sci USA.
2009 Mar. 17; 106(7):2136-41. [1021] Surma M A, Herzog R, Vasilj A,
Klose C, Christinat N, Morin-Rivron D, et al. An automated shotgun
lipidomics platform for high throughput, comprehensive, and
quantitative analysis of blood plasma intact lipids. Eur J lipid
Sci Technol. 2015 October; 117(10):1540-9. [1022] Herzog R,
Schwudke D, Schuhmann K, Sampaio J L, Bornstein S R, Schroeder M,
et al. A novel informatics concept for high-throughput shotgun
lipidomics based on the molecular fragmentation query language.
Genome Biol. 2011 Jan. 19; 12(1):R8. [1023] Herzog R, Schuhmann K,
Schwudke D, Sampaio J L, Bornstein S R, Schroeder M, et al.
LipidXplorer: a software for consensual cross-platform lipidomics.
PLoS One. 2012 January; 7(1):e29851.
[1024] Using this assay the chondrisome preparations derived from
human skeletal muscle (sourced from ReproCELL USA), human blood
(sourced from ZenBio; SER-WB-SDS) and human fibroblast (sourced
from National Disease Research Interchange) as described in
examples 1-2a, 1-3a and 1-1 were analyzed for lipid contents. The
protein content for these samples were also determined as detailed
in example A-5 to be able to compare the lipid levels to protein
content.
[1025] First general evaluation the lipid content of the
preparations was determined. Total lipid content was calculated as
the sum of the molar content of all lipids identified normalized to
the protein content of the preparation. The degree of unsaturation
of the lipids in the preparation was also determined by calculating
the molar content of double bonds in found in the fatty acids of
the lipids as a proportion of the total molar content of lipids
(note that this quantity can be greater than 1 as lipids have more
than 1 fatty acid chain which can each have more than 1 double
bonds in the fatty acid backbone). This degree of unsaturation is
expressed as "double bonds/total lipids". Lastly the proportion of
two major classes of lipids was determined as a % of total lipids
on a molar basis. These two classes were the phospholipids and the
phosphosphingolipids. All four of the quantifications are measured
in the table below for general chondrisome preparations and for
preparations from specific tissue sources.
TABLE-US-00022 General Chondrisome Metric Unit Preparation Blood
Fibroblast Muscle total lipid pmol/mg 571382 .+-. 174496 216102
.+-. 66155 884050 .+-. 73422 613995 .+-. 37901 double bonds/
pmol/pmol 2.86 .+-. 0.22 3 .+-. 0.03 2.6 .+-. 0.01 3 .+-. 0.027
total lipid phospholipid/ 100 * pmol/ 84.46 .+-. 18.96 95.7 .+-.
0.37 97.7 .+-. 0.07 60 .+-. 0.17 total lipid pmol
phosphosphingolipid/ 100 * pmol/ 5 .+-. 4.2 9.3 .+-. 0.17 4.6 .+-.
0.11 1 .+-. 0.012 total lipid pmol
[1026] The lipid content of relevant lipid classes was determined
as a percentage of total lipids on a molar basis. This was
calculated for general chondrisome preparations as detailed in the
table below.
TABLE-US-00023 Lipid Class (100 * pmol lipid class/pmol total
lipid) Quantity Ceramide (Cer) content 0.06-0.58 Cardiolipin (CL)
content 0.6-3.91 Lyso-Phosphatidylcholine (LPC) content 0.05-0.47
Lyso-Phosphatidylethanolamine (LPE) content 0.03-0.14
Phosphatidylcholine (PC) content 25.97-56.7
Phosphatidylcholine-ether (PC O-) content 1.5-6.65
Phosphatidylethanolamine (PE) content 7.4-16.55
Phosphatidylethanolamine-ether (PE O-) content 8.72-14.97
Phosphatidylinositol (PI) content 4.1-5.36 Phosphatidylserine (PS)
content 0.84-10.64 Sphingomyelin (SM) content 1.03-9.43
Triacylglycerol (TAG) content 0.25-38.9
[1027] Eicosanoids (e.g. prostaglandins, thromboxanes and
leukotrienes) are potent cell signaling molecules that are produce
by the metabolism of arachidonic acid. The source of this
arachidonic acid for cells is the breakdown and removal of this
fatty acid from phosphatidylethanolamine (PE) and/or
phosphatidylcholine (PC). When these lipids are partially
hydrolysed and stripped of one of the polyunsaturated arachidonic
acid chains they become LPE and/or LPC respectively. The molar
ratio of these two lipid metabolites is indicative of cellular
arachidonic synthesis capability and this value was calculated and
is displayed in the table below.
TABLE-US-00024 general chondrisome metric preparation blood
fibroblast muscle PE:LPE 151.32 .+-. 88.8 .+-. 0.64 145.9 .+-.
25.28 219.2 .+-. 13.125 55.8 PC:LPC 316.62 .+-. 103.9 .+-. 20.5
343.1 .+-. 35.68 502.8 .+-. 6.92 103.88
[1028] Lipid fatty acid chain length and degree of unsaturation
play roles in the membrane fluidity, preferred membrane structural
state and overall membrane morphology and fission/fusion
capabilities. For these reasons the proportion of long and
unsaturated fatty acids associated with PEs and PCs as a molar
proportion for each respective class was calculated. Specifically,
fatty acid chain lengths of 18 carbons with >0 double bonds and
20 carbons with 4 double bonds (arachidonic acid) were considered
and their quantification is displayed in the table below.
TABLE-US-00025 general chondrisome metric unit preparation blood
fibroblast muscle PE 18:n (n > 0) pmol AA/pmol lipid 0.05 .+-.
0.03 0.04 .+-. 0.001 0.09 .+-. 0 0.04 .+-. 0.095 content class PE
20:4 content pmol AA/pmol lipid 0.05 .+-. 0.03 0.08 .+-. 0.004 0.03
.+-. 0 0.04 .+-. 0.034 class PC 18:n (n > 0) pmol AA/pmol lipid
0.31 .+-. 0.04 0.3 .+-. 0.02 0.3 .+-. 0.01 0.3 .+-. 0.005 content
class PC 20:4 content pmol AA/pmol lipid 0.06 .+-. 0.04 0.1 .+-.
0.005 0.1 .+-. 0.005 0.013 .+-. 0.0002 class
Example C-3: Contaminating and Non-Contaminating Protein Levels
[1029] This assay was performed to determine the proteomics makeup
of the sample preparation and to determine the proportion of
proteins that come from non-mitochondrial sources and are thus
labeled contaminants. Chondrisome preparations were generated from
human skeletal muscle (sourced from ReproCELL USA), human blood
(sourced from ZenBio; SER-WB-SDS), human platelets (sourced from
ZenBio; SER-PRP-SDS) and human fibroblast (sourced from National
Disease Research Interchange) as described in examples 1-2a, 1-3a,
1-3b and 1-1 respectively without final buffer resuspension.
Rather, the chondrisome pellet was frozen for shipment to the
proteomics analysis center.
[1030] The chondrisome samples were then thawed for protein
extraction and analysis. First they were resuspended in 200 .mu.l
of lysis buffer (7M Urea, 2M Thiourea, 4% (w/v) Chaps in 50 mM Tris
pH 8.0) and incubated for 15 minutes at room temperature with
occasional vortexing. Mixtures were then lysed by sonication for 5
minutes in an ice bath and spun down for 5 minutes at 13000 RPM.
Protein content was determined by Pierce 660 colorimetric assay and
100 .mu.g protein of each sample was transferred to a new tube and
the volume was adjusted to 100 .mu.l with 50 mM Tris pH 8. Proteins
were reduced for 15 minutes at 65 Celsius with 10 mM DTT and
alkylated with 15 mM iodoacetamide for 30 minutes at room
temperature in the dark. Proteins were precipitated with gradual
addition of 6 volumes of cold (-20 Celsius) acetone and incubated
over night at -80 Celsius. Protein pellets were washed 3 times with
cold (-20 Celsius) methanol. Proteins were resuspended in 100 .mu.l
50 mM Tris pH 8. 3.33 .mu.g of Trypsin/LysC was added to the
proteins for a first 4 h of digestion at 37 Celsius with agitation.
Samples were diluted to 1 ml with 50 mM Tris pH 8 and 0.1% sodium
deoxycholate was added with another 3.3 .mu.g of Trypsin/LysC for
digestion over night at 37 Celsius with agitation. Digestion was
stopped and sodium deoxycholate was removed by the addition of 2%
v/v formic acid. Samples were vortexed and cleared by
centrifugation for 1 minute at 13 000 RPM. Peptides were purified
by reversed phase solid phase extraction (SPE) and dried down.
Samples were reconstituted in 20 .mu.l of 3% DMSO, 0.2% formic acid
in water and analyzed by LC-MS. To have quantitative measurements a
protein standard was also run on the instrument. Standard peptides
(Pierce.TM. 6 Protein Digest, equimolar, LC-MS grade, #88342) were
diluted to 4, 8, 20, 40 and 100 fmol/ul and were analyzed by
LC-MS/MS. The average AUC (area under the curve) of the 5 best
peptides per protein (3 MS/MS transition/peptide) was calculated
for each concentration to generate a standard curve.
[1031] Acquisition was performed with a ABSciex TripleTOF 5600
(ABSciex, Foster City, Calif., USA) equipped with an electrospray
interface with a 25 .mu.m iD capillary and coupled to an Eksigent
.mu.UHPLC (Eksigent, Redwood City, Calif., USA). Analyst TF 1.6
software was used to control the instrument and for data processing
and acquisition. The source voltage was set to 5.2 kV and
maintained at 225.degree. C., curtain gas was set at 27 psi, gas
one at 12 psi and gas two at 10 psi. Acquisition was performed in
Information Dependant Acquisition (IDA) mode for the protein
database and in SWATH acquisition mode for the samples. Separation
was performed on a reversed phase HALO C18-ES column 0.3 .mu.m
i.d., 2.7 .mu.m particles, 150 mm long (Advance Materials
Technology, Wilmington, Del.) which was maintained at 60.degree. C.
Samples were injected by loop overfilling into a 5 .mu.L loop. For
the 120 minute (samples) LC gradient, the mobile phase consisted of
the following solvent A (0.2% v/v formic acid and 3% DMSO v/v in
water) and solvent B (0.2% v/v formic acid and 3% DMSO in EtOH) at
a flow rate of 3 .mu.L/min.
[1032] For the absolute quantification of the proteins, we
generated a standard curve (5 points, R2>0.99) using the sum of
the AUC of the 5 best peptides (3 MS/MS ion per peptide) per
protein. To generate a database for the analysis of the samples, we
ran the DIAUmpire algorithm on each of the 12 samples and combined
the output MGF files into one database. This database was used in
the Peakview software (ABSciex) to quantify the proteins in each of
the samples, using 5 transition/peptide and 5 peptide/protein
maximum. A peptide was considered as adequately measured if the
score computed by Peakview was superior to 1.5 or had a FDR <1%.
The sum of the AUC of each of the adequately measured peptide was
mapped on the standard curve, and is reported as fmol.
[1033] The resulting protein quantification data was then analyzed
to determine protein levels and proportions of know classes of
proteins as follows: enzymes were identified as proteins that were
annotated with an Enzyme Commission (EC) number; ER associated
proteins were identified as proteins that had a Gene Ontology (GO;
http://www.geneontology.org) cellular compartment classification of
ER and not mitochondria; exosome associated proteins were
identified as proteins that had a Gene Ontology cellular
compartment classification of exosomes and not mitochondria;
MitoCarta proteins were identified as proteins that were identified
as mitochondrial in the MitoCarta database (Calvo et al., NAR 2015l
doi:10.1093/nar/gkv1003); and lastly the mtDNA encoded protein
content was determined from the 4 mtDNA encoded proteins that were
observed in the data (MT-CO2, MT-ATP6, MT-ND5, and MT-ND6). The
molar ratios of each of these categories were determined as the sum
of the molar quantities of all the proteins in each class divided
by the sum of the molar quantities of all identified proteins in
each sample.
TABLE-US-00026 General Chondrisome Category Unit Prep Muscle Blood
Platelet Fibroblast Enzyme mol/mol 0.13-0.33 0.324 .+-. 0.008 0.192
.+-. 0.006 0.137 .+-. 0.005 0.286 .+-. 0.004 ER mol/mol 0.01-0.17
0.007 .+-. 0.0004 0.02 .+-. 0.002 0.037 .+-. 0.002 0.163 .+-. 0.009
Exosome mol/mol 0.11-0.58 0.124 .+-. 0.009 0.547 .+-. 0.03 0.46
.+-. 0.004 0.346 .+-. 0.007 Mitocarta mol/mol 0.04-0.62 0.6 .+-.
0.03 0.056 .+-. 0.02 0.09 .+-. 0.002 0.312 .+-. 0.008 mtDNA mol/mol
0.001-0.04 0.027 .+-. 0.01 0.001 .+-. 0.0002 0.002 .+-. 0.0001
0.002 .+-. 0.0008 encoded
Example C-4: Genetic Quality
[1034] This assay was performed to determine the substantial
presence of known disease causing chondrisome mutations. This was
done by generating a barcoded next generation sequencing library
using commercially available kits and protocols (BiooScientific
NEXTflex.TM. mtDNA-Seq Kit for Illumina Sequencing). Human
fibroblasts were grown and chondrisomes were isolated as described
in example 1-1. The sequencing library was prepared with the
appropriate amplification ends for the next generation sequencing
instrument to be used. Critically the average depth of coverage
that as achieved over the entire chondrisome genome must be greater
than 200.times. and at no single location should coverage drop
below 100.times.. The output reads were filtered for quality where
sequences with >10 consecutive nucleotides with Q<20 were
eliminated. The disease associated mtDNA content of the chondrisome
preparation was assessed by determining the proportion of reads
that contained the wild type sequence at sites of confirmed disease
associated tRNA, rRNA or protein coding mutation sites. These
disease associations are based on Mitomap (MITOMAP: A Human
Mitochondrial Genome Database. http://www.mitomap.org, 2016)
determination of confirmed mutations and their position is based on
the Revised Cambridge Reference Sequence (rCRS; GenBank accession
number NC_012920). Using this assay the chondrisome preparation was
determined to be substantially clear of known disease causing
mutations with >90% of reads mapping to the WT sequence at all
sites indicated in Table 5.
TABLE-US-00027 TABLE 5 Disease causing mutations. Position Locus
Disease WT Mutant tRNA and rRNA mutations 583 MT-TF MELAS/MM &
EXIT G A 1494 MT-RNR1 DEAF C T 1555 MT-RNR1 DEAF A G 1606 MT-TV
AMDF G A 1644 MT-TV HCM + MELAS G A 3243 MT-TL1
MELAS/LS/DMDF/MIDD/SNHL/CPEO/MM/ A G FSGS/ASD/Cardiac + multi-organ
dysfunction 3256 MT-TL1 MELAS C T 3260 MT-TL1 MMC/MELAS A G 3271
MT-TL1 MELAS/DM T C 3291 MT-TL1 MELAS/Myopathy/Deafness + Cognitive
Impairment T C 3302 MT-TL1 MM A G 3303 MT-TL1 MMC C T 4298 MT-TI
CPEO/MS G A 4300 MT-TI MICM A G 4308 MT-TI CPEO G A 4332 MT-TQ
Encephalopathy/MELAS G A 5537 MT-TW Leigh Syndrome A AT 5650 MT-TA
Myopathy G A 5703 MT-TN CPEO/MM G A 7445 MT-TS1 SNHL A G precursor
7471 MT-TS1 PEM/AMDF/Motor neuron disease-like C CC 7497 MT-TS1
MM/EXIT G A 7511 MT-TS1 SNHL T C 8344 MT-TK MERRF; Other -
LD/Depressive mood disorder/ A G leukoencephalopathy/HiCM 8356
MT-TK MERRF T C 8363 MT-TK MICM + DEAF/MERRF/Autism/LS/ G A Ataxia
+ Lipomas 10010 MT-TG PEM T C 12147 MT-TH
MERRF-MELAS/Enchephalopathy G A 12315 MT-TL2 CPEO/KSS G A 14674
MT-TE Reversible COX deficiency myopathy T C 14709 MT-TE MM +
DMDF/Encephalomyopathy/ T C Dementia + diabetes + ophthalmoplegia
Protein coding gene mutations 3376 MT-ND1 LHON MELAS overlap G A
3460 MT-ND1 LHON G A 3635 MT-ND1 LHON G A 3697 MT-ND1 MELAS/LS/LDYT
G A 3700 MT-ND1 LHON G A 3733 MT-ND1 LHON G A 3890 MT-ND1
Progressive encephalomyopathy/LS/optic atrophy G A 4171 MT-ND1 LHON
C A 7445 MT-CO1 SNHL A G 8528 MT-ATP8/6 Infantile cardiomyopathy T
C 8993 MT-ATP6 NARP/Leigh Disease/MILS/other T C 8993 MT-ATP6
NARP/Leigh Disease/MILS/other T G 9176 MT-ATP6 Leigh
Disease/Spastic Paraplegia T G 9176 MT-ATP6 FBSN/Leigh Disease T C
9185 MT-ATP6 Leigh Disease/Ataxia syndromes/NARP-like disease T C
10158 MT-ND3 Leigh Disease T C 10191 MT-ND3 Leigh
Disease/Leigh-like Disease/ESOC T C 10197 MT-ND3 Leigh
Disease/Dystonia/Stroke/LDYT G A 10663 MT-ND4L LHON T C 11777
MT-ND4 Leigh Disease C A 11778 MT-ND4 LHON/Progressive Dystonia G A
12706 MT-ND5 Leigh Disease T C 13051 MT-ND5 LHON G A 13513 MT-ND5
Leigh Disease/MELAS/LHON-MELAS Overlap G A Syndrome 13514 MT-ND5
Leigh Disease/MELAS A G 14459 MT-ND6 LDYT/Leigh Disease G A 14482
MT-ND6 LHON C G 14482 MT-ND6 LHON C A 14484 MT-ND6 LHON T C 14487
MT-ND6 Dystonia/Leigh Disease/Ataxia/Ptosis/Epilepsy T C 14495
MT-ND6 LHON A G 14568 MT-ND6 LHON C T 14849 MT-CYB EXIT/Septo-Optic
Dysplasia T C 14864 MT-CYB MELAS T C 15579 MT-CYB Multisystem
Disorder, EXIT A G The disease abbreviations refer to the
following: LHON Leber Hereditary Optic Neuropathy AD Alzeimer's
Disease ADPD Alzeimer's Disease and Parkinsons's Disease NARP
Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa;
alternate phenotype at this locus is reported as Leigh Disease
MELAS Mitochondrial Encephalomyopathy, Lactic Acidosis, and
Stroke-like episodes MERRF Myoclonic Epilepsy and Ragged Red Muscle
Fibers CPEO Chronic Progressive External Ophthalmoplegia DM
Diabetes Mellitus CIPO Chronic Intestinal Pseudoobstruction with
myopathy and Ophthalmoplegia PEM Progressive encephalopathy MM
Mitochondrial Myopathy LIMM Lethal Infantile Mitochondrial Myopathy
MMC Maternal Myopathy and Cardiomyopathy FICP Fatal Infantile
Cardiomyopathy Plus, a MELAS-associated cardiomyopathy LDYT Leber's
hereditary optic neuropathy and DYsTonia MHCM Maternally inherited
Hypertrophic CardioMyopathy KSS Kearns Sayre Syndrome DMDF Diabetes
Mellitus + DeaFness DEAF Maternally inherited DEAFness or
aminoglycoside-induced DEAFness SNHL SensoriNeural Hearing Loss
Example C-5: Contaminating Nuclear DNA Levels
[1035] The amount of contaminating nuclear DNA (nDNA) in the
chondrisome preparation was examined using semi-quantitative
real-time PCR (RT-PCR). Chondrisomes were isolated from human
skeletal muscle punch biopsies, platelets, or cultured fibroblasts
using the procedures explained in Examples 1-2a and 1-3b and 1-1
respectively. From these preparations, total DNA was isolated using
a Qiagen DNeasy blood and tissue kit (catalog number 69504),
followed by determination of DNA concentration using a Thermo
Scientific NanoDrop.
[1036] After completion of the DNA isolation procedure, RT-PCR was
carried out using Applied Biosystems PCR Master Mix (catalog number
4309155) in a 20 tit total reaction volume using the following
reaction template:
[1037] SYBR Green Master Mix: 10 .mu.L
[1038] 0.45 .mu.M Forward Primer: 1 .mu.L
[1039] 0.45 .mu.M Reverse Primer: 1 .mu.L
[1040] DNA Template: 10 ng
[1041] PCR-Grade Water: Variable
[1042] Forward and reverse primers were produced and acquired by
Integrated DNA Technologies. The table below details the primer
pairs and their associated sequences:
TABLE-US-00028 Forward Primer Reverse Primer Target Sequence
(5'.fwdarw. 3') Sequence (5'.fwdarw. 3') Human mtDNA CAC CCA AGA
ACA GGG TTT GT TGG CCA TGG GTA TGT TGT TA Human nDNA TGC TGT CTC
CAT GTT TGA TGT TCT CTG CTC CCC ACC TCT ATC T AAG T
[1043] An Applied Biosystems 7900HT Real-Time PCR system was used
to perform the amplification and detection with the following
protocol:
[1044] Denaturation, 94.degree. C. 2 min
[1045] 40 Cycles of the following sequence: [1046] Denaturation,
94.degree. C. 15 sec [1047] Annealing, Extension, 60.degree. C. 1
min
[1048] The ratio of mtDNA to nDNA was calculated using the relative
cycle threshold values and the following formula:
mtDNA.sub.relative=2.times.2.sup.(Ct nuclear-Ct mito),
where Ct nuclear denotes the cycle threshold for nDNA and Ct mito
denotes mtDNA, respectively determined at a fluorescence level of
0.014179736. The following table presents the mtDNA copy number
relative to the nuclear DNA copy number:
TABLE-US-00029 Source: Relative mtDNA level over nuclear DNA Human
Fibroblast 64741 .+-. 23551 Human Platelet 246312 .+-. 151024 Human
Muscle 2893 .+-. 773
Example C-6: Endotoxin Levels
[1049] This assay was performed using the commercially available
kit for the determination of endotoxin levels (Pierce LAL
Chromogenic Endotoxin Quantification Kit, ThermoFisher 88282). The
manufacturer's protocols were followed and were briefly as follows.
Critically, all materials (e.g., pipette tips, glass tubes,
microcentrifuge tubes and disposable 96-well microplates) must be
endotoxin-free. The assay microplate loaded in a heating block to
maintain a temperature of 37 C. While at 37 C 50 .mu.L of each
standard (X,Y,Z) or chondrisome preparation sample replicate was
loaded into the appropriate wells. The plate was covered and
incubated for 5 minutes at 37 C.50 .mu.L of LAL buffer was added to
each well and the plate was covered and gently shaken on a plate
shaker for 10 seconds then incubated at 37 C for 10 minutes. 100
.mu.L of substrate solution was added to each well. Again cover,
and shake for 10 seconds and incubate at 37 C for 6 minutes. 50
.mu.L of Stop Reagent (25% acetic acid) was added and then shake
the plate on a plate mixer for 10 seconds. Absorbance was measured
at 405-410 nm on a plate reader. Average absorbance of the blank
replicates were subtracted from the average absorbance of all
standard replicates and average of the chondrisome preparation
replicates. A standard curve was generated by performing linear
regression on the standards (The coefficient of determination, r2,
must be .gtoreq.0.98) and the endo toxin level of the chondrisome
preparation was interpolated. If the chondrisome preparation sample
endotoxin concentration was >1.0 EU/mL (out of range) dilute the
sample five-fold in endotoxin-free water and re-test.
[1050] Using this assay the endotoxin level of the chondrisome
preparation (as prepared in example 1-1) derived from human
fibroblasts was determined to be <0.02 EU/ug chondrisome
protein. (actual value 0.015 U/ug).
[1051] Using this assay the endotoxin level of the chondrisome
preparation (as prepared in example 1-3a) derived from human blood
was determined to be <0.03 EU/ug chondrisome protein. (actual
value 0.025 U/ug).
E Blood Derived Preps
Example E-1: Preparation of Chondrisome Containing
Mitoparticles
[1052] Platelet samples were evaluated for starting concentration
of platelets and then centrifuged and resuspended in Tyrodes buffer
to a concentration of 1.times.10 8 platelets/mL. To release
mitoparticles from platelets, platelets were activated with
thrombin at 0.5 U/mL and incubated at room temperature in the dark
for 4 hours. After activation, the preparation was centrifuged at
2000 g to pellet platelets and the supernatant was collected. In
this experiment, the supernatant was distributed into 2 mL
Eppendorf tubes and spun at 18,000 g in bench top microcentrifuge
for 90 minutes to pellet the mitoparticles containing chondrisomes.
The pelleted mitoparticles were then resuspended in Tyrode's buffer
and used for downstream applications.
Example E-2: Concentration of Chondrisome Containing Mitoparticles
from Platelets
[1053] In order to label platelet cellular membranes and
chondrisomes, the resuspended platelets were stained in solution
with 2 uM PKH67 fluorescent cell linker and 200 nM MitoTracker Deep
Red, and incubated at 37.degree. C. for 30 minutes. Appropriate
unstained and single-stained controls were processed as well.
Platelet solutions were then centrifuged at 2000 g to pellet
platelets. The supernatant was removed and the platelets were
resuspended in the same volume of Tyrode's buffer. After
thrombin-activation for 4 hours, fluorescence-activated cell
sorting was performed on a Beckman Coulter MoFlo XDP instrument and
the population of mitoparticles staining double-positive for PKH67
and MitoTracker Deep Red we quantified. PKH67 was excited with a
488 nm laser and emission captured at 513.+-.26 nm. MitoTracker
Deep Red was excited with a 640 nm laser and emission captured at
671.+-.30 nm. Events double-positive for PKH67 and MitoTracker Deep
Red were determined by gating at the minimum level for which each
appropriately unstained sample showed <1% of events positive for
the specific fluorescent marker (i.e. unstained and
single-PKH67-stained samples show <1% events positive for
MitoTracker Deep Red). The number of double-positive events was
counted and normalized to the volume of sample analyzed during the
FACS assessment. The number of double-positive mitoparticles
containing chondrisomes was expressed as the amount per platelet
number. This assay determined that mitoparticles were identified at
in the concentration range of 2-14 MPs per 10 10 platelets. In this
specific experiment there was 7.8 MPs per 10 10 platelets in the
preparation.
Example E-3: Membrane Potential of Chondrisome Containing
Mitoparticles
[1054] The membrane potential of the chondrisome preparation is
quantified using a commercially available dye, TMRE
(tetramethylrhodamine ester, ThermoFisher #T669).
Chondrisome-containing mitoparticles are prepared from human
platelets as described in Example E-1, and stained for platelet
cellular membranes and chondrisomes, followed by thrombin
activation, as outlined in Example E-2. After thrombin activation
for 4 hours, TMRE (30 nM) is added to the platelet solution and
incubated at 37 C for 30 minutes prior to analysis by FACS. A
parallel set of samples is not stained with TMRE (unstained).
Events double-positive for PKH67 and MitoTracker Deep Red are
determined by gating at the minimum level for which each
appropriately unstained sample showed <1% of events positive for
the specific fluorescent marker (i.e. unstained and
single-PKH67-stained samples show <1% events positive for
MitoTracker Deep Red). The gated double-positive events are then
assessed for TMRE intensity (TMRE excited with 543 nm laser and
emission captured at 570.+-.26 nm). A parallel sample of
thrombin-stimulated platelets is treated with 2 uM FCCP for 5
minutes prior to FACS analysis.
[1055] Membrane potential values (in millivolts, mV) are calculated
based on the intensity of TMRE. For both untreated and FCCP-treated
samples, the corrected fluorescence intensity (FI) value is
calculated by subtracting the geometric mean of TMRE fluorescence
intensity for the unstained sample from the geometric mean of the
untreated and FCCP-treated sample. The quantification of membrane
potential of the chondrisomes-containing mitoparticle population is
calculated using the modified Nernst equation below, which can be
used to determine chondrisome membrane potential based on TMRE
fluorescence (as TMRE accumulates in chondrisomes in a Nernstian
fashion).
Chondrisome membrane potential
(mV)=-61.5*log(FI.sub.untreated/FI.sub.FCCP-treated)
[1056] Performing this assay on chondrisome preparations from mouse
skeletal muscle chondrisomes (as described in example 1-2a) is
expected to yield a membrane potential state of -65 mV (Range was
-20 to -150 mV).
Example E-4: Quantification of Platelet Derived Chondrisome
Mitoparticle Delivery to Specific Subcellular Locations
[1057] This example describes delivery of mitoparticles to cultured
cells.
[1058] Chondrisome-containing mitoparticles are generated from
human platelet samples stained with MitoTracker Deep Red and PKH67
Cell Linker and activated with thrombin for 4 hours (example E-2).
The supernatant is distributed into 2 mL eppendorf tubes and spun
at 18,000 g in bench top microcentrifuge for 90 minutes to pellet
the mitoparticles containing chondrisomes. The pelleted
mitoparticles are then resuspended in 200 uL of Tyrode's buffer.
Chondrisome preparation protein content is assessed by BCA (example
A-2) and the preparation remains on ice until the following
protocol is initiated (within 120 minutes from isolation).
[1059] Leigh fibroblasts from Coriell Institute (GM01503) are
cultured in DMEM media supplemented with 10% fetal bovine serum
(ThermoFisher), and seeded at 25,000 cells per well in one well of
a quadrant glass-bottom imaging dish (Greiner Bio-One). After 24
hours, cells are treated with 8 .mu.g of chondrisome-containing
mitoparticles per well in 500 .mu.L media or an equivalent volume
of Tyrode buffer as control. Cells are incubated with mitoparticles
for 24 hours. Prior to imaging cells are incubated with 50 nM
Lysotracker Red for 30 minutes. Cells are then imaged on a Zeiss
LSM 710 confocal microscope with a 63.times. oil immersion
objective while maintained at 37 C and 5% CO2. PKH67 cell linker
dye is subjected to 488 nm laser excitation and emission is
recorded through a band pass 495-530 nm filter. Lysotracker red is
subjected to 543 nm laser excitation and emission is recorded
through a band pass 560 to 610 nm filter. MitoTracker Deep Red is
subjected to 633 nm helium/neon laser excitation and emission is
recorded through a band-pass 650 to 710 nm filter. To observe
individual chondrisomes Z-stack images are acquired in series of 6
slices per cell with a 1 airy unit pinhole ranging in thickness
from 0.5-0.8 .mu.m per slice.
[1060] Colocalization of donor chondrisomes (positive for
MitoTracker Deep Red only) or chondrisome-containing mitoparticles
(double-positive for MitoTracker Deep Red and PKH67) with recipient
cell lysosomes (Lysotracker Red) is calculated by summing all
events where there is greater than 80% pixel overlap between a
given MitoTracker Deep Red-positive chondrisome region and a
Lysotracker green-positive lysosomal regions within an analyzed
z-plane. All z-plane images are processed in ImageJ (NIH) and
mitoparticle, chondrisomes, and lysosomal regions are thresholded
using the Moments threshold algorithm to identify regions after
subtracting background. Using this imaging assay and colocalization
calculation, >20% of the donor chondrisome-containing
mitoparticles (MitoTracker Deep Red and PKH67) are found to be
targeted to endogenous cellular lysosomes. Analogous imaging and
localization quantification protocols determine the expected
targeting levels to other subcellular locations as detailed in the
table below.
TABLE-US-00030 Colocalization Subcellular Mitoparticle
Quantification Target Preparation Stain Target Cell Stain Method
Proportion Cytosol 200 nM 50 nM Lysotracker Fraction of >5%
MitoTracker Deep Red for 30 minutes MitoTracker Deep Red and PKH67
Red-positive Cell Linker for 30 events showing no minutes at 37 C.
in significant Tyrode's buffer at colocalization a concentration of
(<80% pixel 0.005-0.05 ug/uL. overlap) with Lysotracker Red
regions. Endogenous 200 nM 15 nM TMRE Fraction of >5%
Mitochondrial MitoTracker Deep (ThermoFisher) for MitoTracker Deep
Network Red and PKH67 60 minutes at 37 C. Red-positive Cell Linker
for 30 events showing minutes at 37 C. in significant Tyrode's
buffer at colocalization a concentration of (>80% pixel
0.005-0.05 ug/uL. overlap) with TMRE regions. Lysosome 200 nM 50 nM
Lysotracker Fraction of 5%-90% MitoTracker Deep Red for 30 minutes
MitoTracker Deep Red and PKH67 Red-positive Cell Linker for 30
events showing minutes at 37 C. in significant Tyrode's buffer at
colocalization a concentration of (>80% pixel 0.005-0.05 ug/uL.
overlap) with Lysotracker Red regions. Outer 200 nM Cells are fixed
with Fraction of 5%-90% membrane MitoTracker Deep 4% MitoTracker
Deep Red and PKH67 paraformaldehyde Red-positive Cell Linker for 30
and stained with events showing minutes at 37 C. in rabbit anti-
significant Tyrode's buffer at TOMM20 antibody colocalization a
concentration of for outer (>80% pixel 0.005-0.05 ug/uL.
chondrisome overlap) with membrane, TOMM20- followed by anti-
AlexaFluor 543 rabbit AlexaFluor (red) regions. 543 staining.
F Biological/Functional Characteristics
Example F-1: Apoptosis Induction Level
[1061] An approach to measuring apoptosis was via detection of
fluorescent exclusion dye, propdium iodide. During late-stage
apoptosis or necrosis, the cellular plasma membrane becomes
permeable and propidium iodide is able to transverse the membrane
and stain nuclear DNA of the cells. Live cells with an intact
membrane do not allow propidium iodide uptake and thus show no
nuclear DNA staining.
[1062] Though any cell type can be used for the assay, this
examples pertains specifically to Leigh syndrome fibroblast cells
acquired from Coriell Institute (GM01503) and cultured in DMEM
media supplemented with 10% fetal bovine serum (ThermoFisher).
Cells were seeded at a density of 10,000 cells/well in a 96-well
plate in DMEM media supplemented with 10% FBS. The chondrisome
preparation was generated (see example 1-1 and 1-3b) and the
preparation remains on ice until the quantification assay was
initiated (within 120 minutes from isolation). While on ice, total
protein content of the chondrisome preparation was quantified via
BCA (see example A-5). The chondrisome preparation was added to the
wells at a concentration of 0, 0.5, 1, 2, or 4 .mu.g/well. Cells
were allowed to incubate for 124 hours with or without chondrisomes
in DMEM media supplemented with 10% FBS.
[1063] Following the incubation period, cells were washed once in
phosphate-buffered saline (PBS). PBS containing 1 ug/mL propidium
iodide and 5 uM Hoescht 33342 was added to the wells (200 uL)
according to the manufacturer's protocol (ThermoFisher Cat# P3566
and Cat#62249). Cells were incubated for 10-15 minutes and analyzed
using Celigo Imaging Cell Cytometer (Brooks Life Science Systems).
Blue (377/50 excitation; 470/22 emission) and red (531/40
excitation; 629/53 emission) fluorescence channels was imaged for
each well. Analysis parameters for images acquired by Celigo
Imaging Cell Cytometer were optimized to identify individual cells
based on fluorescence (Algorithm 1, Threshold 1, Precision 2,
Filter Size 25, Separate Touching Objects TRUE). The % cell death
was calculated as the number of propidium iodide-positive cells
normalized to the total number of cells
(Hoescht-positive).times.100. At least 1,000 cells were analyzed
per well, with 3 well replicates per experiment. Cell death was
measured at less than 3% for cells treated with up to 4 ug
chondrisomes from human fibroblasts or human platelets.
Example F-2: Enhancement of Cellular Respiration
[1064] This assay pertains specifically to Leigh syndrome
fibroblast cells acquired from Coriell Institute (GM01503) and
cultured in DMEM media supplemented with 10% fetal bovine serum
(ThermoFisher). Chondrisome preparations were generated (example
1-2a) from leg gastrocnemius muscle from C57bl/6 mice (Charles
River Laboratories). The chondrisome preparation was resuspended in
MSHE buffer (200 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM
EDTA, adjust the pH to 7.4 with KOH). Chondrisome preparation
protein content was assessed by BCA (example A-5) and the
preparation remains on ice until the following protocol was
initiated (within 120 minutes from isolation).
[1065] Leigh fibroblasts were seeded at 12,500 cells per well in a
96-well Seahorse plate (Agilent). After 24 hours, cells were
treated with 4 or 16 .mu.g of chondrisome preparation per well in
200 .mu.L media or an equivalent volume of MSHE buffer as control.
Cells were incubated with chondrisomes for 24 hours and oxygen
consumption rates of fibroblast cells were subsequently measured by
XF96 bioenergetic assay (Agilent).
[1066] Oxygen consumption assays were initiated by removing growth
medium, replacing with low-buffered DMEM minimal medium containing
25 mM glucose and 2 mM glutamine (Agilent) and incubating at
37.degree. C. for 60 minutes to allow temperature and pH to reach
equilibrium. The microplate was then assayed in the XF96
Extracellular Flux Analyzer (Agilent) to measure extracellular flux
changes of oxygen and pH in the media immediately surrounding
adherent cells. After obtaining steady state oxygen consumption
(basal respiration rate) and extracellular acidification rates,
oligomycin (5 .mu.M), which inhibits ATP synthase, and proton
ionophore FCCP (carbonyl cyanide 4-(trifluoromethoxy)
phenylhydrazone; 2 .mu.M), which uncouples chondrisomes, were
injected sequentially through reagent delivery chambers for each
cell well in the microplate to obtain values for maximal oxygen
consumption rates. Finally, 5 .mu.M antimycin A (inhibitor of
chondrisome complex III) was injected to confirm that respiration
changes were due mainly to chondrisome respiration. The minimum
rate of oxygen consumption after antimycin A injection was
subtracted from all oxygen consumption measurements to remove the
non-mitochondrial respiration component. Cell samples that do not
appropriately respond to oligomycin (at least a 25% decrease in
oxygen consumption rate from basal) or FCCP (at least a 50%
increase in oxygen consumption rate after oligomycin) were excluded
from the analysis. Using this assay the fold increase in basal
respiration of the fibroblast from untreated to treated cells was
1.7-fold for cells treated with 4 ug chondrisomes and 2.1-fold for
cells treated with 16 ug chondrisomes.
Example F-3: Subcellular Targeting Levels
[1067] This assay pertains specifically to Leigh syndrome
fibroblast cells acquired from Coriell Institute (GM01503) and
cultured in DMEM media supplemented with 10% fetal bovine serum
(ThermoFisher). Chondrisome preparations were generated (example
1-1) from Hela cells. Prior to chondrisome isolation, Hela cells
were transduced with adenovirus-mito-DsRed (250 viral
particles/cell) and expression was enabled for 72 hours before
chondrisome isolation. After chondrisome isolation, the chondrisome
sample was resuspended in MSHE buffer (200 mM mannitol, 70 mM
sucrose, 10 mM HEPES, 1 mM EDTA, adjust the pH to 7.4 with KOH).
Chondrisome preparation protein content was assessed by BCA
(example A-5) and the preparation remains on ice until the
following protocol was initiated (within 120 minutes from
isolation).
[1068] Leigh fibroblasts were seeded at 25,000 cells per well in
one well of a quadrant glass-bottom imaging dish (Greiner Bio-One).
After 24 hours, cells were treated with 8 .mu.g of Mito-DsRED
chondrisomes per well in 500 .mu.L (described above) media or an
equivalent volume of MSHE buffer as control. Cells were incubated
with chondrisomes for 24 hours. Prior to imaging cells were
incubated with 50 nM Lysotracker Green for 30 minutes. Cells were
then imaged on a Zeiss LSM 710 confocal microscope with a 63.times.
oil immersion objective while maintained at 37 C and 5% CO2.
Lysotracker green was subjected to 488 nm argon laser excitation
and emission was recorded through a band pass 500 to 550 nm filter.
Mito-DsRED was subjected to 543 nm laser excitation and emission
was recorded through a band-pass 550 to 610 nm filter. To observe
individual chondrisomes Z-stack images were acquired in series of 6
slices per cell with a 1 airy unit pinhole ranging in thickness
from 0.5-0.8 .mu.m per slice. At least 3 different fields were
imaged containing 4-30 cells per field, and the resulting analysis
was conducted on 12-120 cells in total.
[1069] Colocalization of donor chondrisomes with recipient cell
lysosomes was calculated by summing all events where there was
greater than 80% pixel overlap between a given Mito-DsRED-positive
chondrisome region and a Lysotracker green-positive lysosomal
regions within an analyzed z-plane. All z-plane images were
processed in ImageJ (NIH) and chondrisomes and lysosomal regions
were thresholded using the Moments threshold algorithm to identify
regions after subtracting background. Using this imaging assay and
colocalization calculation, >70% of the donor chondrisomes
(Mito-DsRED) were found to be targeted to endogenous cellular
lysosomes. Analogous imaging and localization quantification
protocols were used to determine targeting levels to other
subcellular locations as detailed in the table below.
TABLE-US-00031 Determined Subcellular Chondrisome Target Cell
Quantifying Colocalization Target preparation Stain Stain
Colocalization Levels Level Cytosol Expression of Mito- 50 nM
Fraction of Mito-DsRED- <30% DsRED (250 MOI) in Lysotracker
positive events showing donor cell line. Green for 30 no
significant minutes colocalization (<80% pixel overlap) with
Lysotracker Red regions. Endogenous Expression of Mito- 200 nM
Fraction of Mito-DsRED- <10% Mitochondrial DsRED (250 MOI) in
MitoTracker positive events showing Network donor cell line. Green
significant colocalization (ThermoFisher) (>80% pixel overlap)
for 60 minutes at with MitoTracker Green 37 C. regions. Lysosome
Expression of Mito- 50 nM Fraction of Mito-DsRED- >70% DsRED
(250 MOI) in Lysotracker positive events showing donor cell line.
Green for 30 significant colocalization minutes (>80% pixel
overlap) with Lysotracker Red regions. Outer Expression of Mito-
Cells are fixed Fraction of Mito-DsRED- <50% membrane DsRED (250
MOI) in with 4% positive events showing (expected) donor cell line.
paraformaldehyde significant colocalization and stained with
(>80% pixel overlap) rabbit anti- with TOMM20- TOMM20 AlexaFluor
488 (green) antibody for outer regions. chondrisome membrane,
followed by anti- rabbit AlexaFluor 488 staining.
Example F-4: Delivery of a Loaded Cargo
[1070] This assay pertains specifically to Leigh syndrome
fibroblast cells acquired from Coriell Institute (GM01503) and
cultured in DMEM media supplemented with 10% fetal bovine serum
(ThermoFisher). Chondrisome preparations were generated (example
1-2a) from leg gastrocnemius muscle from C57bl/6 mice (Charles
River Laboratories). The chondrisome preparation was resuspended in
MSHE buffer (200 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM
EDTA, adjust the pH to 7.4 with KOH). Chondrisome preparation
protein content was assessed by BCA (example A-5) and the
preparation remains on ice until the following protocol was
initiated (within 120 minutes from isolation). Chondrisome
preparations were stained with 200 nM MitoTracker Deep Red
(ThermoFisher) for 30 minutes at 37 C in MSHE buffer at a
concentration of 0.05 ug/uL. After staining, the chondrisome
preparation was pelleted by centrifugation (10,000 g for 10
minutes), resuspended in 1 mL of MSHE, and the centrifugation and
resuspension was repeated an additional two times to wash away
excess dye. After the last wash, chondrisomes were pelleted and
resuspended in MSHE to a final concentration of 1 ug/uL, and
applied to recipient Leigh fibroblast cells and to empty wells for
control readings. The control wells were imaged immediately to
quantify the signal level corresponding to the MitoTracker loaded
cargo.
[1071] Leigh fibroblasts were seeded at 25,000 cells per well in
one well of a quadrant glass-bottom imaging dish (Greiner Bio-One).
After 24 hours, cells were stained with 200 nM MitoTracker Green
(ThermoFisher) for 60 minutes at 37 C, 5% CO2. Following staining,
cells were washed with DMEM growth medium 3.times. followed by
treatment with 8 .mu.g of MitoTracker Deep Red-stained muscle
chondrisomes per well in 500 .mu.L (described above) media or an
equivalent volume of MSHE buffer as control. Cells were incubated
with chondrisomes for 24 hours. Cells were then imaged on a Zeiss
LSM 710 confocal microscope with a 63.times. oil immersion
objective while maintained at 37 C and 5% CO2. MitoTracker green
was subjected to 488 nm argon laser excitation and emission was
recorded through a band pass 500 to 550 nm filter. MitoTracker Deep
Red was subjected to 633 nm helium/neon laser excitation and
emission was recorded through a band-pass 650 to 710 nm filter. To
observe individual chondrisomes Z-stack images were acquired in
series of 6 slices per cell with a 1 airy unit pinhole ranging in
thickness from 0.5-0.8 .mu.m per slice. At least 3 different fields
were imaged containing 4-30 cells per field, and the resulting
analysis was conducted on 12-120 cells in total.
[1072] Delivery of the loaded chondrisome cargo was confirmed by
observing MitoTracker Deep Red-positive chondrisome cargo within
the recipient cell labeled with MitoTracker green. Images were
processed in ImageJ (NIH) and donor/recipient cell chondrisome
regions were thresholded using the Moments threshold algorithm to
identify regions after subtracting background. Control Leigh
fibroblast cells that did not receive MitoTracker Deep Red-loaded
chondrisome cargo were also imaged with identical settings and
analyzed similarly to confirm that no MitoTracker Deep Red-positive
signal was observed; thus the threshold was set appropriately so
that the number of identified MitoTracker Deep Red-positive
chondrisomes per cell was .ltoreq.1. Uptake of cargo loaded
chondrisomes is defined as positive identification of .gtoreq.2
MitoTracker Deep Red-positive chondrisomes) in a cell. Using this
assay we determined that imaged recipient cells showed uptake of
loaded chondrisomes. In this example we determined that 30% of the
imaged recipient cells showed uptake of donor chondrisomes.
Example F-5: Delivery of an Engineered Cargo
[1073] This assay pertains specifically to Leigh syndrome
fibroblast cells acquired from Coriell Institute (GM01503) and
cultured in DMEM media supplemented with 10% fetal bovine serum
(ThermoFisher). Chondrisome preparations were generated (example
1-1) from Hela cells. Prior to chondrisome isolation, Hela cells
were transduced with adenovirus (250 viral particles/cell) for
chonriosome-targeted DsRED fluorescent protein (mito-DsRED) and
expression was enabled for 72 hours before chondrisome isolation.
After chondrisome isolation, the chondrisome sample was resuspended
in MSHE buffer (200 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM
EDTA, adjust the pH to 7.4 with KOH). Chondrisome preparation
protein content was assessed by BCA (example A-5) and the
preparation remains on ice until the following protocol was
initiated (within 120 minutes from isolation).
[1074] Leigh fibroblasts were seeded at 25,000 cells per well in
one well of a quadrant glass-bottom imaging dish (Greiner Bio-One).
After 24 hours, cells were treated with 8 .mu.g of Mito-DsRED
chondrisomes per well in 500 .mu.L (described above) media or an
equivalent volume of MSHE buffer as control. Cells were incubated
with chondrisomes for 24 hours. Prior to imaging cells were
incubated with 200 nM MitoTracker Green for 60 minutes, followed by
three media washes. Cells were then imaged on a Zeiss LSM 710
confocal microscope with a 63.times. oil immersion objective while
maintained at 37 C and 5% CO2. MitoTracker Green was subjected to
488 nm argon laser excitation and emission was recorded through a
band pass 500 to 550 nm filter. Mito-DsRED was subjected to 543 nm
laser excitation and emission was recorded through a band-pass 550
to 610 nm filter. To observe individual chondrisomes Z-stack images
were acquired in series of 6 slices per cell with a 1 airy unit
pinhole ranging in thickness from 0.5-0.8 .mu.m per slice. At least
3 different fields were imaged containing 4-30 cells per field, and
the resulting analysis was conducted on 12-120 cells in total.
[1075] Delivery of the engineered chondrisome cargo was confirmed
by observing Mito-DsRED-positive chondrisome cargo within the
recipient cell labeled with MitoTracker green. Images were
processed in ImageJ (NIH) and donor/recipient cell chondrisome
regions were thresholded using the Moments threshold algorithm to
identify regions after subtracting background. Control Leigh
fibroblast cells that did not receive Mito-DsRED-engineered
chondrisome cargo were also imaged with identical settings and
analyzed similarly to confirm that no Mito-DsRED-positive signal
was observed; thus the threshold was set appropriately so that the
number of identified Mito-DsRED-positive chondrisomes per cell was
.ltoreq.1. Uptake of chondrisomes with engineered cargo is defined
as positive identification of .gtoreq.2 Mito-DsRED-positive
chondrisomes) in a cell. Using this assay we determined that imaged
recipient cells showed uptake of chondrisomes with engineered
cargo. In this example we determined that 30% of the imaged
recipient cells showed uptake of donor chondrisomes.
Example F-6: Chemical Modulation of Subcellular Chondrisome
Targeting
[1076] This assay pertains specifically to Leigh syndrome
fibroblast cells acquired from Coriell Institute (GM01503) and
cultured in DMEM media supplemented with 10% fetal bovine serum
(ThermoFisher). Chondrisome preparations were generated (example
1-1) from Hela cells. Prior to chondrisome isolation, Hela cells
were transduced with adenovirus (250 viral particles/cell) for
chonriosome-targeted DsRED fluorescent protein (mito-DsRED) and
expression was enabled for 72 hours before chondrisome isolation.
After chondrisome isolation, the chondrisome sample was resuspended
in MSHE buffer (200 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM
EDTA, adjust the pH to 7.4 with KOH). Chondrisome preparation
protein content was assessed by BCA (example A-5) and the
preparation remains on ice until the following protocol was
initiated (within 120 minutes from isolation).
[1077] Leigh fibroblasts were seeded at 25,000 cells per well in
one well of a quadrant glass-bottom imaging dish (Greiner Bio-One).
After 24 hours, cells were treated with 8 .mu.g of Mito-DsRED
chondrisomes per well in 500 .mu.L (described above) media or an
equivalent volume of MSHE buffer as control. Cells were incubated
with chondrisomes for 24 hours. To enhance endosomal escape of the
delivered chondrisome cargo, recipient Leigh fibroblasts were
concomitantly treated with 30 .mu.g/mL chloroquine in order to
inhibit lysosomal acidification/degradation and reduce the fusion
of endosomes with cargo to lysosomes. Cells were incubated with
chondrisomes with or without chloroquine for 24 hours. Prior to
imaging cells were incubated with 200 nM MitoTracker Green for 60
minutes, followed by three media washes. Cells were then imaged on
a Zeiss LSM 710 confocal microscope with a 63.times. oil immersion
objective while maintained at 37 C and 5% CO2. MitoTracker Green
was subjected to 488 nm argon laser excitation and emission was
recorded through a band pass 500 to 550 nm filter. Mito-DsRED was
subjected to 543 nm laser excitation and emission was recorded
through a band-pass 550 to 610 nm filter. To observe individual
chondrisomes Z-stack images were acquired in series of 6 slices per
cell with a 1 airy unit pinhole ranging in thickness from 0.5-0.8
.mu.m per slice.
[1078] Enhanced delivery of the chondrisome cargo was confirmed by
observing Mito-DsRED-positive chondrisome cargo within the
recipient cell labeled with MitoTracker green. Images were
processed in ImageJ (NIH) and donor/recipient cell chondrisome
regions were thresholded using the Moments threshold algorithm to
identify regions after subtracting background. Control Leigh
fibroblast cells that did not receive Mito-DsRED-engineered
chondrisome cargo were also imaged with identical settings and
analyzed similarly to confirm that no Mito-DsRED-positive signal
was observed; thus the threshold was set appropriately so that the
number of identified Mito-DsRED-positive chondrisomes per cell was
.ltoreq.1. In this assay we define positive uptake by a recipient
cell as identification of .gtoreq.2 Mito-DsRED-positive
chondrisomes in a cell. At least 3 different fields were imaged
containing 4-30 cells per field, and the resulting analysis was
conducted on 12-120 cells in total. Using this assay, we determined
there was a 15-45% increase in proportion of cells taking up
chondrisomes from the non-treated to chloroquine treated groups. In
this example we determined there was an increase in cellular uptake
of chondrisomes following treatment with chloroquine: from 30% of
non-chloroquine treated cells taking up donor chondrisomes to 40%
in chloroquine treated, and a 4-fold increase in number of donor
chondrisomes per recipient cell.
Example F-7: Proportion of Delivered Chondrisomes Maintain an
Active Membrane Potential
[1079] This assay pertains specifically to Leigh syndrome
fibroblast cells acquired from Coriell Institute (GM01503) and
cultured in DMEM media supplemented with 10% fetal bovine serum
(ThermoFisher). Chondrisome preparations were generated (example
1-1) from Hela cells. Chondrisome preparation protein content was
assessed by BCA (example A-5) and the preparation remains on ice
until the following protocol was initiated (within 20 minutes from
isolation). Prior to chondrisome isolation, Hela cells were
transduced with adenovirus (250 viral particles/cell) for
chonriosome-targeted DsRED fluorescent protein (mito-DsRED) and
expression was enabled for 72 hours before chondrisome isolation.
After chondrisome isolation, the chondrisome sample was resuspended
in MSHE buffer (200 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM
EDTA, adjust the pH to 7.4 with KOH). Protein concentration of the
chondrisome sample was determined by BCA assay (Pierce).
[1080] Leigh fibroblasts were seeded at 25,000 cells per well in
one well of a quadrant glass-bottom imaging dish (Greiner Bio-One).
After 24 hours, cells were treated with 8 .mu.g of mito-GFP
chondrisomes from Hela cells per well in 500 .mu.L media or an
equivalent volume of MSHE buffer as control. Cells were incubated
with chondrisomes for 24 hours, followed by washing with PBS three
times and growth medium was replaced. Prior to imaging, cells were
stained with tetramethylrhodamine ester (TMRE, 15 nM) for 1 hour.
Cells were washed one time and growth medium with 15 nM TMRE was
replaced. Cells were then imaged on a Zeiss LSM 710 confocal
microscope with a 63.times. oil immersion objective while
maintained at 37 C and 5% CO2. Mito-GFP was subjected to 488 nm
argon laser excitation and emission was recorded through a band
pass 500 to 550 nm filter. TMRE was subjected to 5433 nm
helium/neon laser excitation and emission was recorded through a
band-pass 550 to 610 nm filter. To observe individual chondrisomes
Z-stack images were acquired in series of 6 slices per cell with a
1 airy unit pinhole ranging in thickness from 0.5-0.8 .mu.m per
slice. At least 3 different fields were imaged containing 4-30
cells per field, and the resulting analysis was conducted on 12-120
cells in total.
[1081] Delivery of the chondrisomes with a significant membrane
potential was quantified by measuring the average TMRE fluorescence
intensity of mito-GFP-positive chondrisome cargo that were
localized within the recipient cell network (identified with TMRE).
Images were processed in ImageJ (NIH) and donor/recipient cell
chondrisome regions were thresholded with the Moments threshold
algorithm to identify regions after subtracting background.
Mito-GFP-positive chondrisome regions (donor) were determined to
have a significantly active membrane potential if the average TMRE
fluorescence intensity for the mito-GFP region was >50% of the
average TMRE fluorescence intensity for the entire cell. Control
Leigh fibroblast cells that did not receive mito-GFP-engineered
chondrisome cargo were also imaged with identical settings to
verify that no mito-GFP-positive signal was observed; thus the
threshold was set appropriately so that the number of identified
Mito-DsRED-positive chondrisomes per cell was .ltoreq.1. Using this
assay, we determined that delivery of the chondrisome preparation
resulted in 0.1%-10% of the total delivered chondrisomes having an
active membrane potential.
Example F-8: Persistence of Delivered Chondrisomes
[1082] This assay pertains specifically to Leigh syndrome
fibroblast cells acquired from Coriell Institute (GM01503) and
cultured in DMEM media supplemented with 10% fetal bovine serum
(ThermoFisher). Chondrisome preparations were generated (example
1-1) from Hela cells. Prior to chondrisome isolation, Hela cells
were transduced with adenovirus (250 viral particles/cell) for
chonriosome-targeted DsRED fluorescent protein (mito-DsRED) and
expression was enabled for 72 hours before chondrisome isolation.
After chondrisome isolation, the chondrisome sample was resuspended
in MSHE buffer (200 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM
EDTA, adjust the pH to 7.4 with KOH). Chondrisome preparation
protein content was assessed by BCA (example A-5) and the
preparation remains on ice until the following protocol was
initiated (within 120 minutes from isolation).
[1083] Leigh fibroblasts were seeded at 25,000 cells per well in
one well of a quadrant glass-bottom imaging dish (Greiner Bio-One).
After 24 hours, cells were treated with 8 .mu.g of Mito-DsRED
chondrisomes per well in 500 .mu.L (described above) media or an
equivalent volume of MSHE buffer as control. Cells were incubated
with chondrisomes for 24 hours. Prior to imaging cells were
incubated with 200 nM MitoTracker Green for 60 minutes, followed by
three media washes. Cells were then imaged on a Zeiss LSM 710
confocal microscope with a 63.times. oil immersion objective while
maintained at 37 C and 5% CO2. MitoTracker Green was subjected to
488 nm argon laser excitation and emission was recorded through a
band pass 500 to 550 nm filter. Mito-DsRED was subjected to 543 nm
laser excitation and emission was recorded through a band-pass 550
to 610 nm filter. To observe individual chondrisomes Z-stack images
were acquired in series of 6 slices per cell with a 1 airy unit
pinhole ranging in thickness from 0.5-0.8 .mu.m per slice. Cells
were imaged at 24 hr time intervals over the course of 5 days. At
least 3 different fields were imaged containing 4-30 cells per
field, and the resulting analysis was conducted on 12-120 cells in
total.
[1084] Residence time of the delivered chondrisome cargo was
confirmed by observing Mito-DsRED-positive chondrisome cargo within
the recipient cell labeled with MitoTracker green. Images were
processed in ImageJ (NIH) and donor/recipient cell chondrisome
regions were thresholded using the Moments threshold algorithm to
identify regions after subtracting background. Control Leigh
fibroblast cells that did not receive Mito-DsRED-engineered
chondrisome cargo were also imaged with identical settings and
analyzed similarly to confirm that no Mito-DsRED-positive signal
was observed; thus the threshold was set appropriately so that the
number of identified Mito-DsRED-positive chondrisomes per cell was
.ltoreq.1. The presence of recipient cells showing uptake of donor
chondrisomes (defined as positive identification of .gtoreq.2
Mito-DsRED-positive chondrisomes) was observable up to 5 days after
application of donor chondrisomes to recipient cells.
Example F-9: Quantification of Lipid Utilization
[1085] This assay pertains specifically to INS1 cells cultured in
RPMI media supplemented with 10% fetal bovine serum (ThermoFisher),
11 mM glucose with 500 uM Oleate+Palmitate. Chondrisome
preparations were generated (example 1-4) from brown adipose tissue
from C57bl/6 mice (Charles River Laboratories). Chondrisome
preparation protein content was assessed by BCA (example A-5) and
the preparation remains on ice until the following protocol was
initiated (within 20 minutes from isolation). The chondrisome
sample was resuspended in SHE buffer (250 mM sucrose, 5 mM HEPES, 2
mM EDTA, adjust the pH to 7.2 with KOH). Chondrisomes were
subsequently pelleted by centrifugation (10,000 g for 10 minutes),
resuspended in RPMI media, and applied to recipient INS1 cell at a
concentration of 40 ug per 100,000 cells.
[1086] INS1 cells were seeded at 30,000 cells per well in 96 well
plate. After 24 hours, BAT chondrisome preparations were applied to
the cells. Cells were incubated with or without chondrisomes for 48
hours at 37 C and 5% CO2. Prior to imaging lipid droplets, cells
were incubated with 3.1 uM Nile Red for 15 minutes in RPMI media
while maintained at 37 C and 5% CO2. Cells were then washed with
PBS and imaged on Celigo Imaging Cell Cytometer (Brooks Life
Science Systems). Cellular lipid levels were determined by average
integrated intensity of Nile red signals across the cells over the
entire well. To quantify red fluorescence intensity per cell in
INS1s, cells were imaged using red (531/40 excitation; 629/53
emission) fluorescence channel in each well. Analysis parameters
for images acquired by Celigo Imaging Cell Cytometer were optimized
to identify individual INS1 cells based on fluorescence, and the
average red fluorescence intensity per cell was determined.
Analysis settings were determined to identify fluorescent cells
distinguishable from background fluorescence. Exposure and analysis
settings were kept constant for each condition. Average
fluorescence intensity per cell values were determined by the
average integrated intensity per cell values in order to exclude
error from background pixels included in identified cell regions.
At least 5,000 cells were analyzed per well, with 3 well replicates
per experiment. Using this assay, we find that the delivery of the
chondrisome preparation results in a decrease in cellular lipid
levels by 15-45% relative to the control group that did not receive
chondrisomes (see FIG. 1).
Example F-10: Quantification of Exogenous Protein Delivery
[1087] This assay pertains specifically to HEPG2 cells cultured in
DMEM media supplemented with 10% fetal bovine serum (ThermoFisher).
Chondrisome preparations were generated (example 1-4) from brown
adipose tissue from C57bl/6 mice (Charles River Laboratories).
Chondrisome preparation protein content was assessed by BCA
(example A-5) and the preparation remained on ice until the
following protocol was initiated (within 20 minutes from
isolation). The chondrisome sample was resuspended in SHE buffer
(250 mM sucrose, 5 mM HEPES, 2 mM EDTA, adjust the pH to 7.2 with
KOH). Chondrisomes were subsequently pelleted by centrifugation
(10,000 g for 10 minutes), resuspended in DMEM media, and applied
to recipient HEPG2 cell at a concentration 20 ug per 100,000
cells.
[1088] HEPG2 cells were seeded at 100,000 cells per well in 12 well
plate. After 24 hours, BAT chondrisomes were applied to the cells.
Cells were incubated with chondrisomes for 24 and 48 hours at 37 C
and 5% CO2. Prior to lysing the HEPG2 cells and extracting protein,
DMEM media was collected from each well, cells were detached from
wells by using 0.25% Trypsin. Cells were pelleted by centrifugation
(1500 g for 10 min) and the supernatant media was collected to
remove any exogenous brown adipocyte chondrisomes that had not been
internalized into the recipient HEPG2 cells. The cell pellet was
lysed using RIPA buffer plus protease inhibitor (1:100) on ice.
Cell pellet was incubated on ice for 15-30 minutes before being
centrifuged at 12,000 g for 10 min at 4 C. Cell lysate was then
collected maintained at -20 C for western blot assay.
[1089] Protein samples were made and run through the 4-12% gradient
gel via a standard western blot assay (Trudeau et al., The Journal
of Cell Biology; 214 (1): 25). Membranes were probed for UCP1
(abcam ab10983) and GAPDH antibodies (Cell Signaling 2118S). UCP1
protein level were measured by densitometery relative to GAPDH
protein level, using ImageJ software. FIG. 2 shows that the
recipient cells took up the exogenous proteins. Using this assay we
determined 0.37+/-0.2 and 1.61+/-0.8 of UCP1/GAPDH protein levels
(AU) at 24 hrs and 48 hrs after application of the chondrisomes
respectively.
Example F-11: Increase in Uncoupled Respiration
[1090] This assay pertains specifically to HEPG2 cells cultured in
DMEM media supplemented with 10% fetal bovine serum (ThermoFisher).
Chondrisome preparations were generated (example 1-4) from brown
adipose tissue from C57bl/6 mice (Charles River Laboratories).
Chondrisome preparation protein content was assessed by BCA
(example A-5) and the preparation remained on ice until the
following protocol was initiated (within 20 minutes from
isolation). The chondrisome sample was resuspended in SHE buffer
(250 mM sucrose, 5 mM HEPES, 2 mM EDTA, adjust the pH to 7.2 with
KOH).
[1091] HEPG2 cells were seeded at 12,000 cells per well in a
96-well Seahorse plate (Agilent). After 24 hours, cells were
treated with 4-90-.mu.g of BAT chondrisome protein per 100,000 cell
in 200 .mu.L media. Cells were incubated with chondrisomes for 48
hours and oxygen consumption rates of HEPG2 cells were subsequently
measured by XF96 bioenergetic assay (Agilent).
[1092] Oxygen consumption assays were initiated by removing growth
medium, replacing with low-buffered DMEM minimal medium containing
2.5 mM glucose and 1 mM glutamine (Agilent) and incubating at
37.degree. C. for 30 minutes to allow temperature and pH to reach
equilibrium. The microplate was then assayed in the XF96
Extracellular Flux Analyzer (Agilent) to measure extracellular flux
changes of oxygen and pH in the media immediately surrounding
adherent cells. After obtaining steady state oxygen consumption and
extracellular acidification rates, Palmitate & BSA (0.3 mM
palmitate; 1.2 mM BSA) plus carnitine (0.5 mM), oligomycin (2
.mu.M), which inhibits ATP synthase, and proton ionophore FCCP
(carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; 2 .mu.M),
which uncouples chondrisomes, were injected sequentially through
reagent delivery chambers for each well in the microplate to obtain
values for maximal oxygen consumption rates. Finally, 4 .mu.M
antimycin A (inhibitor of chondrisome complex III) was injected in
order to confirm that respiration changes were due mainly to
chondrisome respiration. The uncoupled respiration ratio (expressed
as %) of anitimycin A normalized oligomycin respiration rate and
antimycin normalized fatty acid respiration rate (100*(Oligomycin
OCR-Antimycin A OCR)/(Palmitate-BSA OCR-Antimycin A OCR)) was
calculated for both cells treated with chondrisomes and control
cells that were untreated. Treated HEPG2 cells were shown to have
an increase in the uncoupled respiration ratio of 10-30% over
untreated cells, see FIG. 3. In this example the increase was shown
to be >10%.
Example F-12: Inhibition of MPTP Opening Following Delivery of the
Chondrisome Preparation
[1093] Quantification of the impact of delivery of chondrisome
preparation to a cell's MPTP state was achieved with a commercially
available kit (MitoProbe Transition Pore Assay Kit, Molecular
Probes M34153). Chondrisome preparations were generated from human
fibroblast (example 1-1) or human platelets (example 1-3b).
Chondrisome preparation protein content was assessed by BCA
(example A-5) and the preparation remains on ice until the
following protocol was initiated (within 120 minutes from
isolation). The assay was performed following the manufacturer's
recommendations but briefly described here.
[1094] Though any cell type can be used for the assay, this example
pertains specifically to Leigh syndrome fibroblast cells acquired
from Coriell Institute (GM01503) and cultured in DMEM media
supplemented with 10% fetal bovine serum (ThermoFisher). Cells were
seeded at a density of 40,000 cells/well in a 24-well plate in DMEM
media supplemented with 10% FBS. 3 wells were for each of:
chondrisome preparation, no chondrisome preparation control. The
chondrisome preparation was added to the 3 test article wells at a
concentration of 2 .mu.g/well. Cells were allowed to incubate for
24 hours in DMEM media supplemented with 10% FBS. Following the
incubation period, cells were washed in phosphate-buffered saline
(PBS). Cells were trypsinized to remove from the cell culture plate
and centrifuged for 200.times.g for 5 minutes at room temperature.
The supernatant was discarded and the cells were resuspended in
prewarmed HBSS/Ca. Cell density was determined via hemocytometer
and adjusted to 10.sup.6 cells/mL. The final volume can be divided
into 3.times.1 ml aliquots in flow cytometry tubes per replicate to
enable 3 chemical treatments for each: one will receive calcein AM
only (tube 1), one will contain calcein AM and CoCl2 (tube 2), and
the final one will contain calcein AM, CoCl2, and ionomycin (tube
3). A sample of the cells containing no added reagents was also
prepared for instrument set up. Tubes 1, 2, and 3, receive 5 .mu.L
of the 2 .mu.M working solution of calcein AM. Tubes 2 and 3
receive 5 .mu.L of CoCl2 (supplied with the kit). Tube 3 receives 5
.mu.L of ionomycin. All tubes were mixed well and incubated at 37 C
for 15 minutes, protected from light. .about.3.5 mL of HBSS/Ca
buffer was added to the tubes and the cells were pelleted by
centrifugation. This step serves to remove excess staining and
quenching reagents. The pellet was resuspended in .about.400 .mu.L
of flow cytometric analysis buffer. After staining, samples on ice
and analyzed within one hour.
[1095] The samples were analyzed using a flow cytometer with 488 nm
excitation and emission filters appropriate for fluorescein. The
change in fluorescence intensity between tubes 2 and 3 indicates
the continuous activation of chondrisome permeability transition
pores. The pore state was quantified by the ratio of the ratio of
tube 3 fluorescence (open pore):tube 2 fluorescence (chondrisome
signal only), with higher values indicating a more open pore. Using
this assay, a 2 ug dose of chondrisomes showed <10% increase in
pore opening relative to untreated cells.
Example F-13: Increased Akt Activation
[1096] Quantification of the impact of delivery of chondrisome
preparation to a cell's AKT activation state was achieved with a
commercially available kit (Akt Activity Assay kit, Abcam,
ab65786). Chondrisome preparations were generated from human
fibroblasts (example 1-1) or human platelets (1-3b). Chondrisome
preparation protein content was assessed by BCA (example A-5) and
the preparation remains on ice until the following protocol was
initiated (within 20 minutes from isolation). The assay was
performed following the manufacturer's recommendations but briefly
described here.
[1097] Though any cell type can be used for this assay, this
particular example pertains specifically to Leigh syndrome
fibroblast cells acquired from Coriell Institute (GM01503) and
cultured in DMEM media supplemented with 10% fetal bovine serum
(ThermoFisher). Cells were seeded at a density of 80,000 cells/well
in a 12-well plate in DMEM media supplemented with 10% FBS.
Duplicate wells were for each of: chondrisome preparation, no
chondrisome preparation control. The chondrisome preparation was
added to the 2 test article wells at a concentration of 8 and 32
.mu.g/well. Cells were allowed to incubate for 24 hours in DMEM
media supplemented with 10% FBS. Following the incubation period,
cells were washed in ice-cold phosphate-buffered saline (PBS).
Cells were lysed by adding 100 uL of ice-cold Kinase Extraction
Buffer and scraping with a cell scraper to collect the lysate. The
collected samples were incubated on ice for 5 minutes followed by
centrifugation at 13,000 rpm for 10 minutes at 4 C. The supernatant
was transferred to a new tube as the cell lysate, and duplicate
samples were combined to make one sample per group.
[1098] 2 uL of Akt Specific Antibody was added to the 200 uL of
cell lysate and incubated at room temperature on a rotator for 45
minutes. Next, 50 uL of Protein A-Sepharose slurry was added to
each sample followed by incubation at room temperature on a rotator
for 1 hour. The samples were centrifuged at 15,000 rpm for 2
minutes, and the supernatant was aspirated. The pelleted beads were
washed once by adding 0.5 mL of Kinase Extraction buffer, followed
by centrifugation to pellet the beads and aspiration of the
supernatant. The pelleted beads were washed once more by adding 0.5
mL Kinase Assay buffer, followed by centrifugation to pellet the
beads and aspiration of the supernatant. 50 uL of Kinase Assay
Buffer was added to the pelleted beads to resuspend. Subsequently 2
uL of GSK-3.alpha. Protein/ATP Mixture was added and the sample was
incubated at 30 C for 2 hours. The beads were pelleted by
centrifugation and 45 uL of 1.times. NuPAGE LDS Sample Buffer
(ThermoFisher #NP0007, diluted in Kinase Assay Buffer) was added to
the beads to resuspend them. The samples were then boiled at 95 C
for 3 minutes, followed by centrifugation to pellet the beads. The
supernatant was transferred to a new microcentrifuge tube and used
as the protein samples for Western blot analysis.
[1099] Protein samples were run through the 4-12% gradient gel via
a standard western blot assay (see Example F-10) and transferred to
a PVDF membrane. After blocking in 3% BSA in tris-buffered saline
with 0.2% Tween-20 (TBST), the membrane was probed with rabbit
anti-Phospho-GSK-3.alpha.(Ser 21) Specific Antibody at a 1:1000
dilution in blocking buffer. Following three washes with TBST, the
membrane was incubated with 1:3000 anti-rabbit IgG, HRP-linked
antibody (Cell Signaling #7074S) diluted in blocking buffer and
then washed three times with TBST before exposing to a
chemiluminescent SuperSignal West Femto Maximum Sensitivity
Substrate (Thermo Scientific #34095) to detect the protein signals.
Total Akt levels in the samples was confirmed by stripping the
membrane with 6.2M guanidinium hydrochloride and reprobing with Akt
antibody (provided with kit). The densitometric values were used
for adjustment of any differences in loading. Densitometric
analysis of the Western blot signals was performed at
non-saturating exposures and analyzed using the ImageJ gel analyzer
function. Representative bands are shown from Leigh fibroblasts
treated with 8 or 32 ug of human fibroblast or platelet
chondrisomes for 24 hours. Treatment with 32 ug isolated
chondrisomes increased phospho-GSK-3.alpha./total Akt levels in
Leigh fibroblast cells 200.+-.80% (see FIG. 4).
Example F-14: Modulation of Cellular Nicotinamide Adenine
Dinucleotide Pools
[1100] Modulation of reductive stress in cells that have been
treated with donor chondrisomes can be determined using
commercially-available NAD/NADH kits, such as the abcam
colorimetric assay (catalog number ab65348). Briefly, cells can be
treated with chondrisomes isolated from human tissue, whole blood,
blood-derived products, or cultured fibroblasts for 24-72 hours, as
described in previous examples. Upon cessation of treatment, cells
are harvested, washed in PBS, and pelleted by centrifuging at
2,000.times.g for 5 min. The supernatant is discarded, and the cell
pellet is treated with the provided NAD/NADH extraction buffer and
two freeze/thaw cycles (20 minutes on dry ice followed by 10
minutes at room temperature). The extract is then centrifuged at
12,000.times.g for 5 minutes at 4.degree. C. to pellet any
insoluble material. The resulting supernatant contains the
extracted NAD/NADH, which is then transferred to a separate
microcentrifuge tube.
[1101] Within the cellular extract, there are potentially numerous
NADH-consuming enzymes that need to be removed prior to proceeding
with the assay. To accomplish this removal, the extracts are
filtered through 10 kDa spin columns, also available through abcam
(catalog number ab93349). The samples are pipetted into the spin
columns, followed by centrifugation at 10,000.times.g for 10
minutes at 4.degree. C. The filtrate is then collected and placed
on ice.
[1102] In order to measure total nicotinamide adenine dinucleotide
pools (i.e., NAD.sup.++NADH), the filtrate can be assayed as is.
However, in order to assay NADH alone, all NAD.sup.+ must be
decomposed from the sample. To accomplish this, an aliquot (200
.mu.L) of the cellular extract is pipetted into a microcentrifuge
tube and heated at 60.degree. C. for 30 minutes in a heating block,
leading to the selective decomposition of NAD.sup.+. Samples are
then cooled on ice.
[1103] The total nicotinamide adenine dinucleotide and NADH samples
are pipetted into a 96-well microplate (1-50 .mu.L/well) along with
a NADH standard curve and incubated with the provided NAD Cycling
Buffer and Cycling Enzyme Mix for 5 minutes. The NADH Develop is
then added to each well, followed by incubation at room temperature
for 1-4 hours. During this incubation, multiple readings can be
acquired in a plate-reading spectrophotometer set to measure
absorbance at 450 nm in kinetic mode. The sample will reach a
plateau absorbance value, which can be used in subsequent
calculations.
[1104] To perform the analysis, the absorbance from the sample
alone (no treatment with NADH Developer) should be subtracted from
the Developer-treated sample. The corrected absorbance values for
the samples are compared against the standard curve and the
concentration of total nicotinamide adenine dinucleotide pools and
NADH calculated as follows:
Total NAD Concentration (Total NAD/Sample Volume).times.Dil.
Factor
NADH Concentration (NADH/Sample Volume).times.Dil. Factor
[1105] The total NAD and NADH concentrations are then be normalized
to the amount of cellular protein, derived from the BCA assay
performed following the freeze/thaw extraction step. Treatment with
donor chondrisomes should decrease total NAD/NADH ratio compared to
the untreated control cells by 5-25%.
Example F-15: Improved Functional Cardiac Metrics
[1106] This example assesses the impact of a chondrisome
preparation on acute myocardial ischemia in a rabbit model.
[1107] The chondrisome preparation was generated as follows:
pectoral muscle tissue was obtained from 2 6 mm biopsy samples from
a healthy donor rabbit and kept at 4 C throughout the protocol.
Solid tissue was minced into small pieces in PBS without any
chelators (e.g. EGTA) and then tissue pieces were transferred into
isolation buffer (IB; 300 mM sucrose, 10 mM K-HEPES, and 1 mM
K-EGTA). The tissue was then homogenized using the "m-mitotissue"
mitochondrial isolation program on the Miltenyl GentleMACS
Dissociator. 1 mg of bacterial protease (subtilisin A) was added to
the homogenate and then incubated on ice for 10 minutes. The
solution was then centrifuged at 750 g for 4 minutes and the
supernatant was sequentially filtered through to 40 um, 40 um and
10 um filters. The filtrate was then centrifuged at 10,000 g for 10
minutes to concentrate the chondrisomes. The filtrate was removed
and the resulting chondrisome pellet was resuspended in respiration
buffer (RB; 250 mM sucrose, 2 mM KH PO 10 mM MgCl 20 mM K-HEPES
Buffer (pH 7.2) and 0.5 mM KEGTA (pH 8.0)) and then diluted
10.times. in the same RB. No BSA was used in any part of the
isolation process.
[1108] The rabbit model of acute myocardial ischemia was prepared
as follows: Male New Zealand White rabbits (2-2.5 kg) were
anesthetized with isoflurane and anesthesia depth checked to ensure
ear pinch and blink reflex, and jaw tone were lost. Rabbits were
intubated and mechanically ventilated with room air. Core
temperature was monitored continually and maintained at
>36.degree. C. using a heating pad. To define baseline
cardiovascular function and hemodynamic parameters, a Doppler
echocardiography and electrocardiograms (ECGs) were be recorded.
Blood draws for biomarkers quantification were be taken at 0 hrs, 6
hrs and 24 hrs. Pulse oximetry probe on the ear measures heart rate
and level of oxygenation. Following baseline acquisitions, surgery
was initiated by making a 2-3 cm left intercostal thoracotomy (4th
intercostal space) to expose the heart. A snare occluder was be
placed around the first antero-lateral branch of the LAD 5 to 10 mm
distal from the apex (to obtain as close as possible to 30% of LV
AAR required). Heparin was injected (3 mg/kg IV) via a catheter
placed in the marginal ear vein before snaring the LAD to prevent
the forming of blood clots inside the coronary artery while
temporarily occluded. The coronary artery was occluded at time 0,
defined by ECG ST segment elevation indicative of myocardial
ischaemia (MI).
[1109] Experimental protocol: Following 29 min of regional ischemia
the chondrisome preparation (or vehicle) was injected into the area
at risk in a consistent pattern that covers the blanched area. The
chondrisome preparation (between 50-75 minutes following tissue
biopsy) was injected at a dose of 6 ug/kg+/-2 ug/kg. Hearts
received 8.times.0.1 ml injections via a sterile 1-ml insulin
syringe with a 28-gauge needle. After 30 minutes of acute MI, the
occluder was be re-opened to allow reperfusion for a period of 24
h. All closures performed under anesthesia Animals were weaned from
anesthesia and allowed to recover on a warming pad until fully
recovered (awake and able to walk on their own). Thirty minutes
prior to the end of the procedure, pain prophylaxis medication was
administered. At the end of the 24 h reperfusion period, the
rabbits were re-anesthetized with isoflurane. Cardiovascular and
hemodynamic parameters were measured via Doppler echocardiography,
and ECG recording, and blood draws performed. At the end of those
procedures, the rabbits were sacrificed and the hearts excised and
mounted into a Langendorff apparatus to allow retrograde perfusion
of the heart with stains and dyes. Once the blood was flushed out,
the LAD was re-occluded with the snare and Evans blue was perfused
through the aorta to stain the perfused zone with deep blue color,
hence delineating the "At risk" zone as the non-stained area. The
hearts were then removed from the Langendorff device. The hearts
were cut into transverse slices sections and incubated in TTC for
15 minutes at 37 C. The surviving tissue will turn deep red. Dead
tissue will be grey-white. The area at risk of the myocardium was
determined by negative staining with Evan's blue, and the infarcted
myocardium was gray-white. Morphometric readouts were used to
calculate the left ventricular area, risk area, and infarct.
Infarct size was then expressed as a percentage of the risk
area.
[1110] Mean values were calculated for the 8 animals per group
(vehicle and chondrisome preparation). Using this model the
following efficacy signals were observed with the delivery of the
chondrisome preparation:
[1111] Echocardiography measures of cardiac function, Chondrisome
vs. vehicle: [1112] Improvement of fractional shortening relative
to baseline [1113] No change in end diastolic volume relative to
baseline [1114] Improvement of end systolic volume relative to
baseline [1115] Improvement of stroke volume relative to baseline
[1116] Improvement of ejection fraction relative to baseline [1117]
Improvement of cardiac output relative to baseline [1118]
Improvement of cardiac index relative to baseline
TABLE-US-00032 [1118] % change from baseline Vehicle Chondrisome
Fractional Shortening -27 .+-. 9 -17 .+-. 14 End diastolic volume
-9 .+-. 22 -10 .+-. 19 End systolic volume 48 .+-. 39 37 .+-. 62
Stroke Volume -41 .+-. 19 -31 .+-. 22 Ejection Fraction -36 .+-. 15
-23 .+-. 23 Cardiac Output -36 .+-. 22 -29 .+-. 20 Cardiac Index
-36 .+-. 22 -29 .+-. 20
[1119] Staining assessment of infarcted area: [1120] Decrease in
infarcted area relative to area at risk [1121] Vehicle: group
Infarct area as % of area at risk of the left ventricle: 46%.+-.12%
[1122] Chondrisome group: Infarct area as % of area at risk of the
left ventricle: 27%.+-.7%
[1123] Serum markers of cardiac injury: [1124] Decreased CKNB
levels relative to vehicle at 6 hrs and 24 hrs [1125] Decreased
cTnI at 6 hrs relative to vehicle
TABLE-US-00033 [1125] 6 hr 24 hr Vehicle Chondrisome Vehicle
Chondrisome CKMB (U/L) 500 .+-. 285 295 .+-. 63 624 .+-. 335 358
.+-. 143 cTnI (ng/ml) 5 .+-. 2 3 .+-. 2 2 .+-. 2 2 .+-. 2
Example F-16: Improved Functional Cardiac Metrics
[1126] This example assesses the impact of a chondrisome
preparation on acute myocardial ischemia in a rabbit model.
[1127] Chondrisome preparation: Pectoral muscle tissue was obtained
from 2 6 mm biopsy samples from a healthy rabbit and kept at 4 C
throughout the protocol. Solid tissue was minced into small pieces
in 2 ml MSHE (200 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM
EDTA, adjust the pH to 7.4 with KOH)+0.5% BSA buffer (0.2-1 g
tissue per 8 mL of MSHE+0.5% BSA buffer) using scissors if
necessary and then homogenized with a glass Potter Elvehjem
homogenizer using Teflon pestle operated at 1600 rpm for 15
strokes. The material was centrifuged at 1000 g for 10 min at
4.degree. C. The supernatant was then distributed into 6 2 mL
microcentrifuge tubes and centrifuged at 10,000 g for 10 min at
4.degree. C. The resulting chondrisome pellets were resuspended in
2 mL of MSHE+0.5% BSA buffer and re-centrifuged at 10,000 g for 10
min at 4.degree. C. The final chondrisome pellet was resuspended in
1 ml of delivery buffer (250 mM sucrose, 2 mM KH2PO4, 10 mM MgCl2,
20 mM K-HEPES, 0.5 mM K-EGTA, pH 7.4) and then diluted 10.times. in
the same buffer. Tissue and chondrisome solutions, including
buffers, were kept on ice at all times.
[1128] Animal model: Male New Zealand White rabbits (2-2.5 kg) were
anesthetized with isoflurane and anesthesia depth checked to ensure
ear pinch and blink reflex, and jaw tone were lost. Rabbits were
intubated and mechanically ventilated with room air. Core
temperature was monitored continually and maintained at
>36.degree. C. using a heating pad. To define baseline
cardiovascular function and hemodynamic parameters, a Doppler
echocardiography and electrocardiograms (ECGs) were be recorded.
Blood draws for biomarkers quantification were be taken at 0 hrs, 6
hrs and 24 hrs. Pulse oximetry probe on the ear measures heart rate
and level of oxygenation. Following baseline acquisitions, surgery
was initiated by making a 2-3 cm left intercostal thoracotomy (4th
intercostal space) to expose the heart. A snare occluder was placed
around the first antero-lateral branch of the LAD 5 to 10 mm distal
from the apex (to obtain as close as possible to 30% of LV AAR
required). Heparin was injected (3 mg/kg IV) via a catheter placed
in the marginal ear vein before snaring the LAD to prevent the
forming of blood clots inside the coronary artery while temporarily
occluded. The coronary artery was occluded at time 0, defined by
ECG ST segment elevation indicative of myocardial ischaemia (MI).
Following 29 min of regional ischemia the chondrisome preparation
(or vehicle) was injected into the area at risk in a consistent
pattern that covers the blanched area.
[1129] Experiment: The chondrisome preparation (between 50-75
minutes following tissue biopsy) was injected at a dose of 36+/-5
ug/kg. Hearts received 8.times.0.1 ml injections via a sterile 1-ml
insulin syringe with a 28-gauge needle. After 30 minutes of acute
MI, the occluder was re-opened to allow reperfusion for a period of
24 h. All closures performed under anesthesia Animals were weaned
from anesthesia and allowed to recover on a warming pad until fully
recovered (awake and able to walk on their own). Thirty minutes
prior to the end of the procedure, pain prophylaxis medication was
administered.
[1130] At the end of the 24 h reperfusion period, the rabbits were
re-anesthetized with isoflurane. Cardiovascular and hemodynamic
parameters were measured via Doppler echocardiography, and ECG
recording, and blood draws performed. At the end of those
procedures, the rabbits were sacrificed and the hearts excised and
mounted into a Langendorff apparatus to allow retrograde perfusion
of the heart with stains and dyes. Once the blood was flushed out,
the LAD was re-occluded with the snare and Evans blue was perfused
through the aorta to stain the perfused zone with deep blue color,
hence delineating the "At risk" zone as the non-stained area. The
hearts were then removed from the Langendorff device. The hearts
were cut into transverse slices sections and incubated in TTC for
15 minutes at 37 C. The surviving tissue will turn deep red. Dead
tissue will be grey-white. The area at risk of the myocardium was
determined by negative staining with Evan's blue, and the infarcted
myocardium was gray-white. Morphometric readouts were used to
calculate the left ventricular area, risk area, and infarct.
Infarct size was then expressed as a percentage of the risk
area.
[1131] Mean values were calculated for the 8 animals per group
(vehicle and chondrisome preparation). Using this model the
following efficacy signals were observed with the delivery of the
chondrisome preparation:
[1132] Echocardiography measures of cardiac function, Chondrisome
vs. vehicle: [1133] No change in fractional shortening relative to
baseline [1134] No change in end diastolic volume relative to
baseline [1135] Smaller volume of end systolic volume relative to
baseline for treatment [1136] Improvement of stroke volume relative
to baseline [1137] Improvement of ejection fraction relative to
baseline [1138] Improvement of cardiac output relative to baseline
[1139] Improvement of cardiac index relative to baseline
TABLE-US-00034 [1139] % change from baseline Vehicle Chondrisome
Fractional Shortening -14 .+-. 15 -13 .+-. 10 End diastolic volume
-18 .+-. 19 -22 .+-. 12 End systolic volume 17 .+-. 37 -18 .+-. 31
Stroke Volume -36 .+-. 21 -24 .+-. 12 Ejection Fraction -25 .+-. 17
0 .+-. 14 Cardiac Output -34 .+-. 30 -24 .+-. 17 Cardiac Index -38
.+-. 25 -24 .+-. 17
[1140] Staining assessment of infarcted area: [1141] No change in
infarcted area relative to area at risk [1142] Vehicle: group
Infarct area as % of area at risk of the left ventricle: 54%.+-.18%
[1143] Chondrisome group: Infarct area as % of area at risk of the
left ventricle: 50%.+-.24%
[1144] Serum markers of cardiac injury: [1145] No improvement in
CKNB levels relative to vehicle at 6 hrs and 24 hrs [1146] No
improvement in cTnI at 6 hrs relative to vehicle at 6 hrs and 24
hrs [1147] Greatly reduced serum hydrogen peroxide at 15-minutes
post reperfusion: [1148] Vehicle group: 146.+-.146 uM [1149]
Chondrisome group: 18.+-.4 uM
TABLE-US-00035 [1149] 6 hr 24 hr Vehicle Chondrisome Vehicle
Chondrisome CKMB (U/L) 394 .+-. 122 455 .+-. 166 325 .+-. 89 666
.+-. 565 cTnI (ng/ml) 476 .+-. 188 588 .+-. 290 412 .+-. 387 702
.+-. 802
Example F-17: No Acute Immune Effect
[1150] This example assesses the acute immune response of delivery
of a chondrisome preparation.
[1151] 7.25 mg of chondrisomes (as measured by protein
concentration via the BCA assay in example A-5) per kg of mouse
weight diluted in MSHE (200 mM mannitol, 70 mM sucrose, 10 mM
HEPES, 1 mM EDTA, adjust the pH to 7.4 with KOH) or vehicle control
without chondrisomes were injected intravenously into the tail vein
or subcutaneously into the intrascapular area of 5 week old female
C57Bl/6 mice in a single injection. Mice were monitored
continuously for the first 30 minutes after injection, every 20
minutes for the four subsequent hours, and then 4 times per day for
the next 24 hours. Animals exhibited no adverse reaction to
chondrisomes over this time.
[1152] 24 hours after injection, mice were euthanized via CO.sub.2
inhalation. Following euthanasia, blood was collected via cardiac
puncture in K.sub.2EDTA and processed to plasma. Lungs, liver, and
spleen were harvested and fixed in buffered formalin. The plasma
was evaluated for an innate immune reaction via ELISA using the
Life Technologies Mouse Inflammatory 4-Plex kit on a Luminex Magpix
machine. The ELISA measured the concentrations of IL-1-beta, IL-6,
GM-CSF, and TNF-alpha. The concentration of these cytokines in the
plasma was measured by establishing a standard curve with serial
dilutions of known concentrations of the cytokines supplied by the
manufacturer in the kit. The concentration of cytokines in both
chondrisomes-treated and vehicle-treated animals at each route of
administration was below the level of detection for the ELISA kit,
and thus were not elevated by the chondrisomes or vehicle treatment
(FIG. 5). Specifically, the ELISA kit measured less than 23.56 pg
IL-1-beta, less than 28.13 pg IL-6, less than 15.33 pg GM-CSF, and
less than 23.26 pg TNF-alpha per ml of plasma. Lungs, liver, and
spleen fixed in buffered formalin were processed to 5 micron-thick
histology slides and stained with hematoxylin and eosin stain
(H&E). The histology slides were analyzed for elevated
immune-cell infiltration into the tissue, and there was no
difference between animals injected with chondrisomes or vehicle
(representative images in FIG. 6).
Example F-18: Metabolic Stimulation
[1153] This example assesses the effect of in-vivo delivery of
chondrisomes isolated from brown adipose tissue.
[1154] Vehicle or 1-2 mg of chondrisomes (as measured by protein
concentration via the BCA assay in example A-5) derived from the
brown adipose tissue of C57BL/6J mice (as described in example 1-4)
incubated at 4 degrees for 7 days were injected into the left
periogonadal white adipose pad of 24-week old diet-induced obese
C57BL/6J mice purchased from Jackson Laboratory via 4 injections of
25 .mu.l each. Immediately following the administration of
chondrisomes, the mice were placed into a Comprehensive Lab Animal
Monitoring System (CLAMS). The CLAMS system measures the oxygen
consumption, carbon dioxide production, respiratory exchange ratio,
energy expenditure, food consumption, total activity, and
ambulatory activity of each mouse. The animals were treated with
chondrisomes or vehicle then placed back into the CLAMS chamber
each day for 5 days, and remain in the CLAMS chamber and not
injected for a 6.sup.th and 7.sup.th day. Prior to injections on
the first day, the percent fat and percent lean body composition of
each mouse was measured via MRI, and the body weight was measured
on a weight scale. Over the course of the experiment the animals
were fed a diet that was 60% fat (Diet D12492 made by Research
Diets). At the end of the 7.sup.th day, the animals were removed
from the CLAMS chamber, the percent fat and percent lean body
composition of each mouse was measured via MRI, and the body weight
was measured on a weight scale. The animals were then sacrificed
via cervical dislocation. Blood was drawn via cardiac puncture and
processed to serum. The serum was processed by centrifuging blood
samples at 1.6 g for 10 min at 4 C. The perigonadal white adipose
pad from both the left and right side of the animal were removed
and weighed on an weight scale. The fat pad was cut in half. Half
of the fat pad was fixed in formalin and half was snap-frozen at
-80 degrees. The liver of the animals was also removed and cut in
half. Half of the liver fixed in formalin and half was snap-frozen
at -80 degrees. To determine whether the chondrisome treatment
affected the mass of the injected perigonadal fat pad, the mass of
the fat pad was divided by the mass of the uninjected fat pad from
the same animal.
[1155] Results:
[1156] Mice that received chondrisomes had a lower ratio of
injected to un-injected fat pad mass than mice that received
vehicle control (FIG. 7).
[1157] The concentration of total cholesterol and total
triglycerides in the serum of animals injected in the periogondal
fat pad with brown adipose tissue chondrisomes or vehicle was
measured using IDEXX Dry-Slide Technology. 70 .mu.l of serum was
loaded onto catalyst sample cups and measured on a IDEXX Catalyst
DX Chemistry Analyzer. Animals treated with chondrisomes had
decreased serum cholesterol and serum triglycerides relative to
vehicle treated animals (FIG. 8; FIG. 9).
Example F-19: No Adaptive Immune Effect
[1158] This example assesses the adaptive immune response of
delivery of a chondrisome preparation.
[1159] 5 mg of chondrisomes (as measured by protein concentration
via BCA assay) per kg of mouse weight diluted in MSHE buffer (200
mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM EDTA, pH adjusted to
7.4 with KOH) was injected in a volume of 25 .mu.l into the left
footpad of a 5-week old female C57BL/6J mouse, and 25 .mu.l of the
vehicle (MSHE) control was injected into the right footpad of the
same mouse. Chondrisomes were isolated from leg gastrocnemius
muscle from C57bl/6 mice (syngeneic) or Balb/c mice (allogenic)
using the isolation protocol described in example 1-2a. 7 days
after the injection, half of the mice were sacrificed via CO.sub.2
asphyxiation followed by cervical dislocation and their popliteal
lymph node proximal to each paw was removed and weighed on an
analytical scale.
[1160] The ratio of the weight of chondrisomes-injected to
vehicle-injected popliteal lymph nodes was calculated as a measure
of the immunogenicity of a single treatment. The average ratio of
the weight of lymph nodes from the mice that received syngeneic
chondrisomes was 1.03 and from the mice that received the
allogeneic chondrisomes was 2.09 (FIG. 10). 14 days later (21 days
after the initial injection), 5 mg of chondrisomes (as measured by
protein concentration via the BCA assay) per kg of mouse weight
diluted in MSHE buffer was injected in a volume of 25 .mu.l into
the left footpad of the remaining live mice, and 25 .mu.l of the
vehicle control were injected into the right footpad of the same
mouse. 5 days later (on the 26.sup.th day of the experiment), the
mice were sacrificed via CO.sub.2 asphyxiation followed by cervical
dislocation and their popliteal lymph node was removed and weighed
on an analytical scale.
[1161] The ratio of the weight of chondrisomes-injected to
vehicle-injected paw lymph nodes was calculated to measure whether
there was an adaptive immune response to the treatment as indicated
by an increased weight ratio as compared to the single treatment.
The average ratio of the weight of lymph nodes from the mice that
received syngeneic chondrisomes after the second injection was 1.43
and from the mice that received the allogeneic chondrisomes was
0.99 (FIG. 10). The increase in the ratio of injected to uninjected
lymph node weights observed between the first and second injection
for mice that received syngeneic chondrisomes was not statistically
significant, and the ratio decreased for mice the received
allogeneic chondrisomes. Thus, there was not an adaptive immune
response to chondrisomes.
TABLE-US-00036 SEQUENCES Human UCP1:
MGGLTASDVHPTLGVQLFSAGIAACLADVITFPLDTAKVRLQVQGECPTSSVIRYKGVLG
TITAVVKTEGRMKLYSGLPAGLQRQISSASLRIGLYDTVQEFLTAGKETAPSLGSKILAG
LTTGGVAVFIGQPTEVVKVRLQAQSHLHGIKPRYTGTYNAYRIIATTEGLTGLWKGTTPN
LMRSVIINCTELVTYDLMKEAFVKNNILADDVPCHLVSALIAGFCATAMSSPVDVVKTRF
INSPPGQYKSVPNCAMKVFTNEGPTAFFKGLVPSFLRLGSWNVIMFVCFEQLKRELSKSR
QTMDCAT (SEQ ID NO: 1) Human UCP2: MVGFKATDVP PTATVKFLGA GTAACIADLI
TFPLDTAKVR LQIQGESQGP VRATASAQYR GVMGTILTMV RTEGPRSLYN GLVAGLQRQM
SFASVRIGLY DSVKQFYTKG SEHASIGSRL LAGSTTGALA VAVAQPTDVV KVRFQAQARA
GGGRRYQSTV NAYKTIAREE GFRGLWKGTS PNVARNAIVN CAELVTYDLI KDALLKANLM
TDDLPCHFTS AFGAGFCTTV IASPVDVVKT RYMNSALGQY SSAGHCALTM LQKEGPRAFY
KGFMPSFLRL GSWNVVMFVT YEQLKRALMA ACTSREAPF (SEQ ID NO: 2) Human
UCP3: MVGLKPSDVP PTMAVKFLGA GTAACFADLV TFPLDTAKVR LQIQGENQAV
QTARLVQYRG VLGTILTMVR TEGPCSPYNG LVAGLQRQMS FASIRIGLYD SVKQVYTPKG
ADNSSLTTRI LAGCTTGAMA VTCAQPTDVV KVRFQASIHL GPSRSDRKYS GTMDAYRTIA
REEGVRGLWK GTLPNIMRNA IVNCAEVVTY DILKEKLLDY HLLTDNFPCH FVSAFGAGFC
ATVVASPVDV VKTRYMNSPP GQYFSPLDCM IKMVAQEGPT AFYKGFTPSF LRLGSWNVVM
FVTYEQLKRA LMKVQMLRES PF (SEQ ID NO: 3) Human UCP4: MSVPEEEERL
LPLTQRWPRA SKFLLSGCAA TVAELATFPL DLTKTRLQMQ GEAALARLGD GARESAPYRG
MVRTALGIIE EEGFLKLWQG VTPAIYRHVV YSGGRMVTYE HLREVVFGKS EDEHYPLWKS
VIGGMMAGVI GQFLANPTDL VKVQMQMEGK RKLEGKPLRF RGVHHAFAKI LAEGGIRGLW
AGWVPNIQRA ALVNMGDLTT YDTVKHYLVL NTPLEDNIMT HGLSSLCSGL VASILGTPAD
VIKSRIMNQP RDKQGRGLLY KSSTDCLIQA VQGEGFMSLY KGFLPSWLRM TPWSMVFWLT
YEKIREMSGV SPF (SEQ ID NO: 4) Human UCP5 MGIFPGIILI FLRVKFATAA
VIVSGHQKST TVSHEMSGLN WKPFVYGGLA SIVAEFGTFP VDLTKTRLQV QGQSIDARFK
EIKYRGMFHA LFRICKEEGV LALYSGIAPA LLRQASYGTI KIGIYQSLKR LFVERLEDET
LLINMICGVV SGVISSTIAN PTDVLKIRMQ AQGSLFQGSM IGSFIDIYQQ EGTRGLWRGV
VPTAQRAAIV VGVELPVYDI TKKHLILSGM MGDTILTHFV SSFTCGLAGA LASNPVDVVR
TRMMNQRAIV GHVDLYKGTV DGILKMWKHE GFFALYKGFW PNWLRLGPWN IIFFITYEQL
KRLQI (SEQ ID NO: 5) subunit VIII of human cytochrome c oxidase:
ATGTCCGTCC TGACGCCGCT GCTGCTGCGG GGCTTGACAG GCTCGGCCCG GCGGCTCCCA
GTGCCGCGCG CCAAGATCCA TTCGTTG (SEQ ID NO: 6) human Sirt3:
MVGAGISTPS GIPDFRSPGS GLYSNLQQYD LPYPEAIFEL PFFFHNPKPF FTLAKELYPG
NYKPNVTHYF LRLLHDKGLL LRLYTQNIDG LERVSGIPAS KLVEAHGTFA SATCTVCQRP
FPGEDIRADV MADRVPRCPV CTGVVKPDIV FFGEPLPQRF LLHVVDFPMA DLLLILGTSL
EVEPFASLTE AVRSSVPRLL INRDLVGPLA WHPRSRDVAQ LGDVVHGVES LVELLGWTEE
MRDLVQRETG KLDGPDK (SEQ ID NO: 7) human pyruvate dehydrogenase
kinase: MRLARLLRGA ALAGPGPGLR AAGFSRSFSS DSGSSPASER GVPGQVDFYA
RFSPSPLSMK QFLDFGSVNA CEKTSFMFLR QELPVRLANI MKEISLLPDN LLRTPSVQLV
QSWYIQSLQE LLDFKDKSAE DAKAIYDFTD TVIRIRNRHN DVIPTMAQGV IEYKESFGVD
PVTSQNVQYF LDRFYMSRIS IRMLLNQHSL LFGGKGKGSP SHRKHIGSIN PNCNVLEVIK
DGYENARRLC DLYYINSPEL ELEELNAKSP GQPIQVVYVP SHLYHMVFEL FKNAMRATME
HHANRGVYPP IQVHVTLGNE DLTVKMSDRG GGVPLRKIDR LFNYMYSTAP RPRVETSRAV
PLAGFGYGLP ISRLYAQYFQ GDLKLYSLEG YGTDAVIYIK ALSTDSIERL PVYNKAAWKH
YNTNHEADDW CVPSREPKDM TTFRSA (SEQ ID NO: 8) human O-GlcNAc
transferase: MASSVGNVAD STEPTKRMLS FQGLAELAHR EYQAGDFEAA ERHCMQLWRQ
EPDNTGVLLL LSSIHFQCRR LDRSAHFSTL AIKQNPLLAE AYSNLGNVYK ERGQLQEAIE
HYRHALRLKP DFIDGYINLA AALVAAGDME GAVQAYVSAL QYNPDLYCVR SDLGNLLKAL
GRLEEAKACY LKAIETQPNF AVAWSNLGCV FNAQGEIWLA IHHFEKAVTL DPNFLDAYIN
LGNVLKEARI FDRAVAAYLR ALSLSPNHAV VHGNLACVYY EQGLIDLAID TYRRAIELQP
HFPDAYCNLA NALKEKGSVA EAEDCYNTAL RLCPTHADSL NNLANIKREQ GNIEEAVRLY
RKALEVFPEF AAAHSNLASV LQQQGKLQEA LMHYKEAIRI SPTFADAYSN MGNTLKEMQD
VQGALQCYTR AIQINPAFAD AHSNLASIHK DSGNIPEAIA SYRTALKLKP DFPDAYCNLA
HCLQIVCDWT DYDERMKKLV SIVADQLEKN RLPSVHPHHS MLYPLSHGFR KAIAERHGNL
CLDKINVLHK PPYEHPKDLK LSDGRLRVGY VSSDFGNHPT SHLMQSIPGM HNPDKFEVFC
YALSPDDGTN FRVKVMAEAN HFIDLSQIPC NGKAADRIHQ DGIHILVNMN GYTKGARNEL
FALRPAPIQA MWLGYPGTSG ALFMDYIITD QETSPAEVAE QYSEKLAYMP HTFFIGDHAN
MFPHLKKKAV IDFKSNGHIY DNRIVLNGID LKAFLDSLPD VKIVKMKCPD GGDNADSSNT
ALNMPVIPMN TIAEAVIEMI NRGQIQITIN GFSISNGLAT TQINNKAATG EEVPRTIIVT
TRSQYGLPED AIVYCNFNQL YKIDPSTLQM WANILKRVPN SVLWLLRFPA VGEPNIQQYA
QNMGLPQNRI IFSPVAPKEE HVRRGQLADV CLDTPLCNGH TTGMDVLWAG TPMVTMPGET
LASRVAASQL TCLGCLELIA KNRQEYEDIA VKLGTDLEYL KKVRGKVWKQ RISSPLFNTK
QYTMELERLY LQMWEHYAAG NKPDHMIKPV EVTESA (SEQ ID NO: 9) human OMP25:
MNGRVDYLVTEEEINLTRGPSGLGFNIVGGTDQQYVSNDSGIYVSRIKENGAAALDGRLQEGDKI
LSVNGQDLKNLLHQDAVDLFRNAGYAVSLRVQHRLQVQNGPIGHRGEGDPSGIPIFMVLVPVFA
LTMVAAWAFMRYRQQL, SEQ ID NO: 10 human TOM22:
MAAAVAAAGAGEPQSPDELLPKGDAEKPEEELEEDDDEELDETLSERLWGLTEMFPERVRSAA
GATFDLSLFVAQKMYRFSRAALWIGTTSFMILVLPVVFETEKLQMEQQQQLQQRQILLGPNTGLS
GGMPGALPSLPGKI, SEQ ID NO: 11 human TIM17A:
MEEYAREPCPWRIVDDCGGAFTMGTIGGGIFQAIKGFRNSPVGVNHRLRGSLTAIKTRAPQLGGS
FAVWGGLFSMIDCSMVQVRGKEDPWNSITSGALTGAILAARNGPVAMVGSAAMGGILLALIEG
AGILLTRFASAQFPNGPQFAEDPSQLPSTQLPSSPFGDYRQYQ, SEQ ID NO: 12 human
TIM17B:
MEEYAREPCPWRIVDDCGGAFTMGVIGGGVFQAIKGFRNAPVGIRHRLRGSANAVRIRAPQIGG
SFAVWGGLFSTIDCGLVRLRGKEDPWNSITSGALTGAVLAARSGPLAMVGSAMMGGILLALIEG
VGILLTRYTAQQFRNAPPFLEDPSQLPPKDGTPAPGYPSYQQYH, SEQ ID NO: 13 human
TIM22:
MAAAAPNAGGSAPETAGSAEAPLQYSLLLQYLVGDKRQPRLLEPGSLGGIPSPAKSEEQKMIEK
AMESCAFKAALACVGGFVLGGAFGVFTAGIDTNVGFDPKDPYRTPTAKEVLKDMGQRGMSYA
KNFAIVGAMFSCTECLIESYRGTSDWKNSVISGCITGGAIGFRAGLKAGAIGCGGFAAFSAAIDYY
LR, SEQ ID NO: 14 human TFAM:
MAFLRSMWGVLSALGRSGAELCTGCGSRLRSPFSFVYLPRWFSSVLASCPKKPVSSYLRFSKEQL
PIFKAQNPDAKTTELIRRIAQRWRELPDSKKKIYQDAYRAEWQVYKEEISRFKEQLTPSQIMSLEK
EIMDKHLKRKAMTKKKELTLLGKPKRPRSAYNVYVAERFQEAKGDSPQEKLKTVKENWKNLS
DSEKELYIQHAKEDETRYHNEMKSWEEQMIEVGRKDLLRRTIKKQRKYGAEEC, SEQ ID NO:
15 human PGC-lalpha (peroxisome proliferator-activated receptor
gamma coactivator 1-alpha):
MAWDMCNQDSESVWSDIECAALVGEDQPLCPDLPELDLSELDVNDLDTDSFLGGLKWCSDQSE
IISNQYNNEPSNIFEKIDEENEANLLAVLTETLDSLPVDEDGLPSFDALTDGDVTTDNEASPSSMPD
GTPPPQEAEEPSLLKKLLLAPANTQLSYNECSGLSTQNHANHNHRIRTNPAIVKTENSWSNKAKSI
CQQQKPQRRPCSELLKYLTTNDDPPHTKPTENRNSSRDKCTSKKKSHTQSQSQHLQAKPTTLSLP
LTPESPNDPKGSPFENKTIERTLSVELSGTAGLTPPTTPPHKANQDNPFRASPKLKSSCKTVVPPPS
KKPRYSESSGTQGNNSTKKGPEQSELYAQLSKSSVLTGGHEERKTKRPSLRLFGDHDYCQSINSK
TEILINISQELQDSRQLENKDVSSDWQGQICSSTDSDQCYLRETLEASKQVSPCSTRKQLQDQEIR
AELNKHFGHPSQAVFDDEADKTGELRDSDFSNEQFSKLPMFINSGLAMDGLFDDSEDESDKLSY
PWDGTQSYSLFNVSPSCSSFNSPCRDSVSPPKSLFSQRPQRMRSRSRSFSRHRSCSRSPYSRSRSRS
PGSRSSSRSCYYYESSHYRHRTHRNSPLYVRSRSRSPYSRRPRYDSYEEYQHERLKREEYRREYE
KRESERAKQRERQRQKAIEERRVIYVGKIRPDTTRTELRDRFEVFGEIEECTVNLRDDGDSYGFIT
YRYTCDAFAALENGYTLRRSNETDFELYFCGRKQFFKSNYADLDSNSDDFDPASTKSKYDSLDF
DSLLKEAQRSLRR, SEQ ID NO: 16 FLAG tag: DYKDDDDK (SEQ ID NO: 17)
human Acat1:
MAVLAALLRSGARSRSPLLRRLVQEIRYVERSYVSKPTLKEVVIVSATRTPIGSFLGSLSLLPATKL
GSIAIQGAIEKAGIPKEEVKEAYMGNVLQGGEGQAPTRQAVLGAGLPISTPCTTINKVCASGMKA
IMMASQSLMCGHQDVMVAGGMESMSNVPYVMNRGSTPYGGVKLEDLIVKDGLTDVYNKIHM
GSCAENTAKKLNIARNEQDAYAINSYTRSKAAWEAGKFGNEVIPVTVTVKGQPDVVVKEDEEY
KRVDFSKVPKLKTVFQKENGTVTAANASTLNDGAAALVLMTADAAKRLNVTPLARIVAFADAA
VEPIDFPIAPVYAASMVLKDVGLKKEDIAMWEVNEAFSLVVLANIKMLEIDPQKVNINGGAVSL
GHPIGMSGARIVGHLTHALKQGEYGLASICNGGGGASAMLIQKL, SEQ ID NO: 18 human
GPS2: MPALLERPKLSNAMARALHRHIMMERERKRQEEEEVDKMMEQKMKEEQERRKKKEMEERMS
LEETKEQILKLEEKLLALQEEKHQLFLQLKKVLHEEEKRRRKEQSDLTTLTSAAYQQSLTVHTGT
HLLSMQGSPGGHNRPGTLMAADRAKQMFGPQVLTTRHYVGSAAAFAGTPEHGQFQGSPGGAY
GTAQPPPHYGPTQPAYSPSQQLRAPSAFPAVQYLSQPQPQPYAVHGHFQPTQTGFLQPGGALSLQ
KQMEHANQQTGFSDSSSLRPMHPQALHPAPGLLASPQLPVQMQPAGKSGFAATSQPGPRLPFIQ
HSQNPRFYHK, SEQ ID NO: 19 Human YBX1:
MSSEAETQQPPAAPPAAPALSAADTKPGTTGSGAGSGGPGGLTSAAPAGGDKKVIATKVLGTVK
WFNVRNGYGFINRNDTKEDVFVHQTAIKKNNPRKYLRSVGDGETVEFDVVEGEKGAEAANVTG
PGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNYQNSESGEKNEGSESAPEGQAQQRRPYRR
RRFPPYYMRRPYGRRPQYSNPPVQGEVMEGADNQGAGEQGRPVRQNMYRGYRPRFRRGPPRQ
RQPREDGNEEDKENQGDETQGQQPPQRRYRRNFNYRRRRPENPKPQDGKETKAADPPAENSSA
PEAEQGGAE, SEQ ID NO: 20 Human OPA1:
MWRLRRAAVACEVCQSLVKHSSGIKGSLPLQKLHLVSRSIYHSHHPTLKLQRPQLRTSFQQFSSL
TNLPLRKLKFSPIKYGYQPRRNFWPARLATRLLKLRYLILGSAVGGGYTAKKTFDQWKDMIPDL
SEYKWIVPDIVWEIDEYIDFEKIRKALPSSEDLVKLAPDFDKIVESLSLLKDFFTSGSPEETAFRAT
DRGSESDKHFRKVSDKEKIDQLQEELLHTQLKYQRILERLEKENKELRKLVLQKDDKGIHHRKL
KKSLIDMYSEVLDVLSDYDASYNTQDHLPRVVVVGDQSAGKTSVLEMIAQARIFPRGSGEMMT
RSPVKVTLSEGPHHVALFKDSSREFDLTKEEDLAALRHEIELRMRKNVKEGCTVSPETISLNVKG
PGLQRMVLVDLPGVINTVTSGMAPDTKETIFSISKAYMQNPNAIILCIQDGSVDAERSIVTDLVS
MDPHGRRTIFVLTKVDLAEKNVASPSRIQQIIEGKLFPMKALGYFAVVTGKGNSSESIEAIREYEE
EFFQNSKLLKTSMLKAHQVTTRNLSLAVSDCFWKMVRESVEQQADSFKATRFNLETEWKNNYP
RLRELDRNELFEKAKNEILDEVISLSQVTPKHWEEILQQSLWERVSTHVIENIYLPAAQTMNSGTF
NTTVDIKLKQWTDKQLPNKAVEVAWETLQEEFSRFMTEPKGKEHDDIFDKLKEAVKEESIKRHK
WNDFAEDSLRVIQHNALEDRSISDKQQWDAAIYFMEEALQARLKDTENAIENMVGPDWKKRW
LYWKNRTQEQCVHNETKNELEKMLKCNEEHPAYLASDEITTVRKNLESRGVEVDPSLIKDTWH
QVYRRHFLKTALNHCNLCRRGFYYYQRHFVDSELECNDVVLFWRIQRMLAITANTLRQQLTNT
EVRRLEKNVKEVLEDFAEDGEKKIKLLTGKRVQLAEDLKKVREIQEKLDAFIEALHQEK, SEQ ID
NO: 21 human MFN1:
MAEPVSPLKHFVLAKKAITAIFDQLLEFVTEGSHFVEATYKNPELDRIATEDDLVEMQGYKDKLS
IIGEVLSRRHMKVAFFGRTSSGKSSVINAMLWDKVLPSGIGHITNCFLSVEGTDGDKAYLMTEGS
DEKKSVKTVNQLAHALHMDKDLKAGCLVRVFWPKAKCALLRDDLVLVDSPGTDVTTELDSWI
DKFCLDADVFVLVANSESTLMNTEKHFFHKVNERLSKPNIFILNNRWDASASEPEYMEDVRRQH
MERCLHFLVEELKVVNALEAQNRIFFVSAKEVLSARKQKAQGMPESGVALAEGFHARLQEFQN
FEQIFEECISQSAVKTKFEQHTIRAKQILATVKNIMDSVNLAAEDKRHYSVEEREDQIDRLDFIRN
QMNLLTLDVKKKIKEVTEEVANKVSCAMTDEICRLSVLVDEFCSEFHPNPDVLKIYKSELNKHIE
DGMGRNLADRCTDEVNALVLQTQQEIIENLKPLLPAGIQDKLHTLIPCKKFDLSYNLNYHKLCSD
FQEDIVFPFSLGWSSLVHRFLGPRNAQRVLLGLSEPIFQLPRSLASTPTAPTTPATPDNASQEELMI
TLVTGLASVTSRTSMGIIIVGGVIWKTIGWKLLSVSLTMYGALYLYERLSWTTHAKERAFKQQFV
NYATEKLRMIVSSTSANCSHQVKQQIATTFARLCQQVDITQKQLEEEIARLPKEIDQLEKIQNNSK
LLRNKAVQLENELENFTKQFLPSSNEES, SEQ ID NO: 22 Human MFN2:
MSLLFSRCNSIVTVKKNKRHMAEVNASPLKHFVTAKKKINGIFEQLGAYIQESATFLEDTYRNAE
LDPVTTEEQVLDVKGYLSKVRGISEVLARRHMKVAFFGRTSNGKSTVINAMLWDKVLPSGIGHT
TNCFLRVEGTDGHEAFLLTEGSEEKRSAKTVNQLAHALHQDKQLHAGSLVSVMWPNSKCPLLK
DDLVLMDSPGIDVTTELDSWIDKFCLDADVFVLVANSESTLMQTEKHFFHKVSERLSRPNIFILN
NRWDASASEPEYMEEVRRQHMERCTSFLVDELGVVDRSQAGDRIFFVSAKEVLNARIQKAQGM
PEGGGALAEGFQVRMFEFQNFERRFEECISQSAVKTKFEQHTVRAKQIAEAVRLIMDSLHMAAR
EQQVYCEEMREERQDRLKFIDKQLELLAQDYKLRIKQITEEVERQVSTAMAEEIRRLSVLVDDY
QMDFHPSPVVLKVYKNELHRHIEEGLGRNMSDRCSTAITNSLQTMQQDMIDGLKPLLPVSVRSQI
DMLVPRQCFSLNYDLNCDKLCADFQEDIEFHFSLGWTMLVNRFLGPKNSRRALMGYNDQVQRP
IPLTPANPSMPPLPQGSLTQEEFMVSMVTGLASLTSRTSMGILVVGGVVWKAVGWRLIALSFGLY
GLLYVYERLTWTTKAKERAFKRQFVEHASEKLQLVISYTGSNCSHQVQQELSGTFAHLCQQVD
VTRENLEQEIAAMNKKIEVLDSLQSKAKLLRNKAGWLDSELNMFTHQYLQPSR, SEQ ID NO:
23
Sequence CWU 1
1
231307PRThomo sapien 1Met Gly Gly Leu Thr Ala Ser Asp Val His Pro
Thr Leu Gly Val Gln 1 5 10 15 Leu Phe Ser Ala Gly Ile Ala Ala Cys
Leu Ala Asp Val Ile Thr Phe 20 25 30 Pro Leu Asp Thr Ala Lys Val
Arg Leu Gln Val Gln Gly Glu Cys Pro 35 40 45 Thr Ser Ser Val Ile
Arg Tyr Lys Gly Val Leu Gly Thr Ile Thr Ala 50 55 60 Val Val Lys
Thr Glu Gly Arg Met Lys Leu Tyr Ser Gly Leu Pro Ala 65 70 75 80 Gly
Leu Gln Arg Gln Ile Ser Ser Ala Ser Leu Arg Ile Gly Leu Tyr 85 90
95 Asp Thr Val Gln Glu Phe Leu Thr Ala Gly Lys Glu Thr Ala Pro Ser
100 105 110 Leu Gly Ser Lys Ile Leu Ala Gly Leu Thr Thr Gly Gly Val
Ala Val 115 120 125 Phe Ile Gly Gln Pro Thr Glu Val Val Lys Val Arg
Leu Gln Ala Gln 130 135 140 Ser His Leu His Gly Ile Lys Pro Arg Tyr
Thr Gly Thr Tyr Asn Ala 145 150 155 160 Tyr Arg Ile Ile Ala Thr Thr
Glu Gly Leu Thr Gly Leu Trp Lys Gly 165 170 175 Thr Thr Pro Asn Leu
Met Arg Ser Val Ile Ile Asn Cys Thr Glu Leu 180 185 190 Val Thr Tyr
Asp Leu Met Lys Glu Ala Phe Val Lys Asn Asn Ile Leu 195 200 205 Ala
Asp Asp Val Pro Cys His Leu Val Ser Ala Leu Ile Ala Gly Phe 210 215
220 Cys Ala Thr Ala Met Ser Ser Pro Val Asp Val Val Lys Thr Arg Phe
225 230 235 240 Ile Asn Ser Pro Pro Gly Gln Tyr Lys Ser Val Pro Asn
Cys Ala Met 245 250 255 Lys Val Phe Thr Asn Glu Gly Pro Thr Ala Phe
Phe Lys Gly Leu Val 260 265 270 Pro Ser Phe Leu Arg Leu Gly Ser Trp
Asn Val Ile Met Phe Val Cys 275 280 285 Phe Glu Gln Leu Lys Arg Glu
Leu Ser Lys Ser Arg Gln Thr Met Asp 290 295 300 Cys Ala Thr 305
2309PRThomo sapien 2Met Val Gly Phe Lys Ala Thr Asp Val Pro Pro Thr
Ala Thr Val Lys 1 5 10 15 Phe Leu Gly Ala Gly Thr Ala Ala Cys Ile
Ala Asp Leu Ile Thr Phe 20 25 30 Pro Leu Asp Thr Ala Lys Val Arg
Leu Gln Ile Gln Gly Glu Ser Gln 35 40 45 Gly Pro Val Arg Ala Thr
Ala Ser Ala Gln Tyr Arg Gly Val Met Gly 50 55 60 Thr Ile Leu Thr
Met Val Arg Thr Glu Gly Pro Arg Ser Leu Tyr Asn 65 70 75 80 Gly Leu
Val Ala Gly Leu Gln Arg Gln Met Ser Phe Ala Ser Val Arg 85 90 95
Ile Gly Leu Tyr Asp Ser Val Lys Gln Phe Tyr Thr Lys Gly Ser Glu 100
105 110 His Ala Ser Ile Gly Ser Arg Leu Leu Ala Gly Ser Thr Thr Gly
Ala 115 120 125 Leu Ala Val Ala Val Ala Gln Pro Thr Asp Val Val Lys
Val Arg Phe 130 135 140 Gln Ala Gln Ala Arg Ala Gly Gly Gly Arg Arg
Tyr Gln Ser Thr Val 145 150 155 160 Asn Ala Tyr Lys Thr Ile Ala Arg
Glu Glu Gly Phe Arg Gly Leu Trp 165 170 175 Lys Gly Thr Ser Pro Asn
Val Ala Arg Asn Ala Ile Val Asn Cys Ala 180 185 190 Glu Leu Val Thr
Tyr Asp Leu Ile Lys Asp Ala Leu Leu Lys Ala Asn 195 200 205 Leu Met
Thr Asp Asp Leu Pro Cys His Phe Thr Ser Ala Phe Gly Ala 210 215 220
Gly Phe Cys Thr Thr Val Ile Ala Ser Pro Val Asp Val Val Lys Thr 225
230 235 240 Arg Tyr Met Asn Ser Ala Leu Gly Gln Tyr Ser Ser Ala Gly
His Cys 245 250 255 Ala Leu Thr Met Leu Gln Lys Glu Gly Pro Arg Ala
Phe Tyr Lys Gly 260 265 270 Phe Met Pro Ser Phe Leu Arg Leu Gly Ser
Trp Asn Val Val Met Phe 275 280 285 Val Thr Tyr Glu Gln Leu Lys Arg
Ala Leu Met Ala Ala Cys Thr Ser 290 295 300 Arg Glu Ala Pro Phe 305
3312PRThomo sapien 3Met Val Gly Leu Lys Pro Ser Asp Val Pro Pro Thr
Met Ala Val Lys 1 5 10 15 Phe Leu Gly Ala Gly Thr Ala Ala Cys Phe
Ala Asp Leu Val Thr Phe 20 25 30 Pro Leu Asp Thr Ala Lys Val Arg
Leu Gln Ile Gln Gly Glu Asn Gln 35 40 45 Ala Val Gln Thr Ala Arg
Leu Val Gln Tyr Arg Gly Val Leu Gly Thr 50 55 60 Ile Leu Thr Met
Val Arg Thr Glu Gly Pro Cys Ser Pro Tyr Asn Gly 65 70 75 80 Leu Val
Ala Gly Leu Gln Arg Gln Met Ser Phe Ala Ser Ile Arg Ile 85 90 95
Gly Leu Tyr Asp Ser Val Lys Gln Val Tyr Thr Pro Lys Gly Ala Asp 100
105 110 Asn Ser Ser Leu Thr Thr Arg Ile Leu Ala Gly Cys Thr Thr Gly
Ala 115 120 125 Met Ala Val Thr Cys Ala Gln Pro Thr Asp Val Val Lys
Val Arg Phe 130 135 140 Gln Ala Ser Ile His Leu Gly Pro Ser Arg Ser
Asp Arg Lys Tyr Ser 145 150 155 160 Gly Thr Met Asp Ala Tyr Arg Thr
Ile Ala Arg Glu Glu Gly Val Arg 165 170 175 Gly Leu Trp Lys Gly Thr
Leu Pro Asn Ile Met Arg Asn Ala Ile Val 180 185 190 Asn Cys Ala Glu
Val Val Thr Tyr Asp Ile Leu Lys Glu Lys Leu Leu 195 200 205 Asp Tyr
His Leu Leu Thr Asp Asn Phe Pro Cys His Phe Val Ser Ala 210 215 220
Phe Gly Ala Gly Phe Cys Ala Thr Val Val Ala Ser Pro Val Asp Val 225
230 235 240 Val Lys Thr Arg Tyr Met Asn Ser Pro Pro Gly Gln Tyr Phe
Ser Pro 245 250 255 Leu Asp Cys Met Ile Lys Met Val Ala Gln Glu Gly
Pro Thr Ala Phe 260 265 270 Tyr Lys Gly Phe Thr Pro Ser Phe Leu Arg
Leu Gly Ser Trp Asn Val 275 280 285 Val Met Phe Val Thr Tyr Glu Gln
Leu Lys Arg Ala Leu Met Lys Val 290 295 300 Gln Met Leu Arg Glu Ser
Pro Phe 305 310 4323PRThomo sapien 4Met Ser Val Pro Glu Glu Glu Glu
Arg Leu Leu Pro Leu Thr Gln Arg 1 5 10 15 Trp Pro Arg Ala Ser Lys
Phe Leu Leu Ser Gly Cys Ala Ala Thr Val 20 25 30 Ala Glu Leu Ala
Thr Phe Pro Leu Asp Leu Thr Lys Thr Arg Leu Gln 35 40 45 Met Gln
Gly Glu Ala Ala Leu Ala Arg Leu Gly Asp Gly Ala Arg Glu 50 55 60
Ser Ala Pro Tyr Arg Gly Met Val Arg Thr Ala Leu Gly Ile Ile Glu 65
70 75 80 Glu Glu Gly Phe Leu Lys Leu Trp Gln Gly Val Thr Pro Ala
Ile Tyr 85 90 95 Arg His Val Val Tyr Ser Gly Gly Arg Met Val Thr
Tyr Glu His Leu 100 105 110 Arg Glu Val Val Phe Gly Lys Ser Glu Asp
Glu His Tyr Pro Leu Trp 115 120 125 Lys Ser Val Ile Gly Gly Met Met
Ala Gly Val Ile Gly Gln Phe Leu 130 135 140 Ala Asn Pro Thr Asp Leu
Val Lys Val Gln Met Gln Met Glu Gly Lys 145 150 155 160 Arg Lys Leu
Glu Gly Lys Pro Leu Arg Phe Arg Gly Val His His Ala 165 170 175 Phe
Ala Lys Ile Leu Ala Glu Gly Gly Ile Arg Gly Leu Trp Ala Gly 180 185
190 Trp Val Pro Asn Ile Gln Arg Ala Ala Leu Val Asn Met Gly Asp Leu
195 200 205 Thr Thr Tyr Asp Thr Val Lys His Tyr Leu Val Leu Asn Thr
Pro Leu 210 215 220 Glu Asp Asn Ile Met Thr His Gly Leu Ser Ser Leu
Cys Ser Gly Leu 225 230 235 240 Val Ala Ser Ile Leu Gly Thr Pro Ala
Asp Val Ile Lys Ser Arg Ile 245 250 255 Met Asn Gln Pro Arg Asp Lys
Gln Gly Arg Gly Leu Leu Tyr Lys Ser 260 265 270 Ser Thr Asp Cys Leu
Ile Gln Ala Val Gln Gly Glu Gly Phe Met Ser 275 280 285 Leu Tyr Lys
Gly Phe Leu Pro Ser Trp Leu Arg Met Thr Pro Trp Ser 290 295 300 Met
Val Phe Trp Leu Thr Tyr Glu Lys Ile Arg Glu Met Ser Gly Val 305 310
315 320 Ser Pro Phe 5325PRThomo sapien 5Met Gly Ile Phe Pro Gly Ile
Ile Leu Ile Phe Leu Arg Val Lys Phe 1 5 10 15 Ala Thr Ala Ala Val
Ile Val Ser Gly His Gln Lys Ser Thr Thr Val 20 25 30 Ser His Glu
Met Ser Gly Leu Asn Trp Lys Pro Phe Val Tyr Gly Gly 35 40 45 Leu
Ala Ser Ile Val Ala Glu Phe Gly Thr Phe Pro Val Asp Leu Thr 50 55
60 Lys Thr Arg Leu Gln Val Gln Gly Gln Ser Ile Asp Ala Arg Phe Lys
65 70 75 80 Glu Ile Lys Tyr Arg Gly Met Phe His Ala Leu Phe Arg Ile
Cys Lys 85 90 95 Glu Glu Gly Val Leu Ala Leu Tyr Ser Gly Ile Ala
Pro Ala Leu Leu 100 105 110 Arg Gln Ala Ser Tyr Gly Thr Ile Lys Ile
Gly Ile Tyr Gln Ser Leu 115 120 125 Lys Arg Leu Phe Val Glu Arg Leu
Glu Asp Glu Thr Leu Leu Ile Asn 130 135 140 Met Ile Cys Gly Val Val
Ser Gly Val Ile Ser Ser Thr Ile Ala Asn 145 150 155 160 Pro Thr Asp
Val Leu Lys Ile Arg Met Gln Ala Gln Gly Ser Leu Phe 165 170 175 Gln
Gly Ser Met Ile Gly Ser Phe Ile Asp Ile Tyr Gln Gln Glu Gly 180 185
190 Thr Arg Gly Leu Trp Arg Gly Val Val Pro Thr Ala Gln Arg Ala Ala
195 200 205 Ile Val Val Gly Val Glu Leu Pro Val Tyr Asp Ile Thr Lys
Lys His 210 215 220 Leu Ile Leu Ser Gly Met Met Gly Asp Thr Ile Leu
Thr His Phe Val 225 230 235 240 Ser Ser Phe Thr Cys Gly Leu Ala Gly
Ala Leu Ala Ser Asn Pro Val 245 250 255 Asp Val Val Arg Thr Arg Met
Met Asn Gln Arg Ala Ile Val Gly His 260 265 270 Val Asp Leu Tyr Lys
Gly Thr Val Asp Gly Ile Leu Lys Met Trp Lys 275 280 285 His Glu Gly
Phe Phe Ala Leu Tyr Lys Gly Phe Trp Pro Asn Trp Leu 290 295 300 Arg
Leu Gly Pro Trp Asn Ile Ile Phe Phe Ile Thr Tyr Glu Gln Leu 305 310
315 320 Lys Arg Leu Gln Ile 325 687DNAhomo sapien 6atgtccgtcc
tgacgccgct gctgctgcgg ggcttgacag gctcggcccg gcggctccca 60gtgccgcgcg
ccaagatcca ttcgttg 877257PRThomo sapien 7Met Val Gly Ala Gly Ile
Ser Thr Pro Ser Gly Ile Pro Asp Phe Arg 1 5 10 15 Ser Pro Gly Ser
Gly Leu Tyr Ser Asn Leu Gln Gln Tyr Asp Leu Pro 20 25 30 Tyr Pro
Glu Ala Ile Phe Glu Leu Pro Phe Phe Phe His Asn Pro Lys 35 40 45
Pro Phe Phe Thr Leu Ala Lys Glu Leu Tyr Pro Gly Asn Tyr Lys Pro 50
55 60 Asn Val Thr His Tyr Phe Leu Arg Leu Leu His Asp Lys Gly Leu
Leu 65 70 75 80 Leu Arg Leu Tyr Thr Gln Asn Ile Asp Gly Leu Glu Arg
Val Ser Gly 85 90 95 Ile Pro Ala Ser Lys Leu Val Glu Ala His Gly
Thr Phe Ala Ser Ala 100 105 110 Thr Cys Thr Val Cys Gln Arg Pro Phe
Pro Gly Glu Asp Ile Arg Ala 115 120 125 Asp Val Met Ala Asp Arg Val
Pro Arg Cys Pro Val Cys Thr Gly Val 130 135 140 Val Lys Pro Asp Ile
Val Phe Phe Gly Glu Pro Leu Pro Gln Arg Phe 145 150 155 160 Leu Leu
His Val Val Asp Phe Pro Met Ala Asp Leu Leu Leu Ile Leu 165 170 175
Gly Thr Ser Leu Glu Val Glu Pro Phe Ala Ser Leu Thr Glu Ala Val 180
185 190 Arg Ser Ser Val Pro Arg Leu Leu Ile Asn Arg Asp Leu Val Gly
Pro 195 200 205 Leu Ala Trp His Pro Arg Ser Arg Asp Val Ala Gln Leu
Gly Asp Val 210 215 220 Val His Gly Val Glu Ser Leu Val Glu Leu Leu
Gly Trp Thr Glu Glu 225 230 235 240 Met Arg Asp Leu Val Gln Arg Glu
Thr Gly Lys Leu Asp Gly Pro Asp 245 250 255 Lys 8436PRThomo sapien
8Met Arg Leu Ala Arg Leu Leu Arg Gly Ala Ala Leu Ala Gly Pro Gly 1
5 10 15 Pro Gly Leu Arg Ala Ala Gly Phe Ser Arg Ser Phe Ser Ser Asp
Ser 20 25 30 Gly Ser Ser Pro Ala Ser Glu Arg Gly Val Pro Gly Gln
Val Asp Phe 35 40 45 Tyr Ala Arg Phe Ser Pro Ser Pro Leu Ser Met
Lys Gln Phe Leu Asp 50 55 60 Phe Gly Ser Val Asn Ala Cys Glu Lys
Thr Ser Phe Met Phe Leu Arg 65 70 75 80 Gln Glu Leu Pro Val Arg Leu
Ala Asn Ile Met Lys Glu Ile Ser Leu 85 90 95 Leu Pro Asp Asn Leu
Leu Arg Thr Pro Ser Val Gln Leu Val Gln Ser 100 105 110 Trp Tyr Ile
Gln Ser Leu Gln Glu Leu Leu Asp Phe Lys Asp Lys Ser 115 120 125 Ala
Glu Asp Ala Lys Ala Ile Tyr Asp Phe Thr Asp Thr Val Ile Arg 130 135
140 Ile Arg Asn Arg His Asn Asp Val Ile Pro Thr Met Ala Gln Gly Val
145 150 155 160 Ile Glu Tyr Lys Glu Ser Phe Gly Val Asp Pro Val Thr
Ser Gln Asn 165 170 175 Val Gln Tyr Phe Leu Asp Arg Phe Tyr Met Ser
Arg Ile Ser Ile Arg 180 185 190 Met Leu Leu Asn Gln His Ser Leu Leu
Phe Gly Gly Lys Gly Lys Gly 195 200 205 Ser Pro Ser His Arg Lys His
Ile Gly Ser Ile Asn Pro Asn Cys Asn 210 215 220 Val Leu Glu Val Ile
Lys Asp Gly Tyr Glu Asn Ala Arg Arg Leu Cys 225 230 235 240 Asp Leu
Tyr Tyr Ile Asn Ser Pro Glu Leu Glu Leu Glu Glu Leu Asn 245 250 255
Ala Lys Ser Pro Gly Gln Pro Ile Gln Val Val Tyr Val Pro Ser His 260
265 270 Leu Tyr His Met Val Phe Glu Leu Phe Lys Asn Ala Met Arg Ala
Thr 275 280 285 Met Glu His His Ala Asn Arg Gly Val Tyr Pro Pro Ile
Gln Val His 290 295 300 Val Thr Leu Gly Asn Glu Asp Leu Thr Val Lys
Met Ser Asp Arg Gly 305 310 315 320 Gly Gly Val Pro Leu Arg Lys Ile
Asp Arg Leu Phe Asn Tyr Met Tyr 325 330 335 Ser Thr Ala Pro Arg Pro
Arg Val Glu Thr Ser Arg Ala Val Pro Leu 340 345 350 Ala Gly Phe Gly
Tyr Gly Leu Pro Ile Ser Arg Leu Tyr Ala Gln Tyr 355 360 365 Phe Gln
Gly Asp Leu Lys Leu Tyr Ser Leu Glu Gly Tyr Gly Thr Asp 370 375 380
Ala Val Ile Tyr Ile Lys Ala Leu Ser Thr Asp Ser Ile Glu Arg Leu 385
390 395 400 Pro Val Tyr Asn Lys Ala Ala Trp Lys His Tyr Asn Thr Asn
His Glu 405 410 415 Ala Asp Asp Trp Cys Val Pro Ser Arg Glu Pro Lys
Asp Met Thr Thr 420 425 430 Phe Arg Ser
Ala 435 9 1046PRThomo sapien 9Met Ala Ser Ser Val Gly Asn Val Ala
Asp Ser Thr Glu Pro Thr Lys 1 5 10 15 Arg Met Leu Ser Phe Gln Gly
Leu Ala Glu Leu Ala His Arg Glu Tyr 20 25 30 Gln Ala Gly Asp Phe
Glu Ala Ala Glu Arg His Cys Met Gln Leu Trp 35 40 45 Arg Gln Glu
Pro Asp Asn Thr Gly Val Leu Leu Leu Leu Ser Ser Ile 50 55 60 His
Phe Gln Cys Arg Arg Leu Asp Arg Ser Ala His Phe Ser Thr Leu 65 70
75 80 Ala Ile Lys Gln Asn Pro Leu Leu Ala Glu Ala Tyr Ser Asn Leu
Gly 85 90 95 Asn Val Tyr Lys Glu Arg Gly Gln Leu Gln Glu Ala Ile
Glu His Tyr 100 105 110 Arg His Ala Leu Arg Leu Lys Pro Asp Phe Ile
Asp Gly Tyr Ile Asn 115 120 125 Leu Ala Ala Ala Leu Val Ala Ala Gly
Asp Met Glu Gly Ala Val Gln 130 135 140 Ala Tyr Val Ser Ala Leu Gln
Tyr Asn Pro Asp Leu Tyr Cys Val Arg 145 150 155 160 Ser Asp Leu Gly
Asn Leu Leu Lys Ala Leu Gly Arg Leu Glu Glu Ala 165 170 175 Lys Ala
Cys Tyr Leu Lys Ala Ile Glu Thr Gln Pro Asn Phe Ala Val 180 185 190
Ala Trp Ser Asn Leu Gly Cys Val Phe Asn Ala Gln Gly Glu Ile Trp 195
200 205 Leu Ala Ile His His Phe Glu Lys Ala Val Thr Leu Asp Pro Asn
Phe 210 215 220 Leu Asp Ala Tyr Ile Asn Leu Gly Asn Val Leu Lys Glu
Ala Arg Ile 225 230 235 240 Phe Asp Arg Ala Val Ala Ala Tyr Leu Arg
Ala Leu Ser Leu Ser Pro 245 250 255 Asn His Ala Val Val His Gly Asn
Leu Ala Cys Val Tyr Tyr Glu Gln 260 265 270 Gly Leu Ile Asp Leu Ala
Ile Asp Thr Tyr Arg Arg Ala Ile Glu Leu 275 280 285 Gln Pro His Phe
Pro Asp Ala Tyr Cys Asn Leu Ala Asn Ala Leu Lys 290 295 300 Glu Lys
Gly Ser Val Ala Glu Ala Glu Asp Cys Tyr Asn Thr Ala Leu 305 310 315
320 Arg Leu Cys Pro Thr His Ala Asp Ser Leu Asn Asn Leu Ala Asn Ile
325 330 335 Lys Arg Glu Gln Gly Asn Ile Glu Glu Ala Val Arg Leu Tyr
Arg Lys 340 345 350 Ala Leu Glu Val Phe Pro Glu Phe Ala Ala Ala His
Ser Asn Leu Ala 355 360 365 Ser Val Leu Gln Gln Gln Gly Lys Leu Gln
Glu Ala Leu Met His Tyr 370 375 380 Lys Glu Ala Ile Arg Ile Ser Pro
Thr Phe Ala Asp Ala Tyr Ser Asn 385 390 395 400 Met Gly Asn Thr Leu
Lys Glu Met Gln Asp Val Gln Gly Ala Leu Gln 405 410 415 Cys Tyr Thr
Arg Ala Ile Gln Ile Asn Pro Ala Phe Ala Asp Ala His 420 425 430 Ser
Asn Leu Ala Ser Ile His Lys Asp Ser Gly Asn Ile Pro Glu Ala 435 440
445 Ile Ala Ser Tyr Arg Thr Ala Leu Lys Leu Lys Pro Asp Phe Pro Asp
450 455 460 Ala Tyr Cys Asn Leu Ala His Cys Leu Gln Ile Val Cys Asp
Trp Thr 465 470 475 480 Asp Tyr Asp Glu Arg Met Lys Lys Leu Val Ser
Ile Val Ala Asp Gln 485 490 495 Leu Glu Lys Asn Arg Leu Pro Ser Val
His Pro His His Ser Met Leu 500 505 510 Tyr Pro Leu Ser His Gly Phe
Arg Lys Ala Ile Ala Glu Arg His Gly 515 520 525 Asn Leu Cys Leu Asp
Lys Ile Asn Val Leu His Lys Pro Pro Tyr Glu 530 535 540 His Pro Lys
Asp Leu Lys Leu Ser Asp Gly Arg Leu Arg Val Gly Tyr 545 550 555 560
Val Ser Ser Asp Phe Gly Asn His Pro Thr Ser His Leu Met Gln Ser 565
570 575 Ile Pro Gly Met His Asn Pro Asp Lys Phe Glu Val Phe Cys Tyr
Ala 580 585 590 Leu Ser Pro Asp Asp Gly Thr Asn Phe Arg Val Lys Val
Met Ala Glu 595 600 605 Ala Asn His Phe Ile Asp Leu Ser Gln Ile Pro
Cys Asn Gly Lys Ala 610 615 620 Ala Asp Arg Ile His Gln Asp Gly Ile
His Ile Leu Val Asn Met Asn 625 630 635 640 Gly Tyr Thr Lys Gly Ala
Arg Asn Glu Leu Phe Ala Leu Arg Pro Ala 645 650 655 Pro Ile Gln Ala
Met Trp Leu Gly Tyr Pro Gly Thr Ser Gly Ala Leu 660 665 670 Phe Met
Asp Tyr Ile Ile Thr Asp Gln Glu Thr Ser Pro Ala Glu Val 675 680 685
Ala Glu Gln Tyr Ser Glu Lys Leu Ala Tyr Met Pro His Thr Phe Phe 690
695 700 Ile Gly Asp His Ala Asn Met Phe Pro His Leu Lys Lys Lys Ala
Val 705 710 715 720 Ile Asp Phe Lys Ser Asn Gly His Ile Tyr Asp Asn
Arg Ile Val Leu 725 730 735 Asn Gly Ile Asp Leu Lys Ala Phe Leu Asp
Ser Leu Pro Asp Val Lys 740 745 750 Ile Val Lys Met Lys Cys Pro Asp
Gly Gly Asp Asn Ala Asp Ser Ser 755 760 765 Asn Thr Ala Leu Asn Met
Pro Val Ile Pro Met Asn Thr Ile Ala Glu 770 775 780 Ala Val Ile Glu
Met Ile Asn Arg Gly Gln Ile Gln Ile Thr Ile Asn 785 790 795 800 Gly
Phe Ser Ile Ser Asn Gly Leu Ala Thr Thr Gln Ile Asn Asn Lys 805 810
815 Ala Ala Thr Gly Glu Glu Val Pro Arg Thr Ile Ile Val Thr Thr Arg
820 825 830 Ser Gln Tyr Gly Leu Pro Glu Asp Ala Ile Val Tyr Cys Asn
Phe Asn 835 840 845 Gln Leu Tyr Lys Ile Asp Pro Ser Thr Leu Gln Met
Trp Ala Asn Ile 850 855 860 Leu Lys Arg Val Pro Asn Ser Val Leu Trp
Leu Leu Arg Phe Pro Ala 865 870 875 880 Val Gly Glu Pro Asn Ile Gln
Gln Tyr Ala Gln Asn Met Gly Leu Pro 885 890 895 Gln Asn Arg Ile Ile
Phe Ser Pro Val Ala Pro Lys Glu Glu His Val 900 905 910 Arg Arg Gly
Gln Leu Ala Asp Val Cys Leu Asp Thr Pro Leu Cys Asn 915 920 925 Gly
His Thr Thr Gly Met Asp Val Leu Trp Ala Gly Thr Pro Met Val 930 935
940 Thr Met Pro Gly Glu Thr Leu Ala Ser Arg Val Ala Ala Ser Gln Leu
945 950 955 960 Thr Cys Leu Gly Cys Leu Glu Leu Ile Ala Lys Asn Arg
Gln Glu Tyr 965 970 975 Glu Asp Ile Ala Val Lys Leu Gly Thr Asp Leu
Glu Tyr Leu Lys Lys 980 985 990 Val Arg Gly Lys Val Trp Lys Gln Arg
Ile Ser Ser Pro Leu Phe Asn 995 1000 1005 Thr Lys Gln Tyr Thr Met
Glu Leu Glu Arg Leu Tyr Leu Gln Met 1010 1015 1020 Trp Glu His Tyr
Ala Ala Gly Asn Lys Pro Asp His Met Ile Lys 1025 1030 1035 Pro Val
Glu Val Thr Glu Ser Ala 1040 1045 10145PRThomo sapien 10Met Asn Gly
Arg Val Asp Tyr Leu Val Thr Glu Glu Glu Ile Asn Leu 1 5 10 15 Thr
Arg Gly Pro Ser Gly Leu Gly Phe Asn Ile Val Gly Gly Thr Asp 20 25
30 Gln Gln Tyr Val Ser Asn Asp Ser Gly Ile Tyr Val Ser Arg Ile Lys
35 40 45 Glu Asn Gly Ala Ala Ala Leu Asp Gly Arg Leu Gln Glu Gly
Asp Lys 50 55 60 Ile Leu Ser Val Asn Gly Gln Asp Leu Lys Asn Leu
Leu His Gln Asp 65 70 75 80 Ala Val Asp Leu Phe Arg Asn Ala Gly Tyr
Ala Val Ser Leu Arg Val 85 90 95 Gln His Arg Leu Gln Val Gln Asn
Gly Pro Ile Gly His Arg Gly Glu 100 105 110 Gly Asp Pro Ser Gly Ile
Pro Ile Phe Met Val Leu Val Pro Val Phe 115 120 125 Ala Leu Thr Met
Val Ala Ala Trp Ala Phe Met Arg Tyr Arg Gln Gln 130 135 140 Leu 145
11142PRThomo sapien 11Met Ala Ala Ala Val Ala Ala Ala Gly Ala Gly
Glu Pro Gln Ser Pro 1 5 10 15 Asp Glu Leu Leu Pro Lys Gly Asp Ala
Glu Lys Pro Glu Glu Glu Leu 20 25 30 Glu Glu Asp Asp Asp Glu Glu
Leu Asp Glu Thr Leu Ser Glu Arg Leu 35 40 45 Trp Gly Leu Thr Glu
Met Phe Pro Glu Arg Val Arg Ser Ala Ala Gly 50 55 60 Ala Thr Phe
Asp Leu Ser Leu Phe Val Ala Gln Lys Met Tyr Arg Phe 65 70 75 80 Ser
Arg Ala Ala Leu Trp Ile Gly Thr Thr Ser Phe Met Ile Leu Val 85 90
95 Leu Pro Val Val Phe Glu Thr Glu Lys Leu Gln Met Glu Gln Gln Gln
100 105 110 Gln Leu Gln Gln Arg Gln Ile Leu Leu Gly Pro Asn Thr Gly
Leu Ser 115 120 125 Gly Gly Met Pro Gly Ala Leu Pro Ser Leu Pro Gly
Lys Ile 130 135 140 12171PRThomo sapien 12Met Glu Glu Tyr Ala Arg
Glu Pro Cys Pro Trp Arg Ile Val Asp Asp 1 5 10 15 Cys Gly Gly Ala
Phe Thr Met Gly Thr Ile Gly Gly Gly Ile Phe Gln 20 25 30 Ala Ile
Lys Gly Phe Arg Asn Ser Pro Val Gly Val Asn His Arg Leu 35 40 45
Arg Gly Ser Leu Thr Ala Ile Lys Thr Arg Ala Pro Gln Leu Gly Gly 50
55 60 Ser Phe Ala Val Trp Gly Gly Leu Phe Ser Met Ile Asp Cys Ser
Met 65 70 75 80 Val Gln Val Arg Gly Lys Glu Asp Pro Trp Asn Ser Ile
Thr Ser Gly 85 90 95 Ala Leu Thr Gly Ala Ile Leu Ala Ala Arg Asn
Gly Pro Val Ala Met 100 105 110 Val Gly Ser Ala Ala Met Gly Gly Ile
Leu Leu Ala Leu Ile Glu Gly 115 120 125 Ala Gly Ile Leu Leu Thr Arg
Phe Ala Ser Ala Gln Phe Pro Asn Gly 130 135 140 Pro Gln Phe Ala Glu
Asp Pro Ser Gln Leu Pro Ser Thr Gln Leu Pro 145 150 155 160 Ser Ser
Pro Phe Gly Asp Tyr Arg Gln Tyr Gln 165 170 13172PRThomo sapien
13Met Glu Glu Tyr Ala Arg Glu Pro Cys Pro Trp Arg Ile Val Asp Asp 1
5 10 15 Cys Gly Gly Ala Phe Thr Met Gly Val Ile Gly Gly Gly Val Phe
Gln 20 25 30 Ala Ile Lys Gly Phe Arg Asn Ala Pro Val Gly Ile Arg
His Arg Leu 35 40 45 Arg Gly Ser Ala Asn Ala Val Arg Ile Arg Ala
Pro Gln Ile Gly Gly 50 55 60 Ser Phe Ala Val Trp Gly Gly Leu Phe
Ser Thr Ile Asp Cys Gly Leu 65 70 75 80 Val Arg Leu Arg Gly Lys Glu
Asp Pro Trp Asn Ser Ile Thr Ser Gly 85 90 95 Ala Leu Thr Gly Ala
Val Leu Ala Ala Arg Ser Gly Pro Leu Ala Met 100 105 110 Val Gly Ser
Ala Met Met Gly Gly Ile Leu Leu Ala Leu Ile Glu Gly 115 120 125 Val
Gly Ile Leu Leu Thr Arg Tyr Thr Ala Gln Gln Phe Arg Asn Ala 130 135
140 Pro Pro Phe Leu Glu Asp Pro Ser Gln Leu Pro Pro Lys Asp Gly Thr
145 150 155 160 Pro Ala Pro Gly Tyr Pro Ser Tyr Gln Gln Tyr His 165
170 14194PRThomo sapien 14Met Ala Ala Ala Ala Pro Asn Ala Gly Gly
Ser Ala Pro Glu Thr Ala 1 5 10 15 Gly Ser Ala Glu Ala Pro Leu Gln
Tyr Ser Leu Leu Leu Gln Tyr Leu 20 25 30 Val Gly Asp Lys Arg Gln
Pro Arg Leu Leu Glu Pro Gly Ser Leu Gly 35 40 45 Gly Ile Pro Ser
Pro Ala Lys Ser Glu Glu Gln Lys Met Ile Glu Lys 50 55 60 Ala Met
Glu Ser Cys Ala Phe Lys Ala Ala Leu Ala Cys Val Gly Gly 65 70 75 80
Phe Val Leu Gly Gly Ala Phe Gly Val Phe Thr Ala Gly Ile Asp Thr 85
90 95 Asn Val Gly Phe Asp Pro Lys Asp Pro Tyr Arg Thr Pro Thr Ala
Lys 100 105 110 Glu Val Leu Lys Asp Met Gly Gln Arg Gly Met Ser Tyr
Ala Lys Asn 115 120 125 Phe Ala Ile Val Gly Ala Met Phe Ser Cys Thr
Glu Cys Leu Ile Glu 130 135 140 Ser Tyr Arg Gly Thr Ser Asp Trp Lys
Asn Ser Val Ile Ser Gly Cys 145 150 155 160 Ile Thr Gly Gly Ala Ile
Gly Phe Arg Ala Gly Leu Lys Ala Gly Ala 165 170 175 Ile Gly Cys Gly
Gly Phe Ala Ala Phe Ser Ala Ala Ile Asp Tyr Tyr 180 185 190 Leu Arg
15246PRThomo sapien 15Met Ala Phe Leu Arg Ser Met Trp Gly Val Leu
Ser Ala Leu Gly Arg 1 5 10 15 Ser Gly Ala Glu Leu Cys Thr Gly Cys
Gly Ser Arg Leu Arg Ser Pro 20 25 30 Phe Ser Phe Val Tyr Leu Pro
Arg Trp Phe Ser Ser Val Leu Ala Ser 35 40 45 Cys Pro Lys Lys Pro
Val Ser Ser Tyr Leu Arg Phe Ser Lys Glu Gln 50 55 60 Leu Pro Ile
Phe Lys Ala Gln Asn Pro Asp Ala Lys Thr Thr Glu Leu 65 70 75 80 Ile
Arg Arg Ile Ala Gln Arg Trp Arg Glu Leu Pro Asp Ser Lys Lys 85 90
95 Lys Ile Tyr Gln Asp Ala Tyr Arg Ala Glu Trp Gln Val Tyr Lys Glu
100 105 110 Glu Ile Ser Arg Phe Lys Glu Gln Leu Thr Pro Ser Gln Ile
Met Ser 115 120 125 Leu Glu Lys Glu Ile Met Asp Lys His Leu Lys Arg
Lys Ala Met Thr 130 135 140 Lys Lys Lys Glu Leu Thr Leu Leu Gly Lys
Pro Lys Arg Pro Arg Ser 145 150 155 160 Ala Tyr Asn Val Tyr Val Ala
Glu Arg Phe Gln Glu Ala Lys Gly Asp 165 170 175 Ser Pro Gln Glu Lys
Leu Lys Thr Val Lys Glu Asn Trp Lys Asn Leu 180 185 190 Ser Asp Ser
Glu Lys Glu Leu Tyr Ile Gln His Ala Lys Glu Asp Glu 195 200 205 Thr
Arg Tyr His Asn Glu Met Lys Ser Trp Glu Glu Gln Met Ile Glu 210 215
220 Val Gly Arg Lys Asp Leu Leu Arg Arg Thr Ile Lys Lys Gln Arg Lys
225 230 235 240 Tyr Gly Ala Glu Glu Cys 245 16798PRTHomo sapiens
16Met Ala Trp Asp Met Cys Asn Gln Asp Ser Glu Ser Val Trp Ser Asp 1
5 10 15 Ile Glu Cys Ala Ala Leu Val Gly Glu Asp Gln Pro Leu Cys Pro
Asp 20 25 30 Leu Pro Glu Leu Asp Leu Ser Glu Leu Asp Val Asn Asp
Leu Asp Thr 35 40 45 Asp Ser Phe Leu Gly Gly Leu Lys Trp Cys Ser
Asp Gln Ser Glu Ile 50 55 60 Ile Ser Asn Gln Tyr Asn Asn Glu Pro
Ser Asn Ile Phe Glu Lys Ile 65 70 75 80 Asp Glu Glu Asn Glu Ala Asn
Leu Leu Ala Val Leu Thr Glu Thr Leu 85 90 95 Asp Ser Leu Pro Val
Asp Glu Asp Gly Leu Pro Ser Phe Asp Ala Leu 100 105 110 Thr Asp Gly
Asp Val Thr Thr Asp Asn Glu Ala Ser Pro Ser Ser Met 115 120 125 Pro
Asp Gly Thr Pro Pro Pro Gln Glu Ala Glu Glu Pro Ser Leu Leu 130 135
140 Lys Lys Leu Leu Leu Ala Pro Ala Asn Thr Gln Leu Ser Tyr Asn Glu
145 150 155 160 Cys Ser Gly Leu
Ser Thr Gln Asn His Ala Asn His Asn His Arg Ile 165 170 175 Arg Thr
Asn Pro Ala Ile Val Lys Thr Glu Asn Ser Trp Ser Asn Lys 180 185 190
Ala Lys Ser Ile Cys Gln Gln Gln Lys Pro Gln Arg Arg Pro Cys Ser 195
200 205 Glu Leu Leu Lys Tyr Leu Thr Thr Asn Asp Asp Pro Pro His Thr
Lys 210 215 220 Pro Thr Glu Asn Arg Asn Ser Ser Arg Asp Lys Cys Thr
Ser Lys Lys 225 230 235 240 Lys Ser His Thr Gln Ser Gln Ser Gln His
Leu Gln Ala Lys Pro Thr 245 250 255 Thr Leu Ser Leu Pro Leu Thr Pro
Glu Ser Pro Asn Asp Pro Lys Gly 260 265 270 Ser Pro Phe Glu Asn Lys
Thr Ile Glu Arg Thr Leu Ser Val Glu Leu 275 280 285 Ser Gly Thr Ala
Gly Leu Thr Pro Pro Thr Thr Pro Pro His Lys Ala 290 295 300 Asn Gln
Asp Asn Pro Phe Arg Ala Ser Pro Lys Leu Lys Ser Ser Cys 305 310 315
320 Lys Thr Val Val Pro Pro Pro Ser Lys Lys Pro Arg Tyr Ser Glu Ser
325 330 335 Ser Gly Thr Gln Gly Asn Asn Ser Thr Lys Lys Gly Pro Glu
Gln Ser 340 345 350 Glu Leu Tyr Ala Gln Leu Ser Lys Ser Ser Val Leu
Thr Gly Gly His 355 360 365 Glu Glu Arg Lys Thr Lys Arg Pro Ser Leu
Arg Leu Phe Gly Asp His 370 375 380 Asp Tyr Cys Gln Ser Ile Asn Ser
Lys Thr Glu Ile Leu Ile Asn Ile 385 390 395 400 Ser Gln Glu Leu Gln
Asp Ser Arg Gln Leu Glu Asn Lys Asp Val Ser 405 410 415 Ser Asp Trp
Gln Gly Gln Ile Cys Ser Ser Thr Asp Ser Asp Gln Cys 420 425 430 Tyr
Leu Arg Glu Thr Leu Glu Ala Ser Lys Gln Val Ser Pro Cys Ser 435 440
445 Thr Arg Lys Gln Leu Gln Asp Gln Glu Ile Arg Ala Glu Leu Asn Lys
450 455 460 His Phe Gly His Pro Ser Gln Ala Val Phe Asp Asp Glu Ala
Asp Lys 465 470 475 480 Thr Gly Glu Leu Arg Asp Ser Asp Phe Ser Asn
Glu Gln Phe Ser Lys 485 490 495 Leu Pro Met Phe Ile Asn Ser Gly Leu
Ala Met Asp Gly Leu Phe Asp 500 505 510 Asp Ser Glu Asp Glu Ser Asp
Lys Leu Ser Tyr Pro Trp Asp Gly Thr 515 520 525 Gln Ser Tyr Ser Leu
Phe Asn Val Ser Pro Ser Cys Ser Ser Phe Asn 530 535 540 Ser Pro Cys
Arg Asp Ser Val Ser Pro Pro Lys Ser Leu Phe Ser Gln 545 550 555 560
Arg Pro Gln Arg Met Arg Ser Arg Ser Arg Ser Phe Ser Arg His Arg 565
570 575 Ser Cys Ser Arg Ser Pro Tyr Ser Arg Ser Arg Ser Arg Ser Pro
Gly 580 585 590 Ser Arg Ser Ser Ser Arg Ser Cys Tyr Tyr Tyr Glu Ser
Ser His Tyr 595 600 605 Arg His Arg Thr His Arg Asn Ser Pro Leu Tyr
Val Arg Ser Arg Ser 610 615 620 Arg Ser Pro Tyr Ser Arg Arg Pro Arg
Tyr Asp Ser Tyr Glu Glu Tyr 625 630 635 640 Gln His Glu Arg Leu Lys
Arg Glu Glu Tyr Arg Arg Glu Tyr Glu Lys 645 650 655 Arg Glu Ser Glu
Arg Ala Lys Gln Arg Glu Arg Gln Arg Gln Lys Ala 660 665 670 Ile Glu
Glu Arg Arg Val Ile Tyr Val Gly Lys Ile Arg Pro Asp Thr 675 680 685
Thr Arg Thr Glu Leu Arg Asp Arg Phe Glu Val Phe Gly Glu Ile Glu 690
695 700 Glu Cys Thr Val Asn Leu Arg Asp Asp Gly Asp Ser Tyr Gly Phe
Ile 705 710 715 720 Thr Tyr Arg Tyr Thr Cys Asp Ala Phe Ala Ala Leu
Glu Asn Gly Tyr 725 730 735 Thr Leu Arg Arg Ser Asn Glu Thr Asp Phe
Glu Leu Tyr Phe Cys Gly 740 745 750 Arg Lys Gln Phe Phe Lys Ser Asn
Tyr Ala Asp Leu Asp Ser Asn Ser 755 760 765 Asp Asp Phe Asp Pro Ala
Ser Thr Lys Ser Lys Tyr Asp Ser Leu Asp 770 775 780 Phe Asp Ser Leu
Leu Lys Glu Ala Gln Arg Ser Leu Arg Arg 785 790 795
178PRTArtificial SequenceSynthetic sequence 17Asp Tyr Lys Asp Asp
Asp Asp Lys 1 5 18427PRThomo sapien 18Met Ala Val Leu Ala Ala Leu
Leu Arg Ser Gly Ala Arg Ser Arg Ser 1 5 10 15 Pro Leu Leu Arg Arg
Leu Val Gln Glu Ile Arg Tyr Val Glu Arg Ser 20 25 30 Tyr Val Ser
Lys Pro Thr Leu Lys Glu Val Val Ile Val Ser Ala Thr 35 40 45 Arg
Thr Pro Ile Gly Ser Phe Leu Gly Ser Leu Ser Leu Leu Pro Ala 50 55
60 Thr Lys Leu Gly Ser Ile Ala Ile Gln Gly Ala Ile Glu Lys Ala Gly
65 70 75 80 Ile Pro Lys Glu Glu Val Lys Glu Ala Tyr Met Gly Asn Val
Leu Gln 85 90 95 Gly Gly Glu Gly Gln Ala Pro Thr Arg Gln Ala Val
Leu Gly Ala Gly 100 105 110 Leu Pro Ile Ser Thr Pro Cys Thr Thr Ile
Asn Lys Val Cys Ala Ser 115 120 125 Gly Met Lys Ala Ile Met Met Ala
Ser Gln Ser Leu Met Cys Gly His 130 135 140 Gln Asp Val Met Val Ala
Gly Gly Met Glu Ser Met Ser Asn Val Pro 145 150 155 160 Tyr Val Met
Asn Arg Gly Ser Thr Pro Tyr Gly Gly Val Lys Leu Glu 165 170 175 Asp
Leu Ile Val Lys Asp Gly Leu Thr Asp Val Tyr Asn Lys Ile His 180 185
190 Met Gly Ser Cys Ala Glu Asn Thr Ala Lys Lys Leu Asn Ile Ala Arg
195 200 205 Asn Glu Gln Asp Ala Tyr Ala Ile Asn Ser Tyr Thr Arg Ser
Lys Ala 210 215 220 Ala Trp Glu Ala Gly Lys Phe Gly Asn Glu Val Ile
Pro Val Thr Val 225 230 235 240 Thr Val Lys Gly Gln Pro Asp Val Val
Val Lys Glu Asp Glu Glu Tyr 245 250 255 Lys Arg Val Asp Phe Ser Lys
Val Pro Lys Leu Lys Thr Val Phe Gln 260 265 270 Lys Glu Asn Gly Thr
Val Thr Ala Ala Asn Ala Ser Thr Leu Asn Asp 275 280 285 Gly Ala Ala
Ala Leu Val Leu Met Thr Ala Asp Ala Ala Lys Arg Leu 290 295 300 Asn
Val Thr Pro Leu Ala Arg Ile Val Ala Phe Ala Asp Ala Ala Val 305 310
315 320 Glu Pro Ile Asp Phe Pro Ile Ala Pro Val Tyr Ala Ala Ser Met
Val 325 330 335 Leu Lys Asp Val Gly Leu Lys Lys Glu Asp Ile Ala Met
Trp Glu Val 340 345 350 Asn Glu Ala Phe Ser Leu Val Val Leu Ala Asn
Ile Lys Met Leu Glu 355 360 365 Ile Asp Pro Gln Lys Val Asn Ile Asn
Gly Gly Ala Val Ser Leu Gly 370 375 380 His Pro Ile Gly Met Ser Gly
Ala Arg Ile Val Gly His Leu Thr His 385 390 395 400 Ala Leu Lys Gln
Gly Glu Tyr Gly Leu Ala Ser Ile Cys Asn Gly Gly 405 410 415 Gly Gly
Ala Ser Ala Met Leu Ile Gln Lys Leu 420 425 19327PRThomo sapien
19Met Pro Ala Leu Leu Glu Arg Pro Lys Leu Ser Asn Ala Met Ala Arg 1
5 10 15 Ala Leu His Arg His Ile Met Met Glu Arg Glu Arg Lys Arg Gln
Glu 20 25 30 Glu Glu Glu Val Asp Lys Met Met Glu Gln Lys Met Lys
Glu Glu Gln 35 40 45 Glu Arg Arg Lys Lys Lys Glu Met Glu Glu Arg
Met Ser Leu Glu Glu 50 55 60 Thr Lys Glu Gln Ile Leu Lys Leu Glu
Glu Lys Leu Leu Ala Leu Gln 65 70 75 80 Glu Glu Lys His Gln Leu Phe
Leu Gln Leu Lys Lys Val Leu His Glu 85 90 95 Glu Glu Lys Arg Arg
Arg Lys Glu Gln Ser Asp Leu Thr Thr Leu Thr 100 105 110 Ser Ala Ala
Tyr Gln Gln Ser Leu Thr Val His Thr Gly Thr His Leu 115 120 125 Leu
Ser Met Gln Gly Ser Pro Gly Gly His Asn Arg Pro Gly Thr Leu 130 135
140 Met Ala Ala Asp Arg Ala Lys Gln Met Phe Gly Pro Gln Val Leu Thr
145 150 155 160 Thr Arg His Tyr Val Gly Ser Ala Ala Ala Phe Ala Gly
Thr Pro Glu 165 170 175 His Gly Gln Phe Gln Gly Ser Pro Gly Gly Ala
Tyr Gly Thr Ala Gln 180 185 190 Pro Pro Pro His Tyr Gly Pro Thr Gln
Pro Ala Tyr Ser Pro Ser Gln 195 200 205 Gln Leu Arg Ala Pro Ser Ala
Phe Pro Ala Val Gln Tyr Leu Ser Gln 210 215 220 Pro Gln Pro Gln Pro
Tyr Ala Val His Gly His Phe Gln Pro Thr Gln 225 230 235 240 Thr Gly
Phe Leu Gln Pro Gly Gly Ala Leu Ser Leu Gln Lys Gln Met 245 250 255
Glu His Ala Asn Gln Gln Thr Gly Phe Ser Asp Ser Ser Ser Leu Arg 260
265 270 Pro Met His Pro Gln Ala Leu His Pro Ala Pro Gly Leu Leu Ala
Ser 275 280 285 Pro Gln Leu Pro Val Gln Met Gln Pro Ala Gly Lys Ser
Gly Phe Ala 290 295 300 Ala Thr Ser Gln Pro Gly Pro Arg Leu Pro Phe
Ile Gln His Ser Gln 305 310 315 320 Asn Pro Arg Phe Tyr His Lys 325
20324PRThomo sapien 20Met Ser Ser Glu Ala Glu Thr Gln Gln Pro Pro
Ala Ala Pro Pro Ala 1 5 10 15 Ala Pro Ala Leu Ser Ala Ala Asp Thr
Lys Pro Gly Thr Thr Gly Ser 20 25 30 Gly Ala Gly Ser Gly Gly Pro
Gly Gly Leu Thr Ser Ala Ala Pro Ala 35 40 45 Gly Gly Asp Lys Lys
Val Ile Ala Thr Lys Val Leu Gly Thr Val Lys 50 55 60 Trp Phe Asn
Val Arg Asn Gly Tyr Gly Phe Ile Asn Arg Asn Asp Thr 65 70 75 80 Lys
Glu Asp Val Phe Val His Gln Thr Ala Ile Lys Lys Asn Asn Pro 85 90
95 Arg Lys Tyr Leu Arg Ser Val Gly Asp Gly Glu Thr Val Glu Phe Asp
100 105 110 Val Val Glu Gly Glu Lys Gly Ala Glu Ala Ala Asn Val Thr
Gly Pro 115 120 125 Gly Gly Val Pro Val Gln Gly Ser Lys Tyr Ala Ala
Asp Arg Asn His 130 135 140 Tyr Arg Arg Tyr Pro Arg Arg Arg Gly Pro
Pro Arg Asn Tyr Gln Gln 145 150 155 160 Asn Tyr Gln Asn Ser Glu Ser
Gly Glu Lys Asn Glu Gly Ser Glu Ser 165 170 175 Ala Pro Glu Gly Gln
Ala Gln Gln Arg Arg Pro Tyr Arg Arg Arg Arg 180 185 190 Phe Pro Pro
Tyr Tyr Met Arg Arg Pro Tyr Gly Arg Arg Pro Gln Tyr 195 200 205 Ser
Asn Pro Pro Val Gln Gly Glu Val Met Glu Gly Ala Asp Asn Gln 210 215
220 Gly Ala Gly Glu Gln Gly Arg Pro Val Arg Gln Asn Met Tyr Arg Gly
225 230 235 240 Tyr Arg Pro Arg Phe Arg Arg Gly Pro Pro Arg Gln Arg
Gln Pro Arg 245 250 255 Glu Asp Gly Asn Glu Glu Asp Lys Glu Asn Gln
Gly Asp Glu Thr Gln 260 265 270 Gly Gln Gln Pro Pro Gln Arg Arg Tyr
Arg Arg Asn Phe Asn Tyr Arg 275 280 285 Arg Arg Arg Pro Glu Asn Pro
Lys Pro Gln Asp Gly Lys Glu Thr Lys 290 295 300 Ala Ala Asp Pro Pro
Ala Glu Asn Ser Ser Ala Pro Glu Ala Glu Gln 305 310 315 320 Gly Gly
Ala Glu 21960PRThomo sapien 21Met Trp Arg Leu Arg Arg Ala Ala Val
Ala Cys Glu Val Cys Gln Ser 1 5 10 15 Leu Val Lys His Ser Ser Gly
Ile Lys Gly Ser Leu Pro Leu Gln Lys 20 25 30 Leu His Leu Val Ser
Arg Ser Ile Tyr His Ser His His Pro Thr Leu 35 40 45 Lys Leu Gln
Arg Pro Gln Leu Arg Thr Ser Phe Gln Gln Phe Ser Ser 50 55 60 Leu
Thr Asn Leu Pro Leu Arg Lys Leu Lys Phe Ser Pro Ile Lys Tyr 65 70
75 80 Gly Tyr Gln Pro Arg Arg Asn Phe Trp Pro Ala Arg Leu Ala Thr
Arg 85 90 95 Leu Leu Lys Leu Arg Tyr Leu Ile Leu Gly Ser Ala Val
Gly Gly Gly 100 105 110 Tyr Thr Ala Lys Lys Thr Phe Asp Gln Trp Lys
Asp Met Ile Pro Asp 115 120 125 Leu Ser Glu Tyr Lys Trp Ile Val Pro
Asp Ile Val Trp Glu Ile Asp 130 135 140 Glu Tyr Ile Asp Phe Glu Lys
Ile Arg Lys Ala Leu Pro Ser Ser Glu 145 150 155 160 Asp Leu Val Lys
Leu Ala Pro Asp Phe Asp Lys Ile Val Glu Ser Leu 165 170 175 Ser Leu
Leu Lys Asp Phe Phe Thr Ser Gly Ser Pro Glu Glu Thr Ala 180 185 190
Phe Arg Ala Thr Asp Arg Gly Ser Glu Ser Asp Lys His Phe Arg Lys 195
200 205 Val Ser Asp Lys Glu Lys Ile Asp Gln Leu Gln Glu Glu Leu Leu
His 210 215 220 Thr Gln Leu Lys Tyr Gln Arg Ile Leu Glu Arg Leu Glu
Lys Glu Asn 225 230 235 240 Lys Glu Leu Arg Lys Leu Val Leu Gln Lys
Asp Asp Lys Gly Ile His 245 250 255 His Arg Lys Leu Lys Lys Ser Leu
Ile Asp Met Tyr Ser Glu Val Leu 260 265 270 Asp Val Leu Ser Asp Tyr
Asp Ala Ser Tyr Asn Thr Gln Asp His Leu 275 280 285 Pro Arg Val Val
Val Val Gly Asp Gln Ser Ala Gly Lys Thr Ser Val 290 295 300 Leu Glu
Met Ile Ala Gln Ala Arg Ile Phe Pro Arg Gly Ser Gly Glu 305 310 315
320 Met Met Thr Arg Ser Pro Val Lys Val Thr Leu Ser Glu Gly Pro His
325 330 335 His Val Ala Leu Phe Lys Asp Ser Ser Arg Glu Phe Asp Leu
Thr Lys 340 345 350 Glu Glu Asp Leu Ala Ala Leu Arg His Glu Ile Glu
Leu Arg Met Arg 355 360 365 Lys Asn Val Lys Glu Gly Cys Thr Val Ser
Pro Glu Thr Ile Ser Leu 370 375 380 Asn Val Lys Gly Pro Gly Leu Gln
Arg Met Val Leu Val Asp Leu Pro 385 390 395 400 Gly Val Ile Asn Thr
Val Thr Ser Gly Met Ala Pro Asp Thr Lys Glu 405 410 415 Thr Ile Phe
Ser Ile Ser Lys Ala Tyr Met Gln Asn Pro Asn Ala Ile 420 425 430 Ile
Leu Cys Ile Gln Asp Gly Ser Val Asp Ala Glu Arg Ser Ile Val 435 440
445 Thr Asp Leu Val Ser Gln Met Asp Pro His Gly Arg Arg Thr Ile Phe
450 455 460 Val Leu Thr Lys Val Asp Leu Ala Glu Lys Asn Val Ala Ser
Pro Ser 465 470 475 480 Arg Ile Gln Gln Ile Ile Glu Gly Lys Leu Phe
Pro Met Lys Ala Leu 485 490 495 Gly Tyr Phe Ala Val Val Thr Gly Lys
Gly Asn Ser Ser Glu Ser Ile 500 505 510 Glu Ala Ile Arg Glu Tyr Glu
Glu Glu Phe Phe Gln Asn Ser Lys Leu 515 520 525 Leu Lys Thr Ser Met
Leu Lys Ala His Gln Val Thr Thr Arg Asn Leu 530 535 540 Ser Leu Ala
Val Ser Asp Cys Phe Trp Lys Met Val Arg Glu Ser Val 545 550 555 560
Glu Gln Gln Ala Asp Ser Phe Lys Ala Thr Arg Phe Asn Leu Glu Thr
565
570 575 Glu Trp Lys Asn Asn Tyr Pro Arg Leu Arg Glu Leu Asp Arg Asn
Glu 580 585 590 Leu Phe Glu Lys Ala Lys Asn Glu Ile Leu Asp Glu Val
Ile Ser Leu 595 600 605 Ser Gln Val Thr Pro Lys His Trp Glu Glu Ile
Leu Gln Gln Ser Leu 610 615 620 Trp Glu Arg Val Ser Thr His Val Ile
Glu Asn Ile Tyr Leu Pro Ala 625 630 635 640 Ala Gln Thr Met Asn Ser
Gly Thr Phe Asn Thr Thr Val Asp Ile Lys 645 650 655 Leu Lys Gln Trp
Thr Asp Lys Gln Leu Pro Asn Lys Ala Val Glu Val 660 665 670 Ala Trp
Glu Thr Leu Gln Glu Glu Phe Ser Arg Phe Met Thr Glu Pro 675 680 685
Lys Gly Lys Glu His Asp Asp Ile Phe Asp Lys Leu Lys Glu Ala Val 690
695 700 Lys Glu Glu Ser Ile Lys Arg His Lys Trp Asn Asp Phe Ala Glu
Asp 705 710 715 720 Ser Leu Arg Val Ile Gln His Asn Ala Leu Glu Asp
Arg Ser Ile Ser 725 730 735 Asp Lys Gln Gln Trp Asp Ala Ala Ile Tyr
Phe Met Glu Glu Ala Leu 740 745 750 Gln Ala Arg Leu Lys Asp Thr Glu
Asn Ala Ile Glu Asn Met Val Gly 755 760 765 Pro Asp Trp Lys Lys Arg
Trp Leu Tyr Trp Lys Asn Arg Thr Gln Glu 770 775 780 Gln Cys Val His
Asn Glu Thr Lys Asn Glu Leu Glu Lys Met Leu Lys 785 790 795 800 Cys
Asn Glu Glu His Pro Ala Tyr Leu Ala Ser Asp Glu Ile Thr Thr 805 810
815 Val Arg Lys Asn Leu Glu Ser Arg Gly Val Glu Val Asp Pro Ser Leu
820 825 830 Ile Lys Asp Thr Trp His Gln Val Tyr Arg Arg His Phe Leu
Lys Thr 835 840 845 Ala Leu Asn His Cys Asn Leu Cys Arg Arg Gly Phe
Tyr Tyr Tyr Gln 850 855 860 Arg His Phe Val Asp Ser Glu Leu Glu Cys
Asn Asp Val Val Leu Phe 865 870 875 880 Trp Arg Ile Gln Arg Met Leu
Ala Ile Thr Ala Asn Thr Leu Arg Gln 885 890 895 Gln Leu Thr Asn Thr
Glu Val Arg Arg Leu Glu Lys Asn Val Lys Glu 900 905 910 Val Leu Glu
Asp Phe Ala Glu Asp Gly Glu Lys Lys Ile Lys Leu Leu 915 920 925 Thr
Gly Lys Arg Val Gln Leu Ala Glu Asp Leu Lys Lys Val Arg Glu 930 935
940 Ile Gln Glu Lys Leu Asp Ala Phe Ile Glu Ala Leu His Gln Glu Lys
945 950 955 960 22741PRThomo sapien 22Met Ala Glu Pro Val Ser Pro
Leu Lys His Phe Val Leu Ala Lys Lys 1 5 10 15 Ala Ile Thr Ala Ile
Phe Asp Gln Leu Leu Glu Phe Val Thr Glu Gly 20 25 30 Ser His Phe
Val Glu Ala Thr Tyr Lys Asn Pro Glu Leu Asp Arg Ile 35 40 45 Ala
Thr Glu Asp Asp Leu Val Glu Met Gln Gly Tyr Lys Asp Lys Leu 50 55
60 Ser Ile Ile Gly Glu Val Leu Ser Arg Arg His Met Lys Val Ala Phe
65 70 75 80 Phe Gly Arg Thr Ser Ser Gly Lys Ser Ser Val Ile Asn Ala
Met Leu 85 90 95 Trp Asp Lys Val Leu Pro Ser Gly Ile Gly His Ile
Thr Asn Cys Phe 100 105 110 Leu Ser Val Glu Gly Thr Asp Gly Asp Lys
Ala Tyr Leu Met Thr Glu 115 120 125 Gly Ser Asp Glu Lys Lys Ser Val
Lys Thr Val Asn Gln Leu Ala His 130 135 140 Ala Leu His Met Asp Lys
Asp Leu Lys Ala Gly Cys Leu Val Arg Val 145 150 155 160 Phe Trp Pro
Lys Ala Lys Cys Ala Leu Leu Arg Asp Asp Leu Val Leu 165 170 175 Val
Asp Ser Pro Gly Thr Asp Val Thr Thr Glu Leu Asp Ser Trp Ile 180 185
190 Asp Lys Phe Cys Leu Asp Ala Asp Val Phe Val Leu Val Ala Asn Ser
195 200 205 Glu Ser Thr Leu Met Asn Thr Glu Lys His Phe Phe His Lys
Val Asn 210 215 220 Glu Arg Leu Ser Lys Pro Asn Ile Phe Ile Leu Asn
Asn Arg Trp Asp 225 230 235 240 Ala Ser Ala Ser Glu Pro Glu Tyr Met
Glu Asp Val Arg Arg Gln His 245 250 255 Met Glu Arg Cys Leu His Phe
Leu Val Glu Glu Leu Lys Val Val Asn 260 265 270 Ala Leu Glu Ala Gln
Asn Arg Ile Phe Phe Val Ser Ala Lys Glu Val 275 280 285 Leu Ser Ala
Arg Lys Gln Lys Ala Gln Gly Met Pro Glu Ser Gly Val 290 295 300 Ala
Leu Ala Glu Gly Phe His Ala Arg Leu Gln Glu Phe Gln Asn Phe 305 310
315 320 Glu Gln Ile Phe Glu Glu Cys Ile Ser Gln Ser Ala Val Lys Thr
Lys 325 330 335 Phe Glu Gln His Thr Ile Arg Ala Lys Gln Ile Leu Ala
Thr Val Lys 340 345 350 Asn Ile Met Asp Ser Val Asn Leu Ala Ala Glu
Asp Lys Arg His Tyr 355 360 365 Ser Val Glu Glu Arg Glu Asp Gln Ile
Asp Arg Leu Asp Phe Ile Arg 370 375 380 Asn Gln Met Asn Leu Leu Thr
Leu Asp Val Lys Lys Lys Ile Lys Glu 385 390 395 400 Val Thr Glu Glu
Val Ala Asn Lys Val Ser Cys Ala Met Thr Asp Glu 405 410 415 Ile Cys
Arg Leu Ser Val Leu Val Asp Glu Phe Cys Ser Glu Phe His 420 425 430
Pro Asn Pro Asp Val Leu Lys Ile Tyr Lys Ser Glu Leu Asn Lys His 435
440 445 Ile Glu Asp Gly Met Gly Arg Asn Leu Ala Asp Arg Cys Thr Asp
Glu 450 455 460 Val Asn Ala Leu Val Leu Gln Thr Gln Gln Glu Ile Ile
Glu Asn Leu 465 470 475 480 Lys Pro Leu Leu Pro Ala Gly Ile Gln Asp
Lys Leu His Thr Leu Ile 485 490 495 Pro Cys Lys Lys Phe Asp Leu Ser
Tyr Asn Leu Asn Tyr His Lys Leu 500 505 510 Cys Ser Asp Phe Gln Glu
Asp Ile Val Phe Pro Phe Ser Leu Gly Trp 515 520 525 Ser Ser Leu Val
His Arg Phe Leu Gly Pro Arg Asn Ala Gln Arg Val 530 535 540 Leu Leu
Gly Leu Ser Glu Pro Ile Phe Gln Leu Pro Arg Ser Leu Ala 545 550 555
560 Ser Thr Pro Thr Ala Pro Thr Thr Pro Ala Thr Pro Asp Asn Ala Ser
565 570 575 Gln Glu Glu Leu Met Ile Thr Leu Val Thr Gly Leu Ala Ser
Val Thr 580 585 590 Ser Arg Thr Ser Met Gly Ile Ile Ile Val Gly Gly
Val Ile Trp Lys 595 600 605 Thr Ile Gly Trp Lys Leu Leu Ser Val Ser
Leu Thr Met Tyr Gly Ala 610 615 620 Leu Tyr Leu Tyr Glu Arg Leu Ser
Trp Thr Thr His Ala Lys Glu Arg 625 630 635 640 Ala Phe Lys Gln Gln
Phe Val Asn Tyr Ala Thr Glu Lys Leu Arg Met 645 650 655 Ile Val Ser
Ser Thr Ser Ala Asn Cys Ser His Gln Val Lys Gln Gln 660 665 670 Ile
Ala Thr Thr Phe Ala Arg Leu Cys Gln Gln Val Asp Ile Thr Gln 675 680
685 Lys Gln Leu Glu Glu Glu Ile Ala Arg Leu Pro Lys Glu Ile Asp Gln
690 695 700 Leu Glu Lys Ile Gln Asn Asn Ser Lys Leu Leu Arg Asn Lys
Ala Val 705 710 715 720 Gln Leu Glu Asn Glu Leu Glu Asn Phe Thr Lys
Gln Phe Leu Pro Ser 725 730 735 Ser Asn Glu Glu Ser 740
23757PRThomo sapien 23Met Ser Leu Leu Phe Ser Arg Cys Asn Ser Ile
Val Thr Val Lys Lys 1 5 10 15 Asn Lys Arg His Met Ala Glu Val Asn
Ala Ser Pro Leu Lys His Phe 20 25 30 Val Thr Ala Lys Lys Lys Ile
Asn Gly Ile Phe Glu Gln Leu Gly Ala 35 40 45 Tyr Ile Gln Glu Ser
Ala Thr Phe Leu Glu Asp Thr Tyr Arg Asn Ala 50 55 60 Glu Leu Asp
Pro Val Thr Thr Glu Glu Gln Val Leu Asp Val Lys Gly 65 70 75 80 Tyr
Leu Ser Lys Val Arg Gly Ile Ser Glu Val Leu Ala Arg Arg His 85 90
95 Met Lys Val Ala Phe Phe Gly Arg Thr Ser Asn Gly Lys Ser Thr Val
100 105 110 Ile Asn Ala Met Leu Trp Asp Lys Val Leu Pro Ser Gly Ile
Gly His 115 120 125 Thr Thr Asn Cys Phe Leu Arg Val Glu Gly Thr Asp
Gly His Glu Ala 130 135 140 Phe Leu Leu Thr Glu Gly Ser Glu Glu Lys
Arg Ser Ala Lys Thr Val 145 150 155 160 Asn Gln Leu Ala His Ala Leu
His Gln Asp Lys Gln Leu His Ala Gly 165 170 175 Ser Leu Val Ser Val
Met Trp Pro Asn Ser Lys Cys Pro Leu Leu Lys 180 185 190 Asp Asp Leu
Val Leu Met Asp Ser Pro Gly Ile Asp Val Thr Thr Glu 195 200 205 Leu
Asp Ser Trp Ile Asp Lys Phe Cys Leu Asp Ala Asp Val Phe Val 210 215
220 Leu Val Ala Asn Ser Glu Ser Thr Leu Met Gln Thr Glu Lys His Phe
225 230 235 240 Phe His Lys Val Ser Glu Arg Leu Ser Arg Pro Asn Ile
Phe Ile Leu 245 250 255 Asn Asn Arg Trp Asp Ala Ser Ala Ser Glu Pro
Glu Tyr Met Glu Glu 260 265 270 Val Arg Arg Gln His Met Glu Arg Cys
Thr Ser Phe Leu Val Asp Glu 275 280 285 Leu Gly Val Val Asp Arg Ser
Gln Ala Gly Asp Arg Ile Phe Phe Val 290 295 300 Ser Ala Lys Glu Val
Leu Asn Ala Arg Ile Gln Lys Ala Gln Gly Met 305 310 315 320 Pro Glu
Gly Gly Gly Ala Leu Ala Glu Gly Phe Gln Val Arg Met Phe 325 330 335
Glu Phe Gln Asn Phe Glu Arg Arg Phe Glu Glu Cys Ile Ser Gln Ser 340
345 350 Ala Val Lys Thr Lys Phe Glu Gln His Thr Val Arg Ala Lys Gln
Ile 355 360 365 Ala Glu Ala Val Arg Leu Ile Met Asp Ser Leu His Met
Ala Ala Arg 370 375 380 Glu Gln Gln Val Tyr Cys Glu Glu Met Arg Glu
Glu Arg Gln Asp Arg 385 390 395 400 Leu Lys Phe Ile Asp Lys Gln Leu
Glu Leu Leu Ala Gln Asp Tyr Lys 405 410 415 Leu Arg Ile Lys Gln Ile
Thr Glu Glu Val Glu Arg Gln Val Ser Thr 420 425 430 Ala Met Ala Glu
Glu Ile Arg Arg Leu Ser Val Leu Val Asp Asp Tyr 435 440 445 Gln Met
Asp Phe His Pro Ser Pro Val Val Leu Lys Val Tyr Lys Asn 450 455 460
Glu Leu His Arg His Ile Glu Glu Gly Leu Gly Arg Asn Met Ser Asp 465
470 475 480 Arg Cys Ser Thr Ala Ile Thr Asn Ser Leu Gln Thr Met Gln
Gln Asp 485 490 495 Met Ile Asp Gly Leu Lys Pro Leu Leu Pro Val Ser
Val Arg Ser Gln 500 505 510 Ile Asp Met Leu Val Pro Arg Gln Cys Phe
Ser Leu Asn Tyr Asp Leu 515 520 525 Asn Cys Asp Lys Leu Cys Ala Asp
Phe Gln Glu Asp Ile Glu Phe His 530 535 540 Phe Ser Leu Gly Trp Thr
Met Leu Val Asn Arg Phe Leu Gly Pro Lys 545 550 555 560 Asn Ser Arg
Arg Ala Leu Met Gly Tyr Asn Asp Gln Val Gln Arg Pro 565 570 575 Ile
Pro Leu Thr Pro Ala Asn Pro Ser Met Pro Pro Leu Pro Gln Gly 580 585
590 Ser Leu Thr Gln Glu Glu Phe Met Val Ser Met Val Thr Gly Leu Ala
595 600 605 Ser Leu Thr Ser Arg Thr Ser Met Gly Ile Leu Val Val Gly
Gly Val 610 615 620 Val Trp Lys Ala Val Gly Trp Arg Leu Ile Ala Leu
Ser Phe Gly Leu 625 630 635 640 Tyr Gly Leu Leu Tyr Val Tyr Glu Arg
Leu Thr Trp Thr Thr Lys Ala 645 650 655 Lys Glu Arg Ala Phe Lys Arg
Gln Phe Val Glu His Ala Ser Glu Lys 660 665 670 Leu Gln Leu Val Ile
Ser Tyr Thr Gly Ser Asn Cys Ser His Gln Val 675 680 685 Gln Gln Glu
Leu Ser Gly Thr Phe Ala His Leu Cys Gln Gln Val Asp 690 695 700 Val
Thr Arg Glu Asn Leu Glu Gln Glu Ile Ala Ala Met Asn Lys Lys 705 710
715 720 Ile Glu Val Leu Asp Ser Leu Gln Ser Lys Ala Lys Leu Leu Arg
Asn 725 730 735 Lys Ala Gly Trp Leu Asp Ser Glu Leu Asn Met Phe Thr
His Gln Tyr 740 745 750 Leu Gln Pro Ser Arg 755
* * * * *
References