U.S. patent application number 15/842829 was filed with the patent office on 2018-06-28 for compositions and methods for nucleic acid and/or protein payload delivery.
The applicant listed for this patent is Ligandal, Inc.. Invention is credited to Christian Foster, Andre Ronald Watson.
Application Number | 20180179553 15/842829 |
Document ID | / |
Family ID | 62488586 |
Filed Date | 2018-06-28 |
United States Patent
Application |
20180179553 |
Kind Code |
A1 |
Watson; Andre Ronald ; et
al. |
June 28, 2018 |
COMPOSITIONS AND METHODS FOR NUCLEIC ACID AND/OR PROTEIN PAYLOAD
DELIVERY
Abstract
Provided are methods and compositions for nanoparticle delivery
of payloads (e.g., nucleic acid and/or protein payloads) to cells.
In some embodiments, a subject nanoparticle includes a core and a
sheddable layer encapsulating the core, where the core includes (i)
an anionic polymer composition; (ii) a cationic polymer
composition; (iii) a cationic polypeptide composition; and (iv) a
nucleic acid and/or protein payload; and where: (a) the anionic
polymer composition includes polymers of D-isomers of an anionic
amino acid and polymers of L-isomers of an anionic amino acid,
and/or (b) the cationic polymer composition comprises polymers of
D-isomers of a cationic amino acid and polymers of L-isomers of a
cationic amino acid. In some cases, the polymers of D-isomers of an
anionic and/or cationic amino acid are present at a ratio, relative
to the polymers of L-isomers, in a range of from 10:1 to 1:10.
Inventors: |
Watson; Andre Ronald; (San
Francisco, CA) ; Foster; Christian; (Oakland,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ligandal, Inc. |
Berkeley |
CA |
US |
|
|
Family ID: |
62488586 |
Appl. No.: |
15/842829 |
Filed: |
December 14, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62434344 |
Dec 14, 2016 |
|
|
|
62443567 |
Jan 6, 2017 |
|
|
|
62443522 |
Jan 6, 2017 |
|
|
|
62517346 |
Jun 9, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/22 20130101; C12N
15/87 20130101; C12N 2310/3517 20130101; A61K 9/5146 20130101; C12N
15/102 20130101; A61K 31/7105 20130101; A61K 47/42 20130101; A61K
48/0016 20130101; A61K 48/0041 20130101; A61P 37/00 20180101; A61P
35/00 20180101; A61K 47/549 20170801; A61K 31/40 20130101; A61K
47/64 20170801; A61K 48/0075 20130101; B82Y 5/00 20130101; A61K
9/0085 20130101; C12N 15/85 20130101; C12N 15/111 20130101; C12N
2310/14 20130101; A61P 3/10 20180101; A61K 47/6455 20170801; C12N
2310/20 20170501; C12N 2320/32 20130101; A61K 47/34 20130101; A61K
47/645 20170801; A61K 9/0019 20130101; A61K 47/6807 20170801 |
International
Class: |
C12N 15/85 20060101
C12N015/85; A61K 47/34 20060101 A61K047/34; A61K 47/64 20060101
A61K047/64; A61K 48/00 20060101 A61K048/00; C12N 15/10 20060101
C12N015/10; A61K 9/51 20060101 A61K009/51; A61P 35/00 20060101
A61P035/00; A61P 3/10 20060101 A61P003/10; A61P 37/00 20060101
A61P037/00 |
Claims
1. A nanoparticle, comprising a core and a sheddable layer
encapsulating the core, wherein the core comprises: (i) an anionic
polymer composition; (ii) a cationic polymer composition; (iii) a
cationic polypeptide composition; and (iv) a nucleic acid and/or
protein payload, wherein (a) said anionic polymer composition
comprises polymers of D-isomers of an anionic amino acid and
polymers of L-isomers of an anionic amino acid; and/or (b) said
cationic polymer composition comprises polymers of D-isomers of a
cationic amino acid and polymers of L-isomers of a cationic amino
acid.
2. The nanoparticle of claim 1, wherein said anionic polymer
composition comprises a first anionic polymer selected from
poly(D-glutamic acid) (PDE) and poly(D-aspartic acid) (PDD); and
comprises a second anionic polymer selected from poly(L-glutamic
acid) (PLE) and poly(L-aspartic acid) (PLD).
3. The nanoparticle of claim 1, wherein said cationic polymer
composition comprises a first cationic polymer selected from
poly(D-arginine), poly(D-lysine), poly(D-histidine),
poly(D-ornithine), and poly(D-citrulline); and comprises a second
cationic polymer selected from poly(L-arginine), poly(L-lysine),
poly(L-histidine), poly(L-ornithine), and poly(L-citrulline).
4. The nanoparticle of claim 1, wherein said polymers of D-isomers
of an anionic amino acid are present at a ratio, relative to said
polymers of L-isomers of an anionic amino acid, in a range of from
10:1 to 1:10.
5. The nanoparticle of claim 1, wherein said polymers of D-isomers
of a cationic amino acid are present at a ratio, relative to said
polymers of L-isomers of a cationic amino acid, in a range of from
10:1 to 1:10.
6. The nanoparticle of claim 1, wherein the sheddable layer is an
anionic coat or a cationic coat.
7. The nanoparticle of claim 1, wherein the sheddable layer is pH
and/or glutathione sensitive.
8. The nanoparticle of claim 1, wherein the sheddable layer
comprises one or more of: silica, a peptoid, a polycysteine,
calcium, calcium phosphate, calcium sulfate, manganese, manganese
phosphate, manganese sulfate, magnesium, magnesium phosphate,
magnesium sulfate, iron, iron phosphate, and iron sulfate.
9. (canceled)
10. The nanoparticle of claim 1, further comprising a surface coat
surrounding the sheddable layer.
11. The nanoparticle of claim 10, wherein the surface coat
comprises a cationic or anionic component that interacts
electrostatically with the sheddable layer.
12. The nanoparticle of claim 10, wherein the surface coat
comprises one or more of: a polymer of a cationic amino acid, a
poly(arginine), an anchoring domain, a cationic anchoring domain,
an anionic anchoring domain, a cell penetrating peptide, a
glycoprotein, a heparin sulfate proteoglycan, and a targeting
ligand.
13. The nanoparticle of claim 1, wherein the surface coat is
zwitterionic and multivalent.
14. The nanoparticle of claim 1, wherein the surface coat comprises
one or more targeting ligands.
15. The nanoparticle of claim 14, wherein at least one of the one
or more targeting ligands is conjugated to a cationic anchoring
domain that interacts with the sheddable layer.
16. The nanoparticle of claim 15, wherein the cationic anchoring
domain is selected from RRRRRRRRR (SEQ ID NO: 15) and HHHHHH (SEQ
ID NO: 16).
17. The nanoparticle of claim 15, wherein the anchoring domain is
conjugated to the at least one of the one or more targeting ligands
via a linker.
18. (canceled)
19. The nanoparticle of claim 17, wherein the linker is a
polypeptide.
20. The nanoparticle of claim 17, wherein the linker is conjugated
to the targeting ligand via sulfhydryl or amine-reactive chemistry,
and/or the linker is conjugated to the anchoring domain via
sulfhydryl or amine-reactive chemistry.
21. The nanoparticle of claim 17, wherein said at least one of the
one or more targeting ligands comprises a cysteine residue and is
conjugated to the linker via the cysteine residue.
22. The nanoparticle of claim 14, wherein said one or more
targeting ligands provides for targeted binding to a family B
G-protein coupled receptor (GPCR).
23. The nanoparticle of claim 22, wherein said targeting ligand
comprises a cysteine substitution, at one or more internal amino
acid positions, relative to a corresponding wild type amino acid
sequence.
24. The nanoparticle of claim 22, wherein said targeting ligand
comprises an amino acid sequence having 85% or more identity to the
amino acid sequence HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID
NO: 1).
25. The nanoparticle of claim 24, wherein said targeting ligand
comprises a cysteine substitution at one or more of positions L10,
S11, and K12 of the amino acid sequence set forth in (SEQ ID NO:
1).
26. The nanoparticle of claim 25, wherein said targeting ligand
comprises the amino acid sequence
HGEGTFTSDLCKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 2).
27. The nanoparticle of claim 14, wherein the surface coat
comprises one or more targeting ligands that provides for targeted
binding to a cell surface protein selected from c-Kit, CD27, and
CD150.
28. The nanoparticle of claim 14, wherein the surface coat
comprises one or more targeting ligands selected from the group
consisting of: rabies virus glycoprotein (RVG) fragment,
ApoE-transferrin, lactoferrin, melanoferritin, ovotransferritin,
L-selectin, E-selectin, P-selectin, PSGL-1, ESL-1, CD44, death
receptor-3 (DR3), LAMP1, LAMP2, Mac2-BP, stem cell factor (SCF),
CD70, SH2 domain-containing protein 1A (SH2D1A), a exendin-4, GLP1,
a targeting ligand that targets .alpha.5.beta.1, RGD, a Transferrin
ligand, an FGF fragment, succinic acid, a bisphosphonate, CD90,
CD45f, CD34, a hematopoietic stem cell chemotactic lipid,
sphingosine, ceramide, sphingosine-1-phosphate,
ceramide-1-phosphate, and an active targeting fragment of any of
the above.
29. The nanoparticle of claim 14, wherein the surface coat
comprises stem cell factor (SCF) or a targeting fragment thereof,
CD70 or a targeting fragment thereof, and SH2 domain-containing
protein 1A (SH2D1A) or a targeting fragment thereof.
30. (canceled)
31. The nanoparticle of claim 14, wherein the surface coat
comprises two or more different targeting ligands.
32. The nanoparticle of claim 1, wherein the cationic polypeptide
composition comprises a polypeptide that comprises a nuclear
localization signal (NLS) and/or a histone tail peptide (HTP).
33. (canceled)
34. The nanoparticle of claim 1, wherein the cationic polypeptide
composition comprises a histone tail peptide (HTP).
35. The nanoparticle of claim 34, wherein the HTP is conjugated to
a cationic amino acid polymer.
36-37. (canceled)
38. The nanoparticle of claim 1, wherein said cationic polypeptide
composition comprises histone peptides having a branched
structure.
39. The nanoparticle of claim 1, wherein the payload comprises one
or more of: (i) a CRISPR/Cas guide RNA, (ii) a DNA molecule
encoding a CRISPR/Cas guide RNA, (iii) a nucleic acid molecule
encoding a CRISPR/Cas RNA-guided polypeptide, (iv) a CRISPR/Cas
RNA-guided polypeptide, (v) a CRISPR/Cas guide RNA complexed with a
CRISPR/Cas RNA-guided polypeptide, (vi) a nucleic acid molecule
encoding a zinc finger protein (ZFP), (vii) a ZFP, (viii) a nucleic
acid molecule encoding a transcription activator-like effector
(TALE) protein, (ix) a TALE protein, and (x) a DNA donor
template.
40. The nanoparticle of claim 1, wherein the payload comprises (i)
a CRISPR/Cas guide RNA and/or a DNA molecule encoding said
CRISPR/Cas guide RNA; and (ii) a CRISPR/Cas RNA-guided polypeptide
and/or a nucleic acid molecule encoding said CRISPR/Cas RNA-guided
polypeptide.
41. The nanoparticle of claim 40, wherein the payload further
comprises a DNA donor template.
42. A nanoparticle formulation, comprising: (a) a first
nanoparticle according to claim 1, wherein the payload comprises
one or more of: (i) a CRISPR/Cas guide RNA, (ii) a DNA molecule
encoding a CRISPR/Cas guide RNA, (iii) a nucleic acid molecule
encoding a CRISPR/Cas RNA-guided polypeptide, (iv) a CRISPR/Cas
RNA-guided polypeptide, (v) a CRISPR/Cas guide RNA complexed with a
CRISPR/Cas RNA-guided polypeptide, (vi) a nucleic acid molecule
encoding a zinc finger protein (ZFP), (vii) a ZFP, (viii) a nucleic
acid molecule encoding a transcription activator-like effector
(TALE) protein, and (ix) a TALE protein; and (b) a second
nanoparticle comprising a nucleic acid payload that comprises a DNA
donor template.
43. A multi-layered nanoparticle, comprising: (a) an inner core
comprising a payload comprising a DNA donor template; (b) a first
sheddable layer surrounding the inner core; (c) an intermediate
core surrounding the first sheddable layer, wherein the
intermediate core comprises one or more of: (i) a CRISPR/Cas guide
RNA, (ii) a DNA molecule encoding a CRISPR/Cas guide RNA, (iii) a
nucleic acid molecule encoding a CRISPR/Cas RNA-guided polypeptide,
(iv) a CRISPR/Cas RNA-guided polypeptide, (v) a CRISPR/Cas guide
RNA complexed with a CRISPR/Cas RNA-guided polypeptide, (vi) a zinc
finger protein (ZFP), (vii) a DNA molecule encoding a ZFP, (viii) a
transcription activator-like effector (TALE) protein, and (ix) a
DNA molecule encoding a TALE protein; and (d) a second sheddable
layer surrounding the intermediate core.
44-49. (canceled)
50. A method of delivering a nucleic acid and/or protein payload to
a target cell, the method comprising: contacting a eukaryotic
target cell with the nanoparticle of claim 1.
51. The method of claim 50, wherein the payload includes a gene
editing tool.
52-61. (canceled)
62. A branched histone molecule, comprising: one or more histone
tail peptides (HTPs) conjugated to side chains of a cationic
polymer.
63-64. (canceled)
65. A ligand-targeted polymeric nanoparticle bearing one or more
guided nuclease payloads and donor strands.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 62/517,346, filed Jun. 9, 2017, of U.S.
Provisional Patent Application No. 62/443,567, filed Jan. 6, 2017,
of U.S. Provisional Patent Application No. 62/443,522, filed Jan.
6, 2017, and of U.S. Provisional Patent Application No. 62/434,344,
filed Dec. 14, 2016, all of which applications are incorporated
herein by reference in their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT
FILE
[0002] A Sequence Listing is provided herewith as a text file,
"LGDL-004_SeqList_ST25.bd" created on Dec. 14, 2017 and having a
size of 128 KB. The contents of the text file are incorporated by
reference herein in their entirety.
INTRODUCTION
[0003] Effective introduction of nucleic acid and/or protein
payloads into cells, e.g., for genome editing and/or altering gene
expression, is an important objective for therapeutic strategies
and for research methodologies. To achieve effective introduction
of a payload, it is important to appropriately package the payload
to protect it from degradation prior to cellular entry, to permit
entry into cells, to direct the payload away from the lysosomal
degradation pathway, and to direct delivery to the appropriate
subcellular compartment. In addition, the timing of release of a
payload from the packaging following cellular entry can influence
the effectiveness of the payload.
[0004] Many nanoparticle-based technologies for payload delivery
offer low levels of cellular transfection and limited effectiveness
upon transfection. There is a need for compositions and methods
that enhance effectiveness of payload delivery to cells.
SUMMARY
[0005] Provided are compositions and methods for delivery of
payloads (e.g., nucleic acid and/or protein payloads) to cells
(e.g., nanoparticle, viral, and non-viral delivery of payloads to
cells). Nanoparticles designed for serum stability, targeted
delivery to specific cell types, biomimicry of endogenous nucleic
acid packaging via histones and nucleosome-like branched polymer,
compartment-specific unpackaging within the nucleus, variable timed
release kinetics, and methods of use thereof, are provided. In some
embodiments, a subject nanoparticle includes a core and a sheddable
layer encapsulating the core (e.g., providing for temporary
stabilization of the core during cell delivery), where the core
includes (i) an anionic polymer composition; (ii) a cationic
polymer composition; (iii) a cationic polypeptide composition; and
(iv) a nucleic acid and/or protein payload; and where: (a) the
anionic polymer composition includes polymers of D-isomers of an
anionic amino acid and polymers of L-isomers of an anionic amino
acid, and/or (b) the cationic polymer composition includes polymers
of D-isomers of a cationic amino acid and polymers of L-isomers of
a cationic amino acid. In some cases, the polymers of D-isomers of
an anionic amino acid are present at a ratio, relative to the
polymers of L-isomers of an anionic amino acid, in a range of from
10:1 to 1:10. In some cases, the polymers of D-isomers of a
cationic amino acid are present at a ratio, relative to said
polymers of L-isomers of a cationic amino acid, in a range of from
10:1 to 1:10.
[0006] In some cases, a nanoparticle of the disclosure includes a
surface coat, which surrounds the sheddable layer. The surface coat
can include a targeting ligand that provides for targeted binding
to a surface molecule of a target cell. In some cases, the
targeting ligand is conjugated (with or without a linker) to an
anchoring domain, e.g., for anchoring the targeting ligand to the
sheddable layer of the nanoparticle.
[0007] Also provided are multi-layered nanoparticles that include a
first payload (e.g., a DNA donor template) as part of the core,
where the core is surrounded by a first sheddable layer, the first
sheddable layer is surrounded by an intermediate layer that
includes a second payload (e.g., a gene editing tool), and the
intermediate layer is surrounded by a second sheddable layer. In
some cases the second sheddable layer is coated with a surface coat
(e.g., a surface coat that includes a targeting ligand.
[0008] Also provided are nanoparticle formulations including two or
more nanoparticles in which the payload of a first nanoparticle
includes a donor DNA template and the payload of a second
nanoparticle includes a gene editing tool (e.g., (i) a CRISPR/Cas
guide RNA; (ii) a DNA encoding a CRISPR/Cas guide RNA; (iii) a DNA
and/or RNA encoding a programmable gene editing protein; and/or
(iv) a programmable gene editing protein).
[0009] Also provided are methods of co-delivery of multiple
payloads (e.g., two or more payloads) as part of the same package.
For example, provided are method of delivering a nucleic acid
and/or protein payload to a target cell, where the method includes
contacting a eukaryotic target cell with a viral or non-viral
delivery vehicle that includes (a) a gene editing tool; and (b) a
nucleic acid or protein agent that induces proliferation of and/or
biases differentiation of the target cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The invention is best understood from the following detailed
description when read in conjunction with the accompanying
drawings. It is emphasized that, according to common practice, the
various features of the drawings are not to-scale. On the contrary,
the dimensions of the various features are arbitrarily expanded or
reduced for clarity. Included in the drawings are the following
figures.
[0011] FIG. 1 depicts results from a fluorimetric assay testing
various parameters (e.g., cation:anion charge ratio) for
condensation of nucleic acid payloads. The result showed, e.g.,
that a charge ratio of 2 works well for the condensation of
plasmids encoding Cas9 and guide RNA molecules.
[0012] FIG. 2 depicts particle size and zeta potential
distributions for nanoparticle cores that were generated. The data
were obtained using a Particle Metrix ZetaView NTA instrument.
[0013] Nanoparticle Size (peak) was 128.8 nm, and Zeta potential
(peak) was +10.5 mV (100%).
[0014] FIG. 3 depicts particle size and zeta potential
distributions for stabilized nanoparticle cores (cores encapsulated
by a sheddable layer). The data were obtained using a Particle
Metrix ZetaView NTA instrument. The stabilized cores had a size of
110.6 nm and zeta potential of -42.1 mV (95%).
[0015] FIG. 4 depicts data showing that nanoparticles with an outer
shell (outer coat) that included RVG9R, which is Rabies Virus
Glycoprotein (RVG) fused to a 9-Arg peptide sequence (as a cationic
anchoring domain), had a characteristic particle size of 115.8 nm
and a zeta potential of -3.1 mV (100%). Optimal outer coating
yields a transition of zeta potential from -50 mV (for the silica
coated core) to between 0 and -10 mV (after adding the outer
shell).
[0016] FIG. 5 depicts results from cell culture experiments in
which different nanoparticles were used to deliver nucleic acid
payloads. The figure compares nanoparticles that include
poly(D-glutamic Acid) as part of the core (in addition to
poly(L-arginine)) to those that do not. The three rows represent
replicates.
[0017] FIG. 6 (panels A-D) depicts microscopy images of neural stem
cells that were contacted with nanoparticles that included
CRISPR/Cas9 expression vectors as the nucleic acid payload. The
core of the nanoparticles included poly(L-arginine) (a cationic
polymer) tagged with a fluorophore (FITC). The endosome and nucleus
were stained using Lysotracker (Red) and Hoescht 3342 (blue)
respectively. Nanoparticles (and Lipofectamine 3000 as a control)
were introduced to cells 16 hours after seeding. Cells were
incubated with Hoescht 3342 and Lysotracker Red prior to imaging.
Panels C-D present bar graphs that quantify colocalization of the
nanoparticle core with the nucleus and with endosomes.
[0018] FIG. 7 (panels A-B) depicts microscopy images of peripheral
blood mononuclear cells (PBMCs) that were transfected with
nanoparticles that included mRNA encoding GFP as a nucleic acid
payload. The images demonstrate that mRNA expression can be
extended to 16 days with nanoparticles that include a core with, at
a defined ratio, a polymer of D-isomers of an anionic amino acid
and a polymer of L-isomers of an anionic amino acid. In this case,
use of a nanoparticle core with a 2:1 ratio of poly(D-Glutamic
acid) to poly(L-Glutamic Acid) resulted in maximum expression at 16
days (panel A=4 days; panel B=16 days).
[0019] FIG. 8 depicts a schematic representation of an example
embodiment of a subject nanoparticle.
[0020] FIG. 9 depicts a schematic representation of an example
embodiment of a subject nanoparticle. In this case, the
nanoparticle is multi-layered, having a core (which includes a
first payload) surrounded by a first sheddable layer, which is
surrounded by an intermediate layer (which includes an additional
payload), which is surrounded by a second sheddable layer, which is
surface coated (i.e., includes an outer shell).
[0021] FIG. 10 (panels A-B) depicts schematic representations of
example configurations of a delivery molecule of a surface coat of
a subject nanoparticle. The delivery molecules depicted include a
targeting ligand conjugated to an anchoring domain that is
interacting electrostatically with a sheddable layer of a
nanoparticle. Note that the targeting ligand can be conjugated at
the N- or C-terminus (left of each panel), but can also be
conjugated at an internal position (right of each panel). The
molecules in panel A include a linker while those in panel B do
not.
[0022] FIG. 11 provides a schematic diagram of a family B GPCR,
highlighting separate domains to be considered when evaluating a
targeting ligand, e.g., for binding to allosteric/affinity
N-terminal domains and orthosteric endosomal-sorting/signaling
domains. (Figure is adapted from Siu, Fai Yiu, et al., Nature
499.7459 (2013): 444-449).
[0023] FIG. 12 provides an example of identifying an internal amino
acid position for insertion and/or substitution (e.g., with a
cysteine residue) for a targeting ligand such that affinity is
maintained and the targeting ligand engages long endosomal
recycling pathways that promote nucleic acid release and limit
nucleic acid degradation. In this case, the targeting ligand is
exendin-4 and amino acid positions 10, 11, and 12 were identified
as sites for possible insertion and/or substitution (e.g., with a
cysteine residue, e.g., an S11C mutation). The figure shows an
alignment of simulated Exendin-4 (SEQ ID NO: 1) to known crystal
structures of glucagon-GCGR (4ERS) and GLP1-GLP1R-ECD complex (PDB:
3IOL), and PDB renderings that were rotated in 3-dimensional
space.
[0024] FIG. 13 shows a tbFGF fragment as part of a ternary
FGF2-FGFR1-HEPARIN complex (1fq9 on PDB). CKNGGFFLRIHPDGRVDGVREKS
(highlighted) (SEQ ID NO: 43) was determined to be important for
affinity to FGFR1.
[0025] FIG. 14 provides an alignment and PDB 3D rendering used to
determine that HFKDPK (SEQ ID NO: 5) is a peptide that can be used
for ligand-receptor orthosteric activity and affinity.
[0026] FIG. 15 provides an alignment and PDB 3D rendering used to
determine that LESNNYNT (SEQ ID NO: 6) is a peptide that can be
used for ligand-receptor orthosteric activity and affinity.
[0027] FIG. 16 provides non-limiting examples nuclear localization
signals (NLSs) that can be used as part of a subject nanoparticle
(e.g., as an NLS-containing peptide; as part of/conjugated to an
NLS-containing peptide, an anionic polymer, a cationic polymer,
and/or a cationic polypeptide; and the like). The figure is adapted
from Kosugi et al., J Biol Chem. 2009 Jan. 2; 284(1):478-85. [Class
1, top to bottom (SEQ ID NOs: 201-221); Class 2, top to bottom (SEQ
ID NOs: 222-224); Class 4, top to bottom (SEQ ID NOs: 225-230);
Class 3, top to bottom (SEQ ID NOs: 231-245); Class 5, top to
bottom (SEQ ID NOs: 246-264)].
[0028] FIG. 17 (panels A-B) depicts schematic representations of
the mouse (panel A) and human (panel B) hematopoietic cell lineage,
and markers that have been identified for various cells within the
lineage.
[0029] FIG. 18 (panels A-B) depicts schematic representations of
miRNA (panel A) and protein (panel B) factors that can be used to
influence cell differentiation and/or proliferation FIG. 19
provides condensation curves on nanoparticles with payload:
VWF-EGFP pDNA with peptide nucleic acid (PNA) Binding Site.
[0030] FIG. 20 provides condensation curves on nanoparticles with
payload: NLS-CAS9-NLS RNP complexed to HBB gRNA.
[0031] FIG. 21 provides condensation curves on nanoparticles with
payload: HBB gRNA.
[0032] FIG. 22 provides condensation curves on nanoparticles with
payload: HBB gRNA.
[0033] FIG. 23 provides condensation curves on nanoparticles with
payload: NLS-CAS9-NLS RNP complexed to HBB gRNA.
[0034] FIG. 24 provides condensation curves on nanoparticles with
payload: VWF-EGFP pDNA with peptide nucleic acid (PNA) Binding
Site.
[0035] FIG. 25 provides condensation curves on nanoparticles with
payload: VWF-EGFP pDNA with peptide nucleic acid (PNA) Binding
Site.
[0036] FIG. 26 provides condensation curves on nanoparticles with
payload: RNP of NLS-CAS9-NLS with HBB gRNA.
[0037] FIG. 27 provides condensation curves on nanoparticles with
payload: VWF-EGFP pDNA with peptide nucleic acid (PNA) Binding
Site.
[0038] FIG. 28 provides condensation curves on nanoparticles with
payload: Cy5_EGFP mRNA.
[0039] FIG. 29 provides condensation curves on nanoparticles with
payload: BLOCK-iT Alexa Fluor 555 siRNA.
[0040] FIG. 30 provides condensation curves on nanoparticles with
payload: NLS-Cas9-EGFP RNP complexed to HBB gRNA.
[0041] FIG. 31 provides data collected when using nanoparticles
with Alexa 555 Block-IT siRNA as payload.
[0042] FIG. 32 provides data collected when using nanoparticles
with ribonuclear protein (RNP) formed by NLS-Cas9-GFP and HBB guide
RNA as payload.
[0043] FIG. 33 provides data collected when using nanoparticles
with Cy5 EGFP mRNA as payload.
[0044] FIG. 34 provides data collected when using nanoparticles
with payload: VWF-EGFP pDNA with Cy5 tagged peptide nucleic acid
(PNA) Binding Site.
[0045] FIG. 35 provides data from a SYBR Gold exclusion assay
showing fluorescence intensity decrease by addition of cationic
polypeptide CD45_mSiglec_(4GS)2_9R_C followed by PLE100 and by
further addition of the cationic polypeptide to RNP.
[0046] FIG. 36 provides data from a SYBR Gold exclusion assay
showing fluorescence intensity variations by addition of cationic
polypeptide CD45_mSiglec_(4GS)2_9R_C followed by PLE100 and by
further addition of the cationic polypeptide to siRNA and SYBR
Gold.
[0047] FIG. 37 provides data from a SYBR Gold exclusion assay
showing fluorescence intensity variations by addition of cationic
polypeptide histone peptide H2A followed by
CD45_mSiglec_(4GS)2_9R_C and by further addition of PLE100 to RNP
of NLS-Cas9-EGFP with HBB gRNA and SYBR Gold.
[0048] FIG. 38 provides data from a SYBR Gold exclusion assay
showing fluorescence intensity variations by addition of cationic
polypeptide histone peptide H4 together with
CD45_mSiglec_(4GS)2_9R_C and by further addition of PLE100 to RNP
of NLS-Cas9-EGFP with HBB gRNA and SYBR Gold.
[0049] FIG. 39 provides data from a SYBR Gold exclusion assay
showing fluorescence intensity variations by addition of cationic
polypeptide CD45_mSiglec_(4GS)2_9R_C fand by further addition of
PLE100 to mRNA.
[0050] FIG. 40 provides data from a SYBR Gold exclusion assay
showing fluorescence intensity variations by addition histone H4
and by further addition of CD45-mSiglec-(4GS)2_9R_c and PLE100 to
mRNA.
[0051] FIG. 41 provides data from a SYBR Gold exclusion assay
showing fluorescence intensity variations by addition histone H2A
and by further addition of CD45-mSiglec-(4GS)2_9R_c and PLE100 to
mRNA.
[0052] FIG. 42 provides data from a SYBR Gold exclusion assay from
intercalation with VWF_EGFP pDNA showing fluorescence intensity
variations by addition of cationic polypeptide
CD45_mSiglec_(4GS)2_9R_C followed by PLE100.
[0053] FIG. 43 provides data from a SYBR Gold exclusion assay from
intercalation with VWF_EGFP pDNA showing fluorescence intensity
variations by addition of histone H4, followed by cationic
polypeptide CD45_mSiglec_(4GS)2_9R_C followed by PLE100.
[0054] FIG. 44 provides data from a SYBR Gold exclusion assay from
intercalation with VWF_EGFP pDNA showing fluorescence intensity
variations by addition of histone H4, followed by cationic
polypeptide CD45_mSiglec_(4GS)2_9R_C followed by PLE100.
[0055] FIG. 45 (panels A-C) provide data related to polyplex size
distribution, silica coated size and zeta potential distribution,
and ligand coated/functionalized particle size and zeta potential
distribution.
[0056] FIG. 46 provides data related to branched histone peptide
conjugate pilot particles.
[0057] FIG. 47 provides data related to project HSC.001.001 (see
Table 5).
[0058] FIG. 48 provides data related to project HSC.001.002 (see
Table 5).
[0059] FIG. 49 provides data related to project HSC.002.01
(Targeting Ligand--ESELLg_mESEL_(4GS)2_9R_N) (see Table 5).
[0060] FIG. 50 provides data related to project HSC.002.02
(Targeting Ligand--ESELLg_mESEL_(4GS)2_9R_C) (see Table 5).
[0061] FIG. 51 provides data related to project HSC.002.03
(Targeting Ligand--CD45_mSiglec_(4GS)2_9R_C) (see Table 5).
[0062] FIG. 52 provides data related to project HSC.002.04
(Targeting Ligand--Cy5mRNA-SiO2-PEG) (see Table 5).
[0063] FIG. 53 provides data related to project BLOOD.002.88
(Targeting Ligand--CD45_mSiglec_(4GS)2_9R_C) (see Table 5).
[0064] FIG. 54 provides data related to project BLOOD.002.89
(Targeting Ligand--CD45_mSiglec_(4GS)2_9R_C) (see Table 5).
[0065] FIG. 55 provides data related to project BLOOD.002.90 (see
Table 5).
[0066] FIG. 56 provides data related to project BLOOD.002.91
(PLR50) (see Table 5).
[0067] FIG. 57 provides data related to project BLOOD.002.92
(Targeting Ligand--CD45_mSiglec_(4GS)2_9R_C) (see Table 5).
[0068] FIG. 58 provides data related to project TCELL.001.1 (see
Table 5).
[0069] FIG. 59 provides data related to project TCELL.001.3 (see
Table 5).
[0070] FIG. 60 provides data related to project TCELL.001.13 (see
Table 5).
[0071] FIG. 61 provides data related to project TCELL.001.14 (see
Table 5).
[0072] FIG. 62 provides data related to project TCELL.001.16 (see
Table 5).
[0073] FIG. 63 provides data related to project TCELL.001.18 (see
Table 5).
[0074] FIG. 64 provides data related to project TCELL.001.28 (see
Table 5).
[0075] FIG. 65 provides data related to project TCELL.001.29 (see
Table 5).
[0076] FIG. 66 provides data related to project TCELL.001.31 (see
Table 5).
[0077] FIG. 67 provides data related to project TCELL.001.33 (see
Table 5).
[0078] FIG. 68 provides data related to project TCELL.001.43 (see
Table 5).
[0079] FIG. 69 provides data related to project TCELL.001.44 (see
Table 5).
[0080] FIG. 70 provides data related to project TCELL.001.46 (see
Table 5).
[0081] FIG. 71 provides data related to project TCELL.001.48 (see
Table 5).
[0082] FIG. 72 provides data related to project TCELL.001.58 (see
Table 5).
[0083] FIG. 73 provides data related to project TCELL.001.59 (see
Table 5).
[0084] FIG. 74 provides data related to project CYNOBM.002.82 (see
Table 5).
[0085] FIG. 75 provides data related to project CYNOBM.002.83 (see
Table 5).
[0086] FIG. 76 provides data related to project CYNOBM.002.84 (see
Table 5).
[0087] FIG. 77 provides data related to project CYNOBM.002.85 (see
Table 5).
[0088] FIG. 78 provides data related to project CYNOBM.002.86 (see
Table 5).
[0089] FIG. 79 provides data related to project CYNOBM.002.76 (see
Table 5).
[0090] FIG. 80 provides data related to project CYNOBM.002.77 (see
Table 5).
[0091] FIG. 81 provides data related to project CYNOBM.002.78 (see
Table 5).
[0092] FIG. 82 provides data related to project CYNOBM.002.79 (see
Table 5).
[0093] FIG. 83 provides data related to project CYNOBM.002.80 (see
Table 5).
[0094] FIG. 84 provides data related to untransfected controls for
CynoBM.002 samples.
[0095] FIG. 85 provides data related to lipofectamine CRISPRMAX
delivery of NLS-Cas9-EGFP BCL11a gRNA RNPs.
[0096] FIG. 86 provides data related to project CynoBM.002 RNP-Only
controls (see Table 5).
[0097] FIG. 87 provides data related to project CynoBM.002.82 (see
Table 5).
[0098] FIG. 88 provides data related to project CynoBM.002.83 (see
Table 5).
[0099] FIG. 89 provides data related to project CYNOBM.002.84 (see
Table 5).
[0100] FIG. 90 provides data related to project CynoBM.002.85 (see
Table 5).
[0101] FIG. 91 provides data related to project CynoBM.002.86 (see
Table 5).
[0102] FIG. 92 provides data related to project CynoBM.002.75 (see
Table 5).
[0103] FIG. 93 provides data related to project CynoBM.002.76 (see
Table 5).
[0104] FIG. 94 provides data related to project CynoBM.002.77 (see
Table 5).
[0105] FIG. 95 provides data related to project CynoBM.002.78 (see
Table 5).
[0106] FIG. 96 provides data related to project CynoBM.002.79 (see
Table 5).
[0107] FIG. 97 provides data related to project CynoBM.002.80 (see
Table 5).
[0108] FIG. 98 provides data related to project CynoBM.002.81 (see
Table 5).
[0109] FIG. 99 provides qualitative images of CynoBM.002 RNP-Only
controls.
[0110] FIG. 100 provides data related to project HSC.004 (see Table
5) high-content screening.
[0111] FIG. 101 provides data related to project TCELL.001 (see
Table 5) high-content screening.
[0112] FIG. 102 provides data related to project TCELL.001 (see
Table 5) lipofectamine CRISPRMAX controls.
[0113] FIG. 103 provides data related to project TCell.001.1 (see
Table 5).
[0114] FIG. 104 provides data related to project TCell.001.2 (see
Table 5).
[0115] FIG. 105 provides data related to project TCell.001.3 (see
Table 5).
[0116] FIG. 106 provides data related to project TCell.001.4 (see
Table 5).
[0117] FIG. 107 provides data related to project TCell.001.5 (see
Table 5).
[0118] FIG. 108 provides data related to project TCell.001.6 (see
Table 5).
[0119] FIG. 109 provides data related to project TCell.001.7 (see
Table 5).
[0120] FIG. 110 provides data related to project TCell.001.8 (see
Table 5).
[0121] FIG. 111 provides data related to project TCell.001.9 (see
Table 5).
[0122] FIG. 112 provides data related to project TCell.001.10 (see
Table 5).
[0123] FIG. 113 provides data related to project TCell.001.11 (see
Table 5).
[0124] FIG. 114 provides data related to project TCell.001.12 (see
Table 5).
[0125] FIG. 115 provides data related to project TCell.001.13 (see
Table 5).
[0126] FIG. 116 provides data related to project TCell.001.14 (see
Table 5).
[0127] FIG. 117 provides data related to project TCell.001.15 (see
Table 5).
[0128] FIG. 118 provides data related to negative controls for
project TCell.001 (see Table 5).
[0129] FIG. 119 provides data related to project Blood.002 (see
Table 5).
[0130] FIG. 120 provides data related to project TCell.001.27 (see
Table 5).
[0131] FIG. 121 depicts charge density plots of CRISPR RNP (a
possible payload), which allows for determining whether an anionic
or cationic peptide/material should be added to form a stable
charged layer on the protein surface.
[0132] FIG. 122 depicts charge density plots of Sleeping Beauty
Transposons (a possible payload), which allows for determining
whether an anionic or cationic peptide/material should be added to
form a stable charged layer on the protein surface.
[0133] FIG. 123 depicts (1) Exemplary anionic peptides (9-10 amino
acids long, approximately to scale to 10 nm diameter CRISPR RNP)
anchoring to cationic sites on the CRISPR RNP surface prior to (2)
addition of cationic anchors as (2a) anchor-linker-ligands or
standalone cationic anchors, with or without addition of (2b)
subsequent multilayering chemistries, co-delivery of multiple
nucleic acid or charged therapeutic agents, or layer stabilization
through cross-linking.
[0134] FIG. 124 depicts examples of orders of addition and
electrostatic matrix compositions based on core templates, which
may include Cas9 RNP or any homogenously or zwitterionically
charged surface.
[0135] FIG. 125 provides a modeled structure of IL2 bound to
IL2R.
[0136] FIG. 126 provides a modeled structure of single chain CD3
antibody fragments.
[0137] FIG. 127 provides a modeled structure of sialoadhesin
N-terminal in complex with N-Acetylneuraminic acid (Neu5Ac).
[0138] FIG. 128 provides a modeled structure of Stem Cell Factor
(SCF).
[0139] FIG. 129 provides example images generated during rational
design of a cKit Receptor Fragment.
[0140] FIG. 130 provides example images generated during rational
design of a cKit Receptor Fragment.
[0141] FIG. 131 provides example images generated during rational
design of a cKit Receptor Fragment.
[0142] FIG. 132 provides circular dichroism data from analyzing the
rationally designed cKit Receptor Fragment.
[0143] FIG. 133 depicts modeling of the stabilized conformation of
the rationally designed cKit Receptor Fragment.
[0144] FIG. 134 depicts an example of a branched histone structure
in which HTPs are conjugated to the side chains of a cationic
polymer backbone. The polymer on the right represents the precursor
backbone molecule and the molecule on the left is an example of a
segment of a branched structure.
DETAILED DESCRIPTION
[0145] As summarized above, provided are compositions and methods
for nanoparticle delivery of payloads (e.g., nucleic acid and/or
protein payloads) to cells. In some embodiments, a subject
nanoparticle includes a core and a sheddable layer encapsulating
the core (e.g., providing for temporary stabilization of the core
during cell delivery), where the core includes (i) an anionic
polymer composition; (ii) a cationic polymer composition; (iii) a
cationic polypeptide composition; and (iv) a nucleic acid and/or
protein payload; and where: (a) the anionic polymer composition
includes polymers of D-isomers of an anionic amino acid and
polymers of L-isomers of an anionic amino acid, and/or (b) the
cationic polymer composition comprises polymers of D-isomers of a
cationic amino acid and polymers of L-isomers of a cationic amino
acid. In some cases, the polymers of D-isomers of an anionic amino
acid are present at a ratio, relative to the polymers of L-isomers
of an anionic amino acid, in a range of from 10:1 to 1:10. In some
cases, the polymers of D-isomers of a cationic amino acid are
present at a ratio, relative to said polymers of L-isomers of a
cationic amino acid, in a range of from 10:1 to 1:10.
[0146] In some cases, a nanoparticle of the disclosure includes a
surface coat, which surrounds the sheddable layer. The surface coat
can include a targeting ligand that provides for targeted binding
to a surface molecule of a target cell. In some cases, the
targeting ligand is conjugated (with or without a linker) to an
anchoring domain, e.g., for anchoring the targeting ligand to the
sheddable layer of the nanoparticle.
[0147] Also provided are multi-layered nanoparticles the include a
first payload (e.g., a DNA donor template) as part of the core,
where the core is surrounded by a first sheddable layer, the first
sheddable layer is surrounded by an intermediate layer that
includes a second payload (e.g., a gene editing tool), and the
intermediate layer is surround by a second sheddable layer. In some
cases the second sheddable layer is coated with a surface coat
(e.g., a surface coat that includes a targeting ligand.
[0148] Also provided are nanoparticle formulations including two or
more nanoparticles in which the payload of a first nanoparticle
includes a donor DNA template and the payload of a second
nanoparticle includes a gene editing tool (e.g., (i) a CRISPR/Cas
guide RNA; (ii) a DNA encoding a CRISPR/Cas guide RNA; (iii) a DNA
and/or RNA encoding a programmable gene editing protein; and/or
(iv) a programmable gene editing protein).
[0149] Before the present methods and compositions are described,
it is to be understood that this invention is not limited to the
particular methods or compositions described, as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting, since the scope of the
present invention will be limited only by the appended claims.
[0150] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0151] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, some potential and preferred methods and materials are
now described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. It
is understood that the present disclosure supersedes any disclosure
of an incorporated publication to the extent there is a
contradiction.
[0152] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order that is logically possible.
[0153] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and reference to "the endonuclease" includes reference to one or
more endonucleases and equivalents thereof, known to those skilled
in the art, and so forth. It is further noted that the claims may
be drafted to exclude any element, e.g., any optional element. As
such, this statement is intended to serve as antecedent basis for
use of such exclusive terminology as "solely," "only" and the like
in connection with the recitation of claim elements, or use of a
"negative" limitation.
[0154] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication.
Further, the dates of publication provided may be different from
the actual publication dates which may need to be independently
confirmed.
Methods and Compositions
[0155] Provided are methods and compositions for delivering a
nucleic acid, protein, and/or ribonucleoprotein payload to a cell.
In some embodiments a subject nanoparticle includes (i) a core that
is encapsulated by (ii) a sheddable layer, and the sheddable layer
is in some cases surrounded by (iii) a surface coat, which can
include a targeting ligand. In addition to the description below,
international patent application publication number WO2015042585 is
hereby incorporated by reference in its entirety.
i. Nanoparticle Core
[0156] The core of a subject nanoparticle includes an anionic
polymer composition (e.g., poly(glutamic acid)), a cationic polymer
composition (e.g., poly(arginine), a cationic polypeptide
composition (e.g., a histone tail peptide), and a payload (e.g.,
nucleic acid and/or protein payload). In some cases the core is
generated by condensation of a cationic amino acid polymer and
payload in the presence of an anionic amino acid polymer (and in
some cases in the presence of a cationic polypeptide of a cationic
polypeptide composition). In some embodiments, condensation of the
components that make up the core can mediate increased transfection
efficiency compared to conjugates of cationic polymers with a
payload. Inclusion of an anionic polymer in a nanoparticle core may
prolong the duration of intracellular residence of the nanoparticle
and release of payload.
[0157] For the cationic and anionic polymer compositions of the
core, ratios of D-isomer polymers to L-isomer polymers can be
controlled in order to control the timed release of payload, where
increased ration of D-isomer polymers to L-isomer polymers leads to
increased stability (reduced payload release rate), which for
example can enable longer lasting gene expression from a payload
delivered by a subject nanoparticle. In some cases modifying the
ratio of D-to-L isomer polypeptides within the nanoparticle core
can cause gene expression profiles (e.g., expression of a protein
encoded by a payload molecule) to be on the order of from 1-90 days
(e.g. from 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-25, 1-20, 1-15,
1-10, 3-90, 3-80, 3-70, 3-60, 3-50, 3-40, 3-30, 3-25, 3-20, 3-15,
3-10, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15,
or 5-10 days). The control of payload release (e.g., when
delivering a gene editing tool), can be particularly effective for
performing genomic edits e.g., in some cases where
homology-directed repair is desired.
[0158] In some embodiments, a nanoparticle includes a core and a
sheddable layer encapsulating the core, where the core includes:
(a) an anionic polymer composition; (b) a cationic polymer
composition; (c) a cationic polypeptide composition; and (d) a
nucleic acid and/or protein payload, where one of (a) and (b)
includes a D-isomer polymer of an amino acid, and the other of (a)
and (b) includes an L-isomer polymer of an amino acid, and where
the ratio of the D-isomer polymer to the L-isomer polymer is in a
range of from 10:1 to 1.5:1 (e.g., from 8:1 to 1.5:1, 6:1 to 1.5:1,
5:1 to 1.5:1, 4:1 to 1.5:1, 3:1 to 1.5:1, 2:1 to 1.5:1, 10:1 to
2:1; 8:1 to 2:1, 6:1 to 2:1, 5:1 to 2:1, 10:1 to 3:1; 8:1 to 3:1,
6:1 to 3:1, 5:1 to 3:1, 10:1 to 4:1; 4:1 to 2:1, 6:1 to 4:1, or
10:1 to 5:1), or from 1:1.5 to 1:10 (e.g., from 1:1.5 to 1:8, 1:1.5
to 1:6, 1:1.5 to 1:5, 1:1.5 to 1:4, 1:1.5 to 1:3, 1:1.5 to 1:2, 1:2
to 1:10, 1:2 to 1:8, 1:2 to 1:6, 1:2 to 1:5, 1:2 to 1:4, 1:2 to
1:3, 1:3 to 1:10, 1:3 to 1:8, 1:3 to 1:6, 1:3 to 1:5, 1:4 to 1:10,
1:4 to 1:8, 1:4 to 1:6, or 1:5 to 1:10). In some such cases, the
ratio of the D-isomer polymer to the L-isomer polymer not 1:1. In
some such cases, the anionic polymer composition includes an
anionic polymer selected from poly(D-glutamic acid) (PDEA) and
poly(D-aspartic acid) (PDDA), where (optionally) the cationic
polymer composition can include a cationic polymer selected from
poly(L-arginine), poly(L-lysine), poly(L-histidine),
poly(L-ornithine), and poly(L-citrulline). In some cases the
cationic polymer composition comprises a cationic polymer selected
from poly(D-arginine), poly(D-lysine), poly(D-histidine),
poly(D-ornithine), and poly(D-citrulline), where (optionally) the
anionic polymer composition can include an anionic polymer selected
from poly(L-glutamic acid) (PLEA) and poly(L-aspartic acid)
(PLDA).
[0159] In some embodiments, a nanoparticle includes a core and a
sheddable layer encapsulating the core, where the core includes:
(i) an anionic polymer composition; (ii) a cationic polymer
composition; (iii) a cationic polypeptide composition; and (iv) a
nucleic acid and/or protein payload, wherein (a) said anionic
polymer composition includes polymers of D-isomers of an anionic
amino acid and polymers of L-isomers of an anionic amino acid;
and/or (b) said cationic polymer composition includes polymers of
D-isomers of a cationic amino acid and polymers of L-isomers of a
cationic amino acid. In some such cases, the anionic polymer
composition comprises a first anionic polymer selected from
poly(D-glutamic acid) (PDEA) and poly(D-aspartic acid) (PDDA); and
comprises a second anionic polymer selected from poly(L-glutamic
acid) (PLEA) and poly(L-aspartic acid) (PLDA). In some cases, the
cationic polymer composition comprises a first cationic polymer
selected from poly(D-arginine), poly(D-lysine), poly(D-histidine),
poly(D-ornithine), and poly(D-citrulline); and comprises a second
cationic polymer selected from poly(L-arginine), poly(L-lysine),
poly(L-histidine), poly(L-ornithine), and poly(L-citrulline). In
some cases, the polymers of D-isomers of an anionic amino acid are
present at a ratio, relative to said polymers of L-isomers of an
anionic amino acid, in a range of from 10:1 to 1:10. In some cases,
the polymers of D-isomers of a cationic amino acid are present at a
ratio, relative to said polymers of L-isomers of a cationic amino
acid, in a range of from 10:1 to 1:10.
Susceptibility of Nanoparticle Components
[0160] In some embodiments, timing of payload release can be
controlled by selecting particular types of proteins, e.g., as part
of the core (e.g., part of a cationic polypeptide composition, part
of a cationic polymer composition, and/or part of an anionic
polymer composition). For example, it may be desirable to delay
payload release for a particular range of time, or until the
payload is present at a particular cellular location (e.g.,
cytosol, nucleus, lysosome, endosome) or under a particular
condition (e.g., low pH, high pH, etc.). As such, in some cases a
protein is used (e.g., as part of the core) that is susceptible to
a specific protein activity (e.g., enzymatic activity), e.g., is a
substrate for a specific protein activity (e.g., enzymatic
activity), and this is in contrast to being susceptible to general
ubiquitous cellular machinery, e.g., general degradation machinery.
A protein that is susceptible to a specific protein activity is
referred to herein as an `enzymatically susceptible protein` (ESP).
Illustrative examples of ESPs include but are not limited to: (i)
proteins that are substrates for matrix metalloproteinase (MMP)
activity (an example of an extracellular activity), e.g., a protein
that includes a motif recognized by an MMP; (ii) proteins that are
substrates for cathepsin activity (an example of an intracellular
endosomal activity), e.g., a protein that includes a motif
recognized by a cathepsin; and (iii) proteins such as histone tails
peptides (HTPs) that are substrates for methyltransferase and/or
acetyltransferase activity (an example of an intracellular nuclear
activity), e.g., a protein that includes a motif that can be
enzymatically methylated/de-methylated and/or a motif that can be
enzymatically acetylated/de-acetylated. For example, in some cases
a nucleic acid payload is condensed with a protein (such as a
histone tails peptide) that is a substrate for acetyltransferase
activity, and acetylation of the protein causes the protein to
release the payload--as such, one can exercise control over payload
release by choosing to use a protein that is more or less
susceptible to acetylation.
[0161] In some cases, a core of a subject nanoparticle includes an
enzymatically neutral polypeptide (ENP), which is a polypeptide
homopolymer (i.e., a protein having a repeat sequence) where the
polypeptide does not have a particular activity and is neutral. For
example, unlike NLS sequences and HTPs, both of which have a
particular activity, ENPs do not.
[0162] In some cases, a core of a subject nanoparticle includes a
enzymatically protected polypeptide (EPP), which is a protein that
is resistant to enzymatic activity. Examples of PPs include but are
not limited to: (i) polypeptides that include D-isomer amino acids
(e.g., D-isomer polymers), which can resist proteolytic
degradation; and (ii) self-sheltering domains such as a
polyglutamine repeat domains (e.g., QQQQQQQQQQ) (SEQ ID NO:
170).
[0163] By controlling the relative amounts of susceptible proteins
(ESPs), neutral proteins (ENPs), and protected proteins (EPPs),
that are part of a subject nanoparticle (e.g., part of the
nanoparticle core), one can control the release of payload. For
example, use of more ESPs can in general lead to quicker release of
payload than use of more EPPs. In addition, use of more ESPs can in
general lead to release of payload that depends upon a particular
set of conditions/circumstances, e.g., conditions/circumstances
that lead to activity of proteins (e.g., enzymes) to which the ESP
is susceptible.
Anionic Polymer Composition
[0164] An anionic polymer composition can include one or more
anionic amino acid polymers. For example, in some cases a subject
anionic polymer composition includes a polymer selected from:
poly(glutamic acid)(PEA), poly(aspartic acid)(PDA), and a
combination thereof. In some cases a given anionic amino acid
polymer can include a mix of aspartic and glutamic acid residues.
Each polymer can be present in the composition as a polymer of
L-isomers or D-isomers, where D-isomers are more stable in a target
cell because they take longer to degrade. Thus, inclusion of
D-isomer poly(amino acids) in the nanoparticle core delays
degradation of the core and subsequent payload release. The payload
release rate can therefore be controlled and is proportional to the
ratio of polymers of D-isomers to polymers of L-isomers, where a
higher ratio of D-isomer to L-isomer increases duration of payload
release (i.e., decreases release rate). In other words, the
relative amounts of D- and L- isomers can modulate the nanoparticle
core's timed release kinetics and enzymatic susceptibility to
degradation and payload release.
[0165] In some cases an anionic polymer composition of a subject
nanoparticle includes polymers of D-isomers and polymers of
L-isomers of an anionic amino acid polymer (e.g., poly(glutamic
acid)(PEA) and poly(aspartic acid)(PDA)). In some cases the D- to
L- isomer ratio is in a range of from 10:1-1:10 (e.g., from
8:1-1:10, 6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10,
10:1-1:8, 8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8,
10:1-1:6, 8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6,
10:1-1:4, 8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4,
10:1-1:3, 8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3,
10:1-1:2, 8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2,
10:1-1:1, 8:1-1:1, 6:1-1:1, 4:1-1:1, 3:1-1:1, or 2:1-1:1).
[0166] Thus, in some cases an anionic polymer composition includes
a first anionic polymer (e.g., amino acid polymer) that is a
polymer of D-isomers (e.g., selected from poly(D-glutamic acid)
(PDEA) and poly(D-aspartic acid) (PDDA)); and includes a second
anionic polymer (e.g., amino acid polymer) that is a polymer of
L-isomers (e.g., selected from poly(L-glutamic acid) (PLEA) and
poly(L-aspartic acid) (PLDA)). In some cases the ratio of the first
anionic polymer (D-isomers) to the second anionic polymer
(L-isomers) is in a range of from 10:1-1:10 (e.g., from 8:1-1:10,
6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10, 10:1-1:8,
8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8, 10:1-1:6,
8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6, 10:1-1:4,
8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4, 10:1-1:3,
8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3, 10:1-1:2,
8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2, 10:1-1:1,
8:1-1:1, 6:1-1:1, 4:1-1:1, 3:1-1:1, or 2:1-1:1)
[0167] In some embodiments, an anionic polymer composition of a
core of a subject nanoparticle includes (e.g., in addition to or in
place of any of the foregoing examples of anionic polymers) a
glycosaminoglycan, a glycoprotein, a polysaccharide,
poly(mannuronic acid), poly(guluronic acid), heparin, heparin
sulfate, chondroitin, chondroitin sulfate, keratan, keratan
sulfate, aggrecan, poly(glucosamine), or an anionic polymer that
comprises any combination thereof.
[0168] In some embodiments, an anionic polymer within the core can
have a molecular weight in a range of from 1-200 kDa (e.g., from
1-150, 1-100, 1-50, 5-200, 5-150, 5-100, 5-50, 10-200, 10-150,
10-100, 10-50, 15-200, 15-150, 15-100, or 15-50 kDa). As an
example, in some cases an anionic polymer includes poly(glutamic
acid) with a molecular weight of approximately 15 kDa.
[0169] In some cases, an anionic amino acid polymer includes a
cysteine residue, which can facilitate conjugation, e.g., to a
linker, an NLS, and/or a cationic polypeptide (e.g., a histone or
HTP). For example, a cysteine residue can be used for crosslinking
(conjugation) via sulfhydryl chemistry (e.g., a disulfide bond)
and/or amine-reactive chemistry. Thus, in some embodiments an
anionic amino acid polymer (e.g., poly(glutamic acid) (PEA),
poly(aspartic acid) (PDA), poly(D-glutamic acid) (PDEA),
poly(D-aspartic acid) (PDDA), poly(L-glutamic acid) (PLEA),
poly(L-aspartic acid) (PLDA)) of an anionic polymer composition
includes a cysteine residue. In some cases the anionic amino acid
polymer includes cysteine residue on the N- and/or C-terminus. In
some cases the anionic amino acid polymer includes an internal
cysteine residue.
[0170] In some cases, an anionic amino acid polymer includes
(and/or is conjugated to) a nuclear localization signal (NLS)
(described in more detail below). Thus, in some embodiments an
anionic amino acid polymer (e.g., poly(glutamic acid) (PEA),
poly(aspartic acid) (PDA), poly(D-glutamic acid) (PDEA),
poly(D-aspartic acid) (PDDA), poly(L-glutamic acid) (PLEA),
poly(L-aspartic acid) (PLDA)) of an anionic polymer composition
includes (and/or is conjugated to) one or more (e.g., two or more,
three or more, or four or more) NLSs. In some cases the anionic
amino acid polymer includes an NLS on the N- and/or C-terminus. In
some cases the anionic amino acid polymer includes an internal
NLS.
[0171] In some cases, an anionic polymer is added prior to a
cationic polymer when generating a subject nanoparticle core.
Cationic Polymer Composition
[0172] A cationic polymer composition can include one or more
cationic amino acid polymers. For example, in some cases a subject
cationic polymer composition includes a polymer selected from:
poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH),
poly(ornithine), poly(citrulline), and a combination thereof. In
some cases a given cationic amino acid polymer can include a mix of
arginine, lysine, histidine, ornithine, and citrulline residues (in
any convenient combination). Each polymer can be present in the
composition as a polymer of L-isomers or D-isomers, where D-isomers
are more stable in a target cell because they take longer to
degrade. Thus, inclusion of D-isomer poly(amino acids) in the
nanoparticle core delays degradation of the core and subsequent
payload release. The payload release rate can therefore be
controlled and is proportional to the ratio of polymers of
D-isomers to polymers of L-isomers, where a higher ratio of
D-isomer to L-isomer increases duration of payload release (i.e.,
decreases release rate). In other words, the relative amounts of D-
and L- isomers can modulate the nanoparticle core's timed release
kinetics and enzymatic susceptibility to degradation and payload
release.
[0173] In some cases a cationic polymer composition of a subject
nanoparticle includes polymers of D-isomers and polymers of
L-isomers of an cationic amino acid polymer (e.g.,
poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH),
poly(ornithine), poly(citrulline)). In some cases the D- to L-
isomer ratio is in a range of from 10:1-1:10 (e.g., from 8:1-1:10,
6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10, 10:1-1:8,
8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8, 10:1-1:6,
8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6, 10:1-1:4,
8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4, 10:1-1:3,
8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3, 10:1-1:2,
8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2, 10:1-1:1,
8:1-1:1, 6:1-1:1, 4:1-1:1, 3:1-1:1, or 2:1-1:1).
[0174] Thus, in some cases a cationic polymer composition includes
a first cationic polymer (e.g., amino acid polymer) that is a
polymer of D-isomers (e.g., selected from poly(D-arginine),
poly(D-lysine), poly(D-histidine), poly(D-ornithine), and
poly(D-citrulline)); and includes a second cationic polymer (e.g.,
amino acid polymer) that is a polymer of L-isomers (e.g., selected
from poly(L-arginine), poly(L-lysine), poly(L-histidine),
poly(L-ornithine), and poly(L-citrulline)). In some cases the ratio
of the first cationic polymer (D-isomers) to the second cationic
polymer (L-isomers) is in a range of from 10:1-1:10 (e.g., from
8:1-1:10, 6:1-1:10, 4:1-1:10, 3:1-1:10, 2:1-1:10, 1:1-1:10,
10:1-1:8, 8:1-1:8, 6:1-1:8, 4:1-1:8, 3:1-1:8, 2:1-1:8, 1:1-1:8,
10:1-1:6, 8:1-1:6, 6:1-1:6, 4:1-1:6, 3:1-1:6, 2:1-1:6, 1:1-1:6,
10:1-1:4, 8:1-1:4, 6:1-1:4, 4:1-1:4, 3:1-1:4, 2:1-1:4, 1:1-1:4,
10:1-1:3, 8:1-1:3, 6:1-1:3, 4:1-1:3, 3:1-1:3, 2:1-1:3, 1:1-1:3,
10:1-1:2, 8:1-1:2, 6:1-1:2, 4:1-1:2, 3:1-1:2, 2:1-1:2, 1:1-1:2,
10:1-1:1, 8:1-1:1, 6:1-1:1, 4:1-1:1, 3:1-1:1, or 2:1-1:1)
[0175] In some embodiments, an cationic polymer composition of a
core of a subject nanoparticle includes (e.g., in addition to or in
place of any of the foregoing examples of cationic polymers)
poly(ethylenimine), poly(amidoamine) (PAMAM), poly(aspartamide),
polypeptoids (e.g., for forming "spiderweb"-like branches for core
condensation), a charge-functionalized polyester, a cationic
polysaccharide, an acetylated amino sugar, chitosan, or a cationic
polymer that comprises any combination thereof (e.g., in linear or
branched forms).
[0176] In some embodiments, an cationic polymer within the core can
have a molecular weight in a range of from 1-200 kDa (e.g., from
1-150, 1-100, 1-50, 5-200, 5-150, 5-100, 5-50, 10-200, 10-150,
10-100, 10-50, 15-200, 15-150, 15-100, or 15-50 kDa). As an
example, in some cases an cationic polymer includes
poly(L-arginine), e.g., with a molecular weight of approximately 29
kDa. As another example, in some cases a cationic polymer includes
linear poly(ethylenimine) with a molecular weight of approximately
25 kDa (PEI). As another example, in some cases a cationic polymer
includes branched poly(ethylenimine) with a molecular weight of
approximately 10 kDa. As another example, in some cases a cationic
polymer includes branched poly(ethylenimine) with a molecular
weight of approximately 70 kDa. In some cases a cationic polymer
includes PAMAM.
[0177] In some cases, a cationic amino acid polymer includes a
cysteine residue, which can facilitate conjugation, e.g., to a
linker, an NLS, and/or a cationic polypeptide (e.g., a histone or
HTP). For example, a cysteine residue can be used for crosslinking
(conjugation) via sulfhydryl chemistry (e.g., a disulfide bond)
and/or amine-reactive chemistry. Thus, in some embodiments a
cationic amino acid polymer (e.g., poly(arginine)(PR),
poly(lysine)(PK), poly(histidine)(PH), poly(ornithine), and
poly(citrulline), poly(D-arginine)(PDR), poly(D-lysine)(PDK),
poly(D-histidine)(PDH), poly(D-ornithine), and poly(D-citrulline),
poly(L-arginine)(PLR), poly(L-lysine)(PLK), poly(L-histidine)(PLH),
poly(L-ornithine), and poly(L-citrulline)) of a cationic polymer
composition includes a cysteine residue. In some cases the cationic
amino acid polymer includes cysteine residue on the N- and/or
C-terminus. In some cases the cationic amino acid polymer includes
an internal cysteine residue.
[0178] In some cases, a cationic amino acid polymer includes
(and/or is conjugated to) a nuclear localization signal (NLS)
(described in more detail below). Thus, in some embodiments a
cationic amino acid polymer (e.g., poly(arginine)(PR),
poly(lysine)(PK), poly(histidine)(PH), poly(ornithine), and
poly(citrulline), poly(D-arginine)(PDR), poly(D-lysine)(PDK),
poly(D-histidine)(PDH), poly(D-ornithine), and poly(D-citrulline),
poly(L-arginine)(PLR), poly(L-lysine)(PLK), poly(L-histidine)(PLH),
poly(L-ornithine), and poly(L-citrulline)) of a cationic polymer
composition includes (and/or is conjugated to) one or more (e.g.,
two or more, three or more, or four or more) NLSs. In some cases
the cationic amino acid polymer includes an NLS on the N- and/or
C-terminus. In some cases the cationic amino acid polymer includes
an internal NLS.
Cationic Polypeptide Composition
[0179] In some embodiments the cationic polypeptide composition of
a nanoparticle can mediate stability, subcellular
compartmentalization, and/or payload release. As one example,
fragments of the N-terminus of histone proteins, referred to
generally as histone tail peptides, within a subject nanoparticle
core are in some case not only capable of being deprotonated by
various histone modifications, such as in the case of histone
acetyltransferase-mediated acetylation, but may also mediate
effective nuclear-specific unpackaging of components (e.g., a
payload) of a nanoparticle core. In some cases a cationic
polypeptide composition includes a histone and/or histone tail
peptide (e.g., a cationic polypeptide can be a histone and/or
histone tail peptide). In some cases a cationic polypeptide
composition includes an NLS-containing peptide (e.g., a cationic
polypeptide can be an NLS-containing peptide). In some cases a
cationic polypeptide composition includes a peptide that includes a
mitochondrial localization signal (e.g., a cationic polypeptide can
be a peptide that includes a mitochondrial localization
signal).
[0180] Histone Tail Peptide (HTPs)
[0181] In some embodiments a cationic polypeptide composition of a
subject nanoparticle includes a histone peptide or a fragment of a
histone peptide, such as an N-terminal histone tail (e.g., a
histone tail of an H1, H2 (e.g., H2A, H2AX, H2B), H3, or H4 histone
protein). A tail fragment of a histone protein is referred to
herein as a histone tail peptide (HTP). Because such a protein (a
histone and/or HTP) can condense with a nucleic acid payload as
part of the core of a subject nanoparticle, a core that includes
one or more histones or HTPs (e.g., as part of the cationic
polypeptide composition) is sometimes referred to herein as a
nucleosome-mimetic core. Histones and/or HTPs can be included as
monomers, and in some cases form dimers, trimers, tetramers and/or
octamers when condensing a nucleic acid payload into a nanoparticle
core. In some cases HTPs are not only capable of being deprotonated
by various histone modifications, such as in the case of histone
acetyltransferase-mediated acetylation, but may also mediate
effective nuclear-specific unpackaging of components of the core
(e.g., release of a payload). Trafficking of a core that includes a
histone and/or HTP may be reliant on alternative endocytotic
pathways utilizing retrograde transport through the Golgi and
endoplasmic reticulum. Furthermore, some histones include an innate
nuclear localization sequence and inclusion of an NLS in the core
can direct the core (including the payload) to the nucleus of a
target cell.
[0182] In some embodiments a subject cationic polypeptide
composition includes a protein having an amino acid sequence of an
H2A, H2AX, H2B, H3, or H4 protein. In some cases a subject cationic
polypeptide composition includes a protein having an amino acid
sequence that corresponds to the N-terminal region of a histone
protein. For example, the fragment (an HTP) can include the first
5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 N-terminal amino acids of
a histone protein. In some cases, a subject HTP includes from 5-50
amino acids (e.g., from 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 8-50,
8-45, 8-40, 8-35, 8-30, 10-50, 10-45, 10-40, 10-35, or 10-30 amino
acids) from the N-terminal region of a histone protein. In some
cases a subject a cationic polypeptide includes from 5-150 amino
acids (e.g., from 5-100, 5-50, 5-35, 5-30, 5-25, 5-20, 8-150,
8-100, 8-50, 8-40, 8-35, 8-30, 10-150, 10-100, 10-50, 10-40, 10-35,
or 10-30 amino acids).
[0183] In some cases a cationic polypeptide (e.g., a histone or
HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4) of a cationic
polypeptide composition includes a post-translational modification
(e.g., in some cases on one or more histidine, lysine, arginine, or
other complementary residues). For example, in some cases the
cationic polypeptide is methylated (and/or susceptible to
methylation/demethylation), acetylated (and/or susceptible to
acetylation/deacetylation), crotonylated (and/or susceptible to
crotonylation/decrotonylation), ubiquitinylated (and/or susceptible
to ubiquitinylation/deubiquitinylation), phosphorylated (and/or
susceptible to phosphorylation/dephosphorylation), SUMOylated
(and/or susceptible to SUMOylation/deSUMOylation), farnesylated
(and/or susceptible to farnesylation/defarnesylation), sulfated
(and/or susceptible to sulfation/desulfation) or otherwise
post-translationally modified. In some cases a cationic polypeptide
(e.g., a histone or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4)
of a cationic polypeptide composition is p300/CBP substrate (e.g.,
see example HTPs below, e.g., SEQ ID NOs: 129-130). In some cases a
cationic polypeptide (e.g., a histone or HTP, e.g., H1, H2, H2A,
H2AX, H2B, H3, or H4) of a cationic polypeptide composition
includes one or more thiol residues (e.g., can include a cysteine
and/or methionine residue) that is sulfated or susceptible to
sulfation (e.g., as a thiosulfate sulfurtransferase substrate). In
some cases a cationic polypeptide (e.g., a histone or HTP, e.g.,
H1, H2, H2A, H2AX, H2B, H3, or H4) of a cationic polypeptide is
amidated on the C-terminus. Histones H2A, H2B, H3, and H4 (and/or
HTPs) may be monomethylated, dimethylated, or trimethylated at any
of their lysines to promote or suppress transcriptional activity
and alter nuclear-specific release kinetics.
[0184] A cationic polypeptide can be synthesized with a desired
modification or can be modified in an in vitro reaction.
Alternatively, a cationic polypeptide (e.g., a histone or HTP) can
be expressed in a cell population and the desired modified protein
can be isolated/purified. In some cases the cationic polypeptide
composition of a subject nanoparticle includes a methylated HTP,
e.g., includes the HTP sequence of H3K4(Me3)--includes the amino
acid sequence set forth as SEQ ID NO: 75 or 88). In some cases a
cationic polypeptide (e.g., a histone or HTP, e.g., H1, H2, H2A,
H2AX, H2B, H3, or H4) of a cationic polypeptide composition
includes a C-terminal amide.
[0185] Examples of Histones and HTPs
[0186] Examples include but are not limited to the following
sequences:
TABLE-US-00001 H2A (SEQ ID NO: 62) SGRGKQGGKARAKAKTRSSR [1-20] (SEQ
ID NO: 63) SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGGG [1-39] (SEQ ID
NO: 64) MSGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAERVGAGAP
VYLAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLL
GKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK [1-130] H2AX (SEQ ID NO: 65)
CKATQASQEY [134-143] (SEQ ID NO: 66) KKTSATVGPKAPSGGKKATQASQEY [KK
120-129] (SEQ ID NO: 67)
MSGRGKTGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGHYAERVGAGAP
VYLAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLL
GGVTIAQGGVLPNIQAVLLPKKTSATVGPKAPSGGKKATQASQEY [1-143] H2B (SEQ ID
NO: 68) PEPA-K(cr)-SAPAPK [1-11 H2BK5(cr)] [cr: crotonylated
(crotonylation)] (SEQ ID NO: 69) PEPAKSAPAPK [1-11] (SEQ ID NO: 70)
AQKKDGKKRKRSRKE [21-35] (SEQ ID NO: 71)
MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKVLKQV
HPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTA
VRLLLPGELAKHAVSEGTKAVTKYTSSK [1-126] H3 (SEQ ID NO: 72) ARTKQTAR
[1-8] (SEQ ID NO: 73) ART-K(Me1)-QTARKS [1-8 H3K4(Me1)] (SEQ ID NO:
74) ART-K(Me2)-QTARKS [1-8 H3K4(Me2)] (SEQ ID NO: 75)
ART-K(Me3)-QTARKS [1-8 H3K4(Me3)] (SEQ ID NO: 76)
ARTKQTARK-pS-TGGKA [1-15 H3pS10] (SEQ ID NO: 77)
ARTKQTARKSTGGKAPRKWC-NH2 [1-18 WC, amide] (SEQ ID NO: 78)
ARTKQTARKSTGG-K(Ac)-APRKQ [1-19 H3K14(Ac)] (SEQ ID NO: 79)
ARTKQTARKSTGGKAPRKQL [1-20] (SEQ ID NO: 80)
ARTKQTAR-K(Ac)-STGGKAPRKQL [1-20 H3K9(Ac)] (SEQ ID NO: 81)
ARTKQTARKSTGGKAPRKQLA [1-21] (SEQ ID NO: 82)
ARTKQTAR-K(Ac)-STGGKAPRKQLA [1-21 H3K9(Ac)] (SEQ ID NO: 83)
ARTKQTAR-K(Me2)-STGGKAPRKQLA [1-21 H3K9(Me1)] (SEQ ID NO: 84)
ARTKQTAR-K(Me2)-STGGKAPRKQLA [1-21 H3K9(Me2)] (SEQ ID NO: 85)
ARTKQTAR-K(Me2)-STGGKAPRKQLA [1-21 H3K9(Me3)] (SEQ ID NO: 86)
ART-K(Me1)-QTARKSTGGKAPRKQLA [1-21 H3K4(Me1)] (SEQ ID NO: 87)
ART-K(Me2)-QTARKSTGGKAPRKQLA [1-21 H3K4(Me2)] (SEQ ID NO: 88)
ART-K(Me3)-QTARKSTGGKAPRKQLA [1-21 H3K4(Me3)] (SEQ ID NO: 89)
ARTKQTAR-K(Ac)-pS-TGGKAPRKQLA [1-21 H3K9(Ac), pS10] (SEQ ID NO: 90)
ART-K(Me3)-QTAR-K(Ac)-pS-TGGKAPRKQLA [1-21 H3K4(Me3), K9(Ac), pS10]
(SEQ ID NO: 91) ARTKQTARKSTGGKAPRKQLAC [1-21 Cys] (SEQ ID NO: 92)
ARTKQTAR-K(Ac)-STGGKAPRKQLATKA [1-24 H3K9(Ac)] (SEQ ID NO: 93)
ARTKQTAR-K(Me3)-STGGKAPRKQLATKA [1-24 H3K9(Me3)] (SEQ ID NO: 94)
ARTKQTARKSTGGKAPRKQLATKAA [1-25] (SEQ ID NO: 95)
ART-K(Me3)-QTARKSTGGKAPRKQLATKAA [1-25 H3K4(Me3)] (SEQ ID NO: 96)
TKQTAR-K(Me1)-STGGKAPR [3-17 H3K9(Me1)] (SEQ ID NO: 97)
TKQTAR-K(Me2)-STGGKAPR [3-17 H3K9(Me2)] (SEQ ID NO: 98)
TKQTAR-K(Me3)-STGGKAPR [3-17 H3K9(Me3)] (SEQ ID NO: 99)
KSTGG-K(Ac)-APRKQ [9-19 H3K14(Ac)] (SEQ ID NO: 100)
QTARKSTGGKAPRKQLASK [5-23] (SEQ ID NO: 101)
APRKQLATKAARKSAPATGGVKKPH [15-39] (SEQ ID NO: 102)
ATKAARKSAPATGGVKKPHRYRPG [21-44] (SEQ ID NO: 103) KAARKSAPA [23-31]
(SEQ ID NO: 104) KAARKSAPATGG [23-34 (SEQ ID NO: 105) KAARKSAPATGGC
[23-34 Cys] (SEQ ID NO: 106) KAAR-K(Ac)-SAPATGG [H3K27(Ac)] (SEQ ID
NO: 107) KAAR-K(Me1)-SAPATGG [H3K27(Me1)] (SEQ ID NO: 108)
KAAR-K(Me2)-SAPATGG [H3K27(Me2)] (SEQ ID NO: 109)
KAAR-K(Me3)-SAPATGG [H3K27(Me3)] (SEQ ID NO: 110)
AT-K(Ac)-AARKSAPSTGGVKKPHRYRPG [21-44 H3K23(Ac)] (SEQ ID NO: 111)
ATKAARK-pS-APATGGVKKPHRYRPG [21-44 pS28] (SEQ ID NO: 112)
ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGV [1-35] (SEQ ID NO: 113)
STGGV-K(Me1)-KPHRY [31-41 H3K36(Me1)] (SEQ ID NO: 114)
STGGV-K(Me2)-KPHRY [31-41 H3K36(Me2)] (SEQ ID NO: 115)
STGGV-K(Me3)-KPHRY [31-41 H3K36(Me3)] (SEQ ID NO: 116)
GTVALREIRRYQ-K(Ac)-STELLIR [44-63 H3K56(Ac)] (SEQ ID NO: 117)
ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGT VALRE [1-50] (SEQ ID
NO: 118) TELLIRKLPFQRLVREIAQDF-K(Me1)-TDLRFQSAAI [H3K79(Me1)] (SEQ
ID NO: 119) EIAQDFKTDLR [73-83] (SEQ ID NO: 120) EIAQDF-K(Ac)-TDLR
[73-83 H3K79(Ac)] (SEQ ID NO: 121) EIAQDF-K(Me3)-TDLR [73-83
H3K79(Me3)] (SEQ ID NO: 122) RLVREIAQDFKTDLRFQSSAV [69-89] (SEQ ID
NO: 123) RLVREIAQDFK-(Me1)-TDLRFQSSAV [69-89 H3K79 (Me1), amide]
(SEQ ID NO: 124) RLVREIAQDFK-(Me2)-TDLRFQSSAV [69-89 H3K79 (Me2),
amide] (SEQ ID NO: 125) RLVREIAQDFK-(Me3)-TDLRFQSSAV [69-89 H3K79
(Me3), amide] (SEQ ID NO: 126) KRVTIMPKDIQLARRIRGERA [116-136] (SEQ
ID NO: 127) MARTKQTARKSTGGKAPRKQLATKVARKSAPATGGVKKPHRYRP
GTVALREIRRYQKSTELLIRKLPFQRLMREIAQDFKTDLRFQSS
AVMALQEACESYLVGLFEDTNLCVIHAKRVTIMPKDIQLARRIR GERA[1-136] H4 (SEQ ID
NO: 128) SGRGKGG [1-7] (SEQ ID NO: 129) RGKGGKGLGKGA [4-12] (SEQ ID
NO: 130) SGRGKGGKGLGKGGAKRHRKV [1-21] (SEQ ID NO: 131)
KGLGKGGAKRHRKVLRDNWC-NH2 [8-25 WC, amide]
(SEQ ID NO: 132) SGRG-K(Ac)-GG-K(Ac)-GLG-K(Ac)-GGA-K(Ac)-RHR
KVLRDNGSGSK [1-25 H4K5, 8, 12, 16(Ac)] (SEQ ID NO: 133)
SGRGKGGKGLGKGGAKRHRK-NH2 [1-20 H4 PRMT7 (protein arginine
methyltransferase 7) Substrate, M1] (SEQ ID NO: 134)
SGRG-K(Ac)-GGKGLGKGGAKRHRK [1-20 H4K5 (Ac)] (SEQ ID NO: 135)
SGRGKGG-K(Ac)-GLGKGGAKRHRK [1-20 H4K8 (Ac)] (SEQ ID NO: 136)
SGRGKGGKGLG-K(Ac)-GGAKRHRK [1-20 H4K12 (Ac)] (SEQ ID NO: 137)
SGRGKGGKGLGKGGA-K(Ac)-RHRK [1-20 H4K16 (Ac)] (SEQ ID NO: 138)
KGLGKGGAKRHRKVLRDNWC-NH2 [1-25 WC, amide] (SEQ ID NO: 139)
MSGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLARRGGVKRI
SGLIYEETRGVLKVFLENVIRDAVTYTEHAKRKTVTAMDVVYALKRQ GRTLYGFGG
[1-103]
[0187] As such, a cationic polypeptide of a subject cationic
polypeptide composition can include an amino acid sequence having
the amino acid sequence set forth in any of SEQ ID NOs: 62-139. In
some cases a cationic polypeptide of subject a cationic polypeptide
composition includes an amino acid sequence having 80% or more
sequence identity (e.g., 85% or more, 90% or more, 95% or more, 98%
or more, 99% or more, or 100% sequence identity) with the amino
acid sequence set forth in any of SEQ ID NOs: 62-139. In some cases
a cationic polypeptide of subject a cationic polypeptide
composition includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 98% or more, 99% or more, or
100% sequence identity) with the amino acid sequence set forth in
any of SEQ ID NOs: 62-139. The cationic polypeptide can include any
convenient modification, and a number of such contemplated
modifications are discussed above, e.g., methylated, acetylated,
crotonylated, ubiquitinylated, phosphorylated, SUMOylated,
farnesylated, sulfated, and the like.
[0188] In some cases a cationic polypeptide of a cationic
polypeptide composition includes an amino acid sequence having 80%
or more sequence identity (e.g., 85% or more, 90% or more, 95% or
more, 98% or more, 99% or more, or 100% sequence identity) with the
amino acid sequence set forth in SEQ ID NO: 94. In some cases a
cationic polypeptide of a cationic polypeptide composition includes
an amino acid sequence having 95% or more sequence identity (e.g.,
98% or more, 99% or more, or 100% sequence identity) with the amino
acid sequence set forth in SEQ ID NO: 94. In some cases a cationic
polypeptide of a cationic polypeptide composition includes the
amino acid sequence set forth in SEQ ID NO: 94. In some cases a
cationic polypeptide of a cationic polypeptide composition includes
the sequence represented by H3K4(Me3) (SEQ ID NO: 95), which
comprises the first 25 amino acids of the human histone 3 protein,
and tri-methylated on the lysine 4 (e.g., in some cases amidated on
the C-terminus).
[0189] In some embodiments a cationic polypeptide (e.g., a histone
or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4) of a cationic
polypeptide composition includes a cysteine residue, which can
facilitate conjugation to: a cationic (or in some cases anionic)
amino acid polymer, a linker, an NLS, and/or other cationic
polypeptides (e.g., in some cases to form a branched histone
structure). For example, a cysteine residue can be used for
crosslinking (conjugation) via sulfhydryl chemistry (e.g., a
disulfide bond) and/or amine-reactive chemistry. In some cases the
cysteine residue is internal. In some cases the cysteine residue is
positioned at the N-terminus and/or C-terminus. In some cases, a
cationic polypeptide (e.g., a histone or HTP, e.g., H1, H2, H2A,
H2AX, H2B, H3, or H4) of a cationic polypeptide composition
includes a mutation (e.g., insertion or substitution) that adds a
cysteine residue. Examples of HTPs that include a cysteine include
but are not limited to:
TABLE-US-00002 (SEQ ID NO: 140) CKATQASQEY-from H2AX (SEQ ID NO:
141) ARTKQTARKSTGGKAPRKQLAC-from H3 (SEQ ID NO: 142)
ARTKQTARKSTGGKAPRKWC (SEQ ID NO: 143) KAARKSAPATGGC-from H3 (SEQ ID
NO: 144) KGLGKGGAKRHRKVLRDNWC-from H4 (SEQ ID NO: 145)
MARTKQTARKSTGGKAPRKQLATKVARKSAPATGGVKKPHRYRPGTVALR
EIRRYQKSTELLIRKLPFQRLMREIAQDFKTDLRFQSSAVMALQEACESY
LVGLFEDTNLCVIHAKRVTIMPKDIQLARRIRGERA-from H3
[0190] In some embodiments a cationic polypeptide (e.g., a histone
or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4) of a cationic
polypeptide composition is conjugated to a cationic (and/or
anionic) amino acid polymer of the core of a subject nanoparticle.
As an example, a histone or HTP can be conjugated to a cationic
amino acid polymer (e.g., one that includes poly(lysine)), via a
cysteine residue, e.g., where the pyridyl disulfide group(s) of
lysine(s) of the polymer are substituted with a disulfide bond to
the cysteine of a histone or HTP.
Modified/Branching Structure
[0191] In some embodiments a cationic polypeptide of a subject a
cationic polypeptide composition has a linear structure. In some
embodiments a cationic polypeptide of a subject a cationic
polypeptide composition has a branched structure.
[0192] For example, in some cases, a cationic polypeptide (e.g.,
HTPs, e.g., HTPs with a cysteine residue) is conjugated (e.g., at
its C-terminus) to the end of a cationic polymer (e.g.,
poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine)),
thus forming an extended linear polypeptide. In some cases, one or
more (two or more, three or more, etc.) cationic polypeptides
(e.g., HTPs, e.g., HTPs with a cysteine residue) are conjugated
(e.g., at their C-termini) to the end(s) of a cationic polymer
(e.g., poly(L-arginine), poly(D-lysine), poly(L-lysine),
poly(D-lysine)), thus forming an extended linear polypeptide. In
some cases the cationic polymer has a molecular weight in a range
of from 4,500-150,000 Da).
[0193] As another example, in some cases, one or more (two or more,
three or more, etc.) cationic polypeptides (e.g., HTPs, e.g., HTPs
with a cysteine residue) are conjugated (e.g., at their C-termini)
to the side-chains of a cationic polymer (e.g., poly(L-arginine),
poly(D-lysine), poly(L-lysine), poly(D-lysine)), thus forming a
branched structure (branched polypeptide). Formation of a branched
structure by components of the nanoparticle core (e.g., components
of a subject cationic polypeptide composition) can in some cases
increase the amount of core condensation (e.g., of a nucleic acid
payload) that can be achieved. Thus, in some cases it is desirable
to used components that form a branched structure. Various types of
branches structures are of interest, and examples of branches
structures that can be generated (e.g., using subject cationic
polypeptides such as HTPs, e.g., HTPs with a cysteine residue;
peptoids, polyamides, and the like) include but are not limited to:
brush polymers, webs (e.g., spider webs), graft polymers,
star-shaped polymers, comb polymers, polymer networks, dendrimers,
and the like.
[0194] As an example, FIG. 134 depicts a brush type of branched
structure. In some cases, a branched structure includes from 2-30
cationic polypeptides (e.g., HTPs) (e.g., from 2-25, 2-20, 2-15,
2-10, 2-5, 4-30, 4-25, 4-20, 4-15, or 4-10 cationic polypeptides),
where each can be the same or different than the other cationic
polypeptides of the branched structure (see, e.g., FIG. 134). In
some cases the cationic polymer has a molecular weight in a range
of from 4,500-150,000 Da). In some cases, 5% or more (e.g., 10% or
more, 20% or more, 25% or more, 30% or more, 40% or more, or 50% or
more) of the side-chains of a cationic polymer (e.g.,
poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine))
are conjugated to a subject cationic polypeptide (e.g., HTP, e.g.,
HTP with a cysteine residue). In some cases, up to 50% (e.g., up to
40%, up to 30%, up to 25%, up to 20%, up to 15%, up to 10%, or up
to 5%) of the side-chains of a cationic polymer (e.g.,
poly(L-arginine), poly(D-lysine), poly(L-lysine), poly(D-lysine))
are conjugated to a subject cationic polypeptide (e.g., HTP, e.g.,
HTP with a cysteine residue). Thus, an HTP can be branched off of
the backbone of a polymer such as a cationic amino acid
polymer.
[0195] In some cases formation of branched structures can be
facilitated using components such as peptoids (polypeptoids),
polyamides, dendrimers, and the like. For example, in some cases
peptoids (e.g., polypeptoids) are used as a component of a
nanoparticle core, e.g., in order to generate a web (e.g., spider
web) structure, which can in some cases facilitate condensation of
the nanoparticle core.
[0196] One or more of the natural or modified polypeptide sequences
herein may be modified with terminal or intermittent arginine,
lysine, or histidine sequences. In one embodiment, each polypeptide
is included in equal amine molarities within a nanoparticle core.
In this embodiment, each polypeptide's C-terminus can be modified
with 5R (5 arginines). In some embodiments, each polypeptide's
C-terminus can be modified with 9R (9 arginines). In some
embodiments, each polypeptide's N-terminus can be modified with 5R
(5 arginines). In some embodiments, each polypeptide's N-terminus
can be modified with 9R (9 arginines). In some cases, an H2A, H2B,
H3 and/or H4 histone fragment (e.g., HTP) are each bridged in
series with a FKFL Cathepsin B proteolytic cleavage domain or RGFFP
Cathepsin D proteolytic cleavage domain. In some cases, an H2A,
H2B, H3 and/or H4 histone fragment (e.g., HTP) can be bridged in
series by a 5R (5 arginines), 9R (9 arginines), 5K (5 lysines), 9K
(9 lysines), 5H (5 histidines), or 9H (9 histidines) cationic
spacer domain. In some cases, one or more H2A, H2B, H3 and/or H4
histone fragments (e.g., HTPs) are disulfide-bonded at their
N-terminus to protamine.
[0197] To illustrate how to generate a branched histone structure,
example methods of preparation are provided. One example of such a
method includes the following: covalent modification of equimolar
ratios of Histone H2AX [134-143], Histone H3 [1-21 Cys], Histone H3
[23-34 Cys], Histone H4 [8-25 WC] and SV40 T-Ag-derived NLS can be
performed in a reaction with 10% pyridyl disulfide modified
poly(L-Lysine) [MW=5400, 18000, or 45000 Da; n=30, 100, or 250]. In
a typical reaction, a 29 .mu.L aqueous solution of 700 .mu.M
Cys-modified histone/NLS (20 nmol) can be added to 57 .mu.L of 0.2
M phosphate buffer (pH 8.0). Second, 14 .mu.L of 100 .mu.M pyridyl
disulfide protected poly(lysine) solution can then be added to the
histone solution bringing the final volume to 100 .mu.L with a 1:2
ratio of pyridyl disulfide groups to Cysteine residues. This
reaction can be carried out at room temperature for 3 h. The
reaction can be repeated four times and degree of conjugation can
be determined via absorbance of pyridine-2-thione at 343 nm.
[0198] As another example, covalent modification of a 0:1, 1:4,
1:3, 1:2, 1:1, 1:2, 1:3, 1:4, or 1:0 molar ratio of Histone H3
[1-21 Cys] peptide and Histone H3 [23-34 Cys] peptide can be
performed in a reaction with 10% pyridyl disulfide modified
poly(L-Lysine) or poly(L-Arginine) [MW=5400, 18000, or 45000 Da;
n=30, 100, or 250]. In a typical reaction, a 29 .mu.L aqueous
solution of 700 .mu.M Cys-modified histone (20 nmol) can be added
to 57 .mu.L of 0.2 M phosphate buffer (pH 8.0). Second, 14 .mu.L of
100 .mu.M pyridyl disulfide protected poly(lysine) solution can
then be added to the histone solution bringing the final volume to
100 .mu.L with a 1:2 ratio of pyridyl disulfide groups to Cysteine
residues. This reaction can be carried out at room temperature for
3 h. The reaction can be repeated four times and degree of
conjugation can be determined via absorbance of pyridine-2-thione
at 343 nm.
[0199] In some cases, an anionic polymer is conjugated to a
targeting ligand.
Nuclear Localization Sequence (NLS)
[0200] In some embodiments a cationic polypeptide (e.g., a histone
or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4) of a cationic
polypeptide composition includes (and/or is conjugated to) one or
more (e.g., two or more, three or more, or four or more) nuclear
localization sequences (NLSs). Thus in some cases the cationic
polypeptide composition of a subject nanoparticle includes a
peptide that includes an NLS. In some cases a histone protein (or
an HTP) of a subject nanoparticle includes one or more (e.g., two
or more, three or more) natural nuclear localization signals
(NLSs). In some cases a histone protein (or an HTP) of a subject
nanoparticle includes one or more (e.g., two or more, three or
more) NLSs that are heterologous to the histone protein (i.e., NLSs
that do not naturally occur as part of the histone/HTP, e.g., an
NLS can be added by humans). In some cases the HTP includes an NLS
on the N- and/or C-terminus.
[0201] In some embodiments a cationic amino acid polymer (e.g.,
poly(arginine)(PR), poly(lysine)(PK), poly(histidine)(PH),
poly(ornithine), poly(citrulline), poly(D-arginine)(PDR),
poly(D-lysine)(PDK), poly(D-histidine)(PDH), poly(D-ornithine),
poly(D-citrulline), poly(L-arginine)(PLR), poly(L-lysine)(PLK),
poly(L-histidine)(PLH), poly(L-ornithine), or poly(L-citrulline))
of a cationic polymer composition includes (and/or is conjugated
to) one or more (e.g., two or more, three or more, or four or more)
NLSs. In some cases the cationic amino acid polymer includes an NLS
on the N- and/or C-terminus. In some cases the cationic amino acid
polymer includes an internal NLS.
[0202] In some embodiments an anionic amino acid polymer (e.g.,
poly(glutamic acid) (PEA), poly(aspartic acid) (PDA),
poly(D-glutamic acid) (PDEA), poly(D-aspartic acid) (PDDA),
poly(L-glutamic acid) (PLEA), or poly(L-aspartic acid) (PLDA)) of
an anionic polymer composition includes (and/or is conjugated to)
one or more (e.g., two or more, three or more, or four or more)
NLSs. In some cases the anionic amino acid polymer includes an NLS
on the N- and/or C-terminus. In some cases the anionic amino acid
polymer includes an internal NLS.
[0203] Any convenient NLS can be used (e.g., conjugated to a
histone, an HTP, a cationic amino acid polymer, an anionic amino
acid polymer, and the like). Examples include, but are not limited
to Class 1 and Class 2 `monopartite NLSs`, as well as NLSs of
Classes 3-5 (see, e.g., FIG. 16, which is adapted from Kosugi et
al., J Biol Chem. 2009 Jan. 2; 284(1):478-85). In some cases, an
NLS has the formula: (K/R) (K/R).sub.X10-12(K/R).sub.3-5. In some
cases, an NLS has the formula: K(K/R)X(K/R).
[0204] In some embodiments a cationic polypeptide of a cationic
polypeptide composition includes one more (e.g., two or more, three
or more, or four or more) NLSs. In some cases the cationic
polypeptide is not a histone protein or histone fragment (e.g., is
not an HTP). Thus, in some cases the cationic polypeptide of a
cationic polypeptide composition is an NLS-containing peptide.
[0205] In some cases, the NLS-containing peptide includes a
cysteine residue, which can facilitate conjugation to: a cationic
(or in some cases anionic) amino acid polymer, a linker, histone
protein for HTP, and/or other cationic polypeptides (e.g., in some
cases as part of a branched histone structure). For example, a
cysteine residue can be used for crosslinking (conjugation) via
sulfhydryl chemistry (e.g., a disulfide bond) and/or amine-reactive
chemistry. In some cases the cysteine residue is internal. In some
cases the cysteine residue is positioned at the N-terminus and/or
C-terminus. In some cases, an NLS-containing peptide of a cationic
polypeptide composition includes a mutation (e.g., insertion or
substitution) (e.g., relative to a wild type amino acid sequence)
that adds a cysteine residue.
[0206] Examples of NLSs that can be used as an NLS-containing
peptide (or conjugated to any convenient cationic polypeptide such
as an HTP or cationic polymer or cationic amino acid polymer or
anionic amino acid polymer) include but are not limited to (some of
which include a cysteine residue):
TABLE-US-00003 (SEQ ID NO: 151) PKKKRKV (T-ag NLS) (SEQ ID NO: 152)
PKKKRKVEDPYC-SV40 T-Ag-derived NLS (SEQ ID NO: 153)
PKKKRKVGPKKKRKVGPKKKRKVGPKKKRKVGC (NLS SV40) (SEQ ID NO: 154)
CYGRKKRRQRRR-N-terminal cysteine of cysteine-TAT (SEQ ID NO: 155)
CSIPPEVKFNKPFVYLI (SEQ ID NO: 156) DRQIKIWFQNRRMKVVKK (SEQ ID NO:
157) PKKKRKVEDPYC-C-term cysteine of an SV40 T-Ag-derived NLS (SEQ
ID NO: 158) PAAKRVKLD [cMyc NLS]
For non-limiting examples of NLSs that can be used, see, e.g.,
Kosugi et al., J Biol Chem. 2009 Jan. 2; 284(1):478-85, e.g., see
FIG. 16 of this disclosure.
Mitochondrial Localization Signal
[0207] In some embodiments a cationic polypeptide (e.g., a histone
or HTP, e.g., H1, H2, H2A, H2AX, H2B, H3, or H4), an anionic
polymer, and/or a cationic polymer of a subject nanoparticle
includes (and/or is conjugated to) one or more (e.g., two or more,
three or more, or four or more) mitochondrial localization
sequences. Any convenient mitochondrial localization sequence can
be used. Examples of mitochondrial localization sequences include
but are not limited to: PEDEIWLPEPESVDVPAKPISTSSMMMP (SEQ ID NO:
149), a mitochondrial localization sequence of SDHB,
mono/di/triphenylphosphonium or other phosphoniums, VAMP 1A, VAMP
1B, the 67 N-terminal amino acids of DGAT2, and the 20 N-terminal
amino acids of Bax.
Payload
[0208] Nanoparticles of the disclosure include a payload, which can
be made of nucleic acid and/or protein. For example, in some cases
a subject nanoparticle is used to deliver a nucleic acid payload
(e.g., a DNA and/or RNA). The nucleic acid payload can be any
nucleic acid of interest, e.g., the nucleic acid payload can be
linear or circular, and can be a plasmid, a viral genome, an RNA
(e.g., a coding RNA such as an mRNA or a non-coding RNA such as a
guide RNA, a short interfering RNA (siRNA), a short hairpin RNA
(shRNA), a microRNA (miRNA), and the like), a DNA, etc. In some
cases, the nucleic payload is an RNAi agent (e.g., an shRNA, an
siRNA, a miRNA, etc.) or a DNA template encoding an RNAi agent. In
some cases, the nucleic acid payload is an siRNA molecule (e.g.,
one that targets an mRNA, one that targets a miRNA). In some cases,
the nucleic acid payload is an LNA molecule (e.g., one that targets
a miRNA). In some cases, the nucleic acid payload is a miRNA. In
some cases the nucleic acid payload includes an mRNA that encodes a
protein of interest (e.g., one or more reprograming and/or
transdifferentiation factors such as Oct4, Sox2, Klf4, c-Myc,
Nanog, and Lin28, e.g., alone or in any desired combination such as
(i) Oct4, Sox2, Klf4, and c-Myc; (ii) Oct4, Sox2, Nanog, and Lin28;
and the like; a gene editing endonuclease; a therapeutic protein;
and the like). In some cases the nucleic acid payload includes a
non-coding RNA (e.g., an RNAi agent, a CRISPR/Cas guide RNA, etc.)
and/or a DNA molecule encoding the non-coding RNA. In some
embodiments a nucleic acid payload includes a nucleic acid (DNA
and/or mRNA) that encodes IL2R.alpha. and IL12R.gamma. (e.g., to
modulate the behavior or survival of a target cell), and in some
cases the payload is released intracellularly from a subject
nanoparticle over the course of from 7-90 days (e.g., from 7-80,
7-60, 7-50, 7-40, 7-35, or 7-30 days). In some embodiments a
nucleic acid payload includes a nucleic acid (DNA and/or mRNA) that
encodes BCL-XL (e.g., to prevent apoptosis of a target cell due to
engagement of Fas or TNF.alpha. receptors). In some embodiments a
nucleic acid payload includes a nucleic acid (DNA and/or mRNA) that
encodes
Foxp3 (e.g., to promote an immune effector phenotype in targeted
T-cells). In some embodiments a nucleic acid payload includes a
nucleic acid (DNA and/or mRNA) that encodes SCF. In some
embodiments a nucleic acid payload includes a nucleic acid (DNA
and/or mRNA) that encodes HoxB4. In some embodiments a nucleic acid
payload includes a nucleic acid (DNA and/or mRNA) that encodes
SIRT6. In some embodiments a nucleic acid payload includes a
nucleic acid molecule (e.g., an siRNA, an LNA, etc.) that targets
(reduces expression of) a microRNA such as miR-155 (see, e.g., MiR
Base accession: MI0000681 and MI0000177). In some embodiments a
nucleic acid payload includes an siRNA that targets ku70 and/or an
siRNA that targets ku80.
[0209] The term "nucleic acid payload" encompasses modified nucleic
acids. Likewise, the terms "RNAi agent" and "siRNA" encompass
modified nucleic acids. For example, the nucleic acid molecule can
be a mimetic, can include a modified sugar backbone, one or more
modified internucleoside linkages (e.g., one or more
phosphorothioate and/or heteroatom internucleoside linkages), one
or more modified bases, and the like. In some embodiments, a
subject payload includes triplex-forming peptide nucleic acids
(PNAs) (see, e.g., McNeer et al., Gene Ther. 2013 June;
20(6):658-69). Thus, in some cases a subject core includes PNAs. In
some cases a subject core includes PNAs and DNAs.
[0210] A subject nucleic acid payload (e.g., an siRNA) can have a
morpholino backbone structure. In some case, a subject nucleic acid
payload (e.g., an siRNA) can have one or more locked nucleic acids
(LNAs). Suitable sugar substituent groups include methoxy
(--O--CH.sub.3), aminopropoxy
(--OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), allyl
(--CH.sub.2--CH.dbd.CH.sub.2), --O-allyl
(--O--CH.sub.2--CH.dbd.CH.sub.2) and fluoro (F). 2'-sugar
substituent groups may be in the arabino (up) position or ribo
(down) position. Suitable base modifications include synthetic and
natural nucleobases such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl (--C.dbd.C--CH.sub.3) uracil and cytosine and
other alkynyl derivatives of pyrimidine bases, 6-azo uracil,
cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,
8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other
8-substituted adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,
8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine
and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases
include tricyclic pyrimidines such as phenoxazine
cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one),
phenothiazine cytidine
(1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a
substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole
cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).
[0211] In some cases, a nucleic acid payload can include a
conjugate moiety (e.g., one that enhances the activity, stability,
cellular distribution or cellular uptake of the nucleic acid
payload). These moieties or conjugates can include conjugate groups
covalently bound to functional groups such as primary or secondary
hydroxyl groups. Conjugate groups include, but are not limited to,
intercalators, reporter molecules, polyamines, polyamides,
polyethylene glycols, polyethers, groups that enhance the
pharmacodynamic properties of oligomers, and groups that enhance
the pharmacokinetic properties of oligomers. Suitable conjugate
groups include, but are not limited to, cholesterols, lipids,
phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and
dyes. Groups that enhance the pharmacodynamic properties include
groups that improve uptake, enhance resistance to degradation,
and/or strengthen sequence-specific hybridization with the target
nucleic acid. Groups that enhance the pharmacokinetic properties
include groups that improve uptake, distribution, metabolism or
excretion of a subject nucleic acid.
[0212] Any convenient polynucleotide can be used as a subject
nucleic acid payload. Examples include but are not limited to:
species of RNA and DNA including mRNA, m1A modified mRNA
(monomethylation at position 1 of Adenosine), siRNA, miRNA,
aptamers, shRNA, AAV-derived nucleic acids and scaffolds,
morpholino RNA, peptoid and peptide nucleic acids, cDNA, DNA
origami, DNA and RNA with synthetic nucleotides, DNA and RNA with
predefined secondary structures, multimers and oligomers of the
aforementioned, and payloads whose sequence may encode other
products such as any protein or polypeptide whose expression is
desired.
[0213] In some cases a payload of a subject nanoparticle includes a
protein. Examples of protein payloads include, but are not limited
to: programmable gene editing proteins (e.g., transcription
activator-like (TAL) effectors (TALEs), TALE nucleases (TALENs),
zinc-finger proteins (ZFPs), zinc-finger nucleases (ZFNs),
DNA-guided polypeptides such as Natronobacterium gregoryi Argonaute
(NgAgo), CRISPR/Cas RNA-guided polypeptides such as Cas9, CasX,
CasY, Cpf1, and the like); transposons (e.g., a Class I or Class II
transposon--e.g., piggybac, sleeping beauty, Tc1/mariner, Tol2,
PIF/harbinger, hAT, mutator, merlin, transib, helitron, maverick,
frog prince, minos, Himar1 and the like); meganucleases (e.g.,
I-Scel, I-Ceul, I-Crel, I-Dmol, I-Chul, I-Dirl, I-Flmul, I-Flmull,
I-Anil, I-ScelV, I-Csml, I-Panl, I-Panl, I-PanMl, I-ScelI, I-Ppol,
I-ScelII, I-Ltrl, I-Gpil, I-GZel, I-Onul, I-HjeMl, I-Msol, I-Tevl,
I-TevlI, I-TevlII, PI-MleI, PI-Mtul, PI-Pspl, PI-Tli I, PI-Tli II,
PI-SceV, and the like); megaTALs (see, e.g., Boissel et al.,
Nucleic Acids Res. 2014 February; 42(4): 2591-2601); SCF; BCL-XL;
Foxp3; HoxB4; and SiRT6. For any of the above proteins, a payload
of a subject nanoparticle can include a nucleic acid (DNA and/or
mRNA) encoding the protein, and/or can include the actual
protein.
[0214] Gene Editing Tools
[0215] In some cases, a nucleic acid payload includes or encodes a
gene editing tool (i.e., a component of a gene editing system,
e.g., a site specific gene editing system such as a programmable
gene editing system). For example, a nucleic acid payload can
include one or more of: (i) a CRISPR/Cas guide RNA, (ii) a DNA
encoding a CRISPR/Cas guide RNA, (iii) a DNA and/or RNA encoding a
programmable gene editing protein such as a zinc finger protein
(ZFP) (e.g., a zinc finger nuclease--ZFN), a transcription
activator-like effector (TALE) protein (e.g., fused to a
nuclease--TALEN), a DNA-guided polypeptide such as Natronobacterium
gregoryi Argonaute (NgAgo), and/or a CRISPR/Cas RNA-guided
polypeptide (e.g., Cas9, CasX, CasY, Cpf1, and the like); (iv) a
DNA donor template; (v) a nucleic acid molecule (DNA, RNA) encoding
a site-specific recombinase (e.g., Cre recombinase, Dre
recombinase, Flp recombinase, KD recombinase, B2 recombinase, B3
recombinase, R recombinase, Hin recombinase, Tre recombinase,
PhiC31 integrase, Bxb1 integrase, R4 integrase, lambda integrase,
HK022 integrase, HP1 integrase, and the like); (vi) a DNA encoding
a resolvase and/or invertase (e.g., Gin, Hin, .gamma..delta.3, Tn3,
Sin, Beta, and the like); and (vii) a transposon and/or a DNA
derived from a transposon (e.g., bacterial transposons such as Tn3,
Tn5, Tn7, Tn9, Tn10, Tn903, Tn1681, and the like; eukaryotic
transposons such as Tc1/mariner super family transposons, PiggyBac
superfamily transposons, hAT superfamily transposons, PiggyBac,
Sleeping Beauty, Frog Prince, Minos, Himar1, and the like). In some
cases a subject nanoparticle is used to deliver a protein payload,
e.g., a gene editing protein such as a ZFP (e.g., ZFN), a TALE
(e.g., TALEN), a DNA-guided polypeptide such as Natronobacterium
gregoryi Argonaute (NgAgo), a CRISPR/Cas RNA-guided polypeptide
(e.g., Cas9, CasX, CasY, Cpf1, and the like), a site-specific
recombinase (e.g., Cre recombinase, Dre recombinase, Flp
recombinase, KD recombinase, B2 recombinase, B3 recombinase, R
recombinase, Hin recombinase, Tre recombinase, PhiC31 integrase,
Bxb1 integrase, R4 integrase, lambda integrase, HK022 integrase,
HP1 integrase, and the like), a resolvase/invertase (e.g., Gin,
Hin, .gamma..delta.3, Tn3, Sin, Beta, and the like); and/or a
transposase (e.g., a transposase related to transposons such as
bacterial transposons such as Tn3, Tn5, Tn7, Tn9, Tn10, Tn903,
Tn1681, and the like; or eukaryotic transposons such as Tc1/mariner
super family transposons, PiggyBac superfamily transposons, hAT
superfamily transposons, PiggyBac, Sleeping Beauty, Frog Prince,
Minos, Himar1, and the like). In some cases, the nanoparticle is
used to deliver a nucleic acid payload and a protein payload, and
in some such cases the payload includes a ribonucleoprotein complex
(RNP).
[0216] Depending on the nature of the system and the desired
outcome, a gene editing system (e.g. a site specific gene editing
system such as a programmable gene editing system) can include a
single component (e.g., a ZFP, a ZFN, a TALE, a TALEN, a
site-specific recombinase, a resolvase/integrase, a transpose, a
transposon, and the like) or can include multiple components. In
some cases a gene editing system includes at least two components.
For example, in some cases a gene editing system (e.g. a
programmable gene editing system) includes (i) a donor template
nucleic acid; and (ii) a gene editing protein (e.g., a programmable
gene editing protein such as a ZFP, a ZFN, a TALE, a TALEN, a
DNA-guided polypeptide such as Natronobacterium gregoryi Argonaute
(NgAgo), a CRISPR/Cas RNA-guided polypeptide such as Cas9, CasX,
CasY, or Cpf1, and the like), or a nucleic acid molecule encoding
the gene editing protein (e.g., DNA or RNA such as a plasmid or
mRNA). As another example, in some cases a gene editing system
(e.g. a programmable gene editing system) includes (i) a CRISPR/Cas
guide RNA, or a DNA encoding the CRISPR/Cas guide RNA; and (ii) a
CRISPR/CAS RNA-guided polypeptide (e.g., Cas9, CasX, CasY, Cpf1,
and the like), or a nucleic acid molecule encoding the RNA-guided
polypeptide (e.g., DNA or RNA such as a plasmid or mRNA). As
another example, in some cases a gene editing system (e.g. a
programmable gene editing system) includes (i) an NgAgo-like guide
DNA; and (ii) a DNA-guided polypeptide (e.g., NgAgo), or a nucleic
acid molecule encoding the DNA-guided polypeptide (e.g., DNA or RNA
such as a plasmid or mRNA). In some cases a gene editing system
(e.g. a programmable gene editing system) includes at least three
components: (i) a donor DNA template; (ii) a CRISPR/Cas guide RNA,
or a DNA encoding the CRISPR/Cas guide RNA; and (iii) a CRISPR/Cas
RNA-guided polypeptide (e.g., Cas9, CasX, CasY, or Cpf1), or a
nucleic acid molecule encoding the RNA-guided polypeptide (e.g.,
DNA or RNA such as a plasmid or mRNA). In some cases a gene editing
system (e.g. a programmable gene editing system) includes at least
three components: (i) a donor DNA template; (ii) an NgAgo-like
guide DNA, or a DNA encoding the NgAgo-like guide DNA; and (iii) a
DNA-guided polypeptide (e.g., NgAgo), or a nucleic acid molecule
encoding the DNA-guided polypeptide (e.g., DNA or RNA such as a
plasmid or mRNA).
[0217] In some embodiments, a subject nanoparticle is used to
deliver a gene editing tool. In other words in some cases the
payload includes one or more gene editing tools. The term "gene
editing tool" is used herein to refer to one or more components of
a gene editing system. Thus, in some cases the payload includes a
gene editing system and in some cases the payload includes one or
more components of a gene editing system (i.e., one or more gene
editing tools). For example, a target cell might already include
one of the components of a gene editing system and the user need
only add the remaining components. In such a case the payload of a
subject nanoparticle does not necessarily include all of the
components of a given gene editing system. As such, in some cases a
payload includes one or more gene editing tools.
[0218] As an illustrative example, a target cell might already
include a gene editing protein (e.g., a ZFP, a TALE, a DNA-guided
polypeptide (e.g., NgAgo), a CRISPR/Cas RNA-guided polypeptide such
Cas9, CasX, CasY, Cpf1, and the like, a site-specific recombinase
such as Cre recombinase, Dre recombinase, Flp recombinase, KD
recombinase, B2 recombinase, B3 recombinase, R recombinase, Hin
recombinase, Tre recombinase, PhiC31 integrase, Bxb1 integrase, R4
integrase, lambda integrase, HK022 integrase, HP1 integrase, and
the like, a resolvase/invertase such as Gin, Hin, .gamma..delta.3,
Tn3, Sin, Beta, and the like, a transposase, etc.) and/or a DNA or
RNA encoding the protein, and therefore the payload can include one
or more of: (i) a donor template; and (ii) a CRISPR/Cas guide RNA,
or a DNA encoding the CRISPR/Cas guide RNA; or an NgAgo-like guide
DNA. Likewise, the target cell may already include a CRISPR/Cas
guide RNA and/or a DNA encoding the guide RNA or an NgAgo-like
guide DNA, and the payload can include one or more of: (i) a donor
template; and (ii) a CRISPR/Cas RNA-guided polypeptide (e.g., Cas9,
CasX, CasY, Cpf1, and the like), or a nucleic acid molecule
encoding the RNA-guided polypeptide (e.g., DNA or RNA such as a
plasmid or mRNA); or a DNA-guided polypeptide (e.g., NgAgo), or a
nucleic acid molecule encoding the DNA-guided polypeptide.
[0219] As would be understood by one of ordinary skill in the art,
a gene editing system need not be a system that `edits` a nucleic
acid. For example, it is well recognized that a gene editing system
can be used to modify target nucleic acids (e.g., DNA and/or RNA)
in a variety of ways without creating a double strand break (DSB)
in the target DNA. For example, in some cases a double stranded
target DNA is nicked (one strand is cleaved), and in some cases
(e.g., in some cases where the gene editing protein is devoid of
nuclease activity, e.g., a CRISPR/Cas RNA-guided polypeptide may
harbor mutations in the catalytic nuclease domains), the target
nucleic acid is not cleaved at all. For example, in some cases a
CRISPR/Cas protein (e.g., Cas9, CasX, CasY, Cpf1) with or without
nuclease activity, is fused to a heterologous protein domain. The
heterologous protein domain can provide an activity to the fusion
protein such as (i) a DNA-modifying activity (e.g., nuclease
activity, methyltransferase activity, demethylase activity, DNA
repair activity, DNA damage activity, deamination activity,
dismutase activity, alkylation activity, depurination activity,
oxidation activity, pyrimidine dimer forming activity, integrase
activity, transposase activity, recombinase activity, polymerase
activity, ligase activity, helicase activity, photolyase activity
or glycosylase activity), (ii) a transcription modulation activity
(e.g., fusion to a transcriptional repressor or activator), or
(iii) an activity that modifies a protein (e.g., a histone) that is
associated with target DNA (e.g., methyltransferase activity,
demethylase activity, acetyltransferase activity, deacetylase
activity, kinase activity, phosphatase activity, ubiquitin ligase
activity, deubiquitinating activity, adenylation activity,
deadenylation activity, SUMOylating activity, deSUMOylating
activity, ribosylation activity, deribosylation activity,
myristoylation activity or demyristoylation activity). As such, a
gene editing system can be used in applications that modify a
target nucleic acid in way that do not cleave the target nucleic
acid, and can also be used in applications that modulate
transcription from a target DNA.
[0220] For additional information related to programmable gene
editing tools (e.g., CRISPR/Cas RNa-guided proteins such as Cas9,
CasX, CasY, and Cpf1, Zinc finger proteins such as Zinc finger
nucleases, TALE proteins such as TALENs, CRISPR/Cas guide RNAs, and
the like) refer to, for example, Dreier, et al., (2001) J Biol Chem
276:29466-78; Dreier, et al., (2000) J Mol Biol 303:489-502; Liu,
et al., (2002) J Biol Chem 277:3850-6); Dreier, et al., (2005) J
Biol Chem 280:35588-97; Jamieson, et al., (2003) Nature Rev Drug
Discov 2:361-8; Durai, et al., (2005) Nucleic Acids Res 33:5978-90;
Segal, (2002) Methods 26:76-83; Porteus and Carroll, (2005) Nat
Biotechnol 23:967-73; Pabo, et al., (2001) Ann Rev Biochem
70:313-40; Wolfe, et al., (2000) Ann Rev Biophys Biomol Struct
29:183-212; Segal and Barbas, (2001) Curr Opin Biotechnol 12:632-7;
Segal, et al., (2003) Biochemistry 42:2137-48; Beerli and Barbas,
(2002) Nat Biotechnol 20:135-41; Carroll, et al., (2006) Nature
Protocols 1:1329; Ordiz, et al., (2002) Proc Natl Acad Sci USA
99:13290-5; Guan, et al., (2002) Proc Natl Acad Sci USA
99:13296-301; Sanjana et al., Nature Protocols, 7:171-192 (2012);
Zetsche et al, Cell. 2015 Oct. 22; 163(3):759-71; Makarova et al,
Nat Rev Microbiol. 2015 November; 13(11):722-36; Shmakov et al.,
Mol Cell. 2015 Nov. 5; 60(3):385-97; Jinek et al., Science. 2012
Aug. 17; 337(6096):816-21; Chylinski et al., RNA Biol. 2013 May;
10(5):726-37; Ma et al., Biomed Res Int. 2013; 2013:270805; Hou et
al., Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15644-9; Jinek
et al., Elife. 2013; 2:e00471; Pattanayak et al., Nat Biotechnol.
2013 September; 31(9):839-43; Qi et al, Cell. 2013 Feb. 28;
152(5):1173-83; Wang et al., Cell. 2013 May 9; 153(4):910-8; Auer
et. al., Genome Res. 2013 Oct. 31; Chen et. al., Nucleic Acids Res.
2013 Nov. 1; 41(20):e19; Cheng et. al., Cell Res. 2013 October;
23(10):1163-71; Cho et. al., Genetics. 2013 November;
195(3):1177-80; DiCarlo et al., Nucleic Acids Res. 2013 April;
41(7):4336-43; Dickinson et. al., Nat Methods. 2013 October;
10(10):1028-34; Ebina et. al., Sci Rep. 2013; 3:2510; Fujii et. al,
Nucleic Acids Res. 2013 Nov. 1; 41(20):e187; Hu et. al., Cell Res.
2013 November; 23(11):1322-5; Jiang et. al., Nucleic Acids Res.
2013 Nov. 1; 41(20):e188; Larson et. al., Nat Protoc. 2013
November; 8(11):2180-96; Mali et. at., Nat Methods. 2013 October;
10(10):957-63; Nakayama et. al., Genesis. 2013 December;
51(12):835-43; Ran et. al., Nat Protoc. 2013 November;
8(11):2281-308; Ran et. al., Cell. 2013 Sep. 12; 154(6):1380-9;
Upadhyay et. al., G3 (Bethesda). 2013 Dec. 9; 3(12):2233-8; Walsh
et. al., Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15514-5; Xie
et. al., Mol Plant. 2013 Oct. 9; Yang et. al., Cell. 2013 Sep. 12;
154(6):1370-9; Briner et al., Mol Cell. 2014 Oct. 23; 56(2):333-9;
Burstein et al., Nature. 2016 Dec. 22-Epub ahead of print; Gao et
al., Nat Biotechnol. 2016 Jul. 34(7):768-73; as well as
international patent application publication Nos. WO2002099084;
WO00/42219; WO02/42459; WO2003062455; WO03/080809; WO05/014791;
WO05/084190; WO08/021207; WO09/042186; WO09/054985; and
WO10/065123; U.S. patent application publication Nos. 20030059767,
20030108880, 20140068797; 20140170753; 20140179006; 20140179770;
20140186843; 20140186919; 20140186958; 20140189896; 20140227787;
20140234972; 20140242664; 20140242699; 20140242700; 20140242702;
20140248702; 20140256046; 20140273037; 20140273226; 20140273230;
20140273231; 20140273232; 20140273233; 20140273234; 20140273235;
20140287938; 20140295556; 20140295557; 20140298547; 20140304853;
20140309487; 20140310828; 20140310830; 20140315985; 20140335063;
20140335620; 20140342456; 20140342457; 20140342458; 20140349400;
20140349405; 20140356867; 20140356956; 20140356958; 20140356959;
20140357523; 20140357530; 20140364333; 20140377868; 20150166983;
and 20160208243; and U.S. Pat. Nos. 6,140,466; 6,511,808; 6,453,242
8,685,737; 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445;
8,865,406; 8,795,965; 8,771,945; and 8,697,359; all of which are
hereby incorporated by reference in their entirety.
[0221] In some embodiments, more than one payload is delivered as
part of the same package (e.g., nanoparticle), e.g., in some cases
different payloads are part of different cores. One advantage of
delivering multiple payloads as part of the same package (e.g.,
nanoparticle) is that the efficiency of each payload is not
diluted. As an illustrative example, if payload A and payload B are
delivered in two separate packages (package A and package B,
respectively), then the efficiencies are multiplicative, e.g., if
package A and package B each have a 1% transfection efficiency, the
chance of delivering payload A and payload B to the same cell is
0.01% (1%.times.1%). However, if payload A and payload B are both
delivered as part of the same package (e.g., part of the same
nanoparticle--package A), then the chance of delivering payload A
and payload B to the same cell is 1%, a 100-fold improvement over
0.01%.
[0222] Likewise, in a scenario where package A and package B each
have a 0.1% transfection efficiency, the chance of delivering
payload A and payload B to the same cell is 0.0001%
(0.1%.times.0.1%). However, if payload A and payload B are both
delivered as part of the same package (e.g., part of the same
nanoparticle--package A) in this scenario, then the chance of
delivering payload A and payload B to the same cell is 0.1%, a
1000-fold improvement over 0.0001%.
[0223] As such, in some embodiments, one or more gene editing tools
(e.g., as described above) is delivered in combination with (e.g.,
as part of the same nanoparticle) a protein (and/or a DNA or mRNA
encoding same) and/or a non-coding RNA that increases genomic
editing efficiency. In some cases, one or more gene editing tools
(e.g., as described above) is delivered in combination with (e.g.,
as part of the same nanoparticle) a protein (and/or a DNA or mRNA
encoding same) and/or a non-coding RNA that controls cell division
and/or differentiation. In some cases, one or more gene editing
tools (e.g., as described above) is delivered in combination with
(e.g., as part of the same nanoparticle) a protein (and/or a DNA or
mRNA encoding same) and/or a non-coding RNA that biases the cell
DNA repair machinery toward non-homologous end joining (NHEJ) or
homology directed repair (HDR).
[0224] As non-limiting examples of the above, in some embodiments
one or more gene editing tools can be delivered in combination with
one or more of: SCF (and/or a DNA or mRNA encoding SCF), HoxB4
(and/or a DNA or mRNA encoding HoxB4), BCL-XL (and/or a DNA or mRNA
encoding BCL-XL), SIRT6 (and/or a DNA or mRNA encoding SIRT6), a
nucleic acid molecule (e.g., an siRNA and/or an LNA) that
suppresses miR-155, a nucleic acid molecule (e.g., an siRNA, an
shRNA, a microRNA) that reduces ku70 expression, and a nucleic acid
molecule (e.g., an siRNA, an shRNA, a microRNA) that reduces ku80
expression.
[0225] For examples of microRNAs that can be delivered in
combination with a gene editing tool, see FIG. 18A. For example,
the following microRNAs can be used for the following purposes: for
blocking differentiation of a pluripotent stem cell toward ectoderm
lineage: miR-430/427/302 (see, e.g., MiR Base accession: MI0000738,
MI0000772, MI0000773, MI0000774, MI0006417, MI0006418, MI0000402,
MI0003716, MI0003717, and MI0003718); for blocking differentiation
of a pluripotent stem cell toward endoderm lineage: miR-109 and/or
miR-24 (see, e.g., MiR Base accession: MI0000080, MI0000081,
MI0000231, and MI0000572); for driving differentiation of a
pluripotent stem cell toward endoderm lineage: miR-122 (see, e.g.,
MiR Base accession: MI0000442 and MI0000256) and/or miR-192 (see,
e.g., MiR Base accession: MI0000234 and MI0000551); for driving
differentiation of an ectoderm progenitor cell toward a
keratinocyte fate: miR-203 (see, e.g., MiR Base accession:
MI0000283, MI0017343, and MI0000246); for driving differentiation
of a neural crest stem cell toward a smooth muscle fate: miR-145
(see, e.g., MiR Base accession: MI0000461, MI0000169, and
MI0021890); for driving differentiation of a neural stem cell
toward a glial cell fate and/or toward a neuron fate: miR-9 (see,
e.g., MiR Base accession: MI0000466, MI0000467, MI0000468,
MI0000157, MI0000720, and MI0000721) and/or miR-124a (see, e.g.,
MiR Base accession: MI0000443, MI0000444, MI0000445, MI0000150,
MI0000716, and MI0000717); for blocking differentiation of a
mesoderm progenitor cell toward a chondrocyte fate: miR-199a (see,
e.g., MiR Base accession: MI0000242, MI0000281, MI0000241, and
MI0000713); for driving differentiation of a mesoderm progenitor
cell toward an osteoblast fate: miR-296 (see, e.g., MiR Base
accession: MI0000747 and MI0000394) and/or miR-2861 (see, e.g., MiR
Base accession: MI0013006 and MI0013007); for driving
differentiation of a mesoderm progenitor cell toward a cardiac
muscle fate: miR-1 (see, e.g., MiR Base accession: MI0000437,
MI0000651, MI0000139, MI0000652, MI0006283); for blocking
differentiation of a mesoderm progenitor cell toward a cardiac
muscle fate: miR-133 (see, e.g., MiR Base accession: MI0000450,
MI0000451, MI0000822, MI0000159, MI0000820, MI0000821, and
MI0021863); for driving differentiation of a mesoderm progenitor
cell toward a skeletal muscle fate: miR-214 (see, e.g., MiR Base
accession: MI0000290 and MI0000698), miR-206 (see, e.g., MiR Base
accession: MI0000490 and MI0000249), miR-1 and/or miR-26a (see,
e.g., MiR Base accession: MI0000083, MI0000750, MI0000573, and
MI0000706); for blocking differentiation of a mesoderm progenitor
cell toward a skeletal muscle fate: miR-133 (see, e.g., MiR Base
accession: MI0000450, MI0000451, MI0000822, MI0000159, MI0000820,
MI0000821, and MI0021863), miR-221 (see, e.g., MiR Base accession:
MI0000298 and MI0000709), and/or miR-222 (see, e.g., MiR Base
accession: MI0000299 and MI0000710); for driving differentiation of
a hematopoietic progenitor cell toward differentiation: miR-223
(see, e.g., MiR Base accession: MI0000300 and MI0000703); for
blocking differentiation of a hematopoietic progenitor cell toward
differentiation: miR-128a (see, e.g., MiR Base accession: MI0000447
and MI0000155) and/or miR-181a (see, e.g., MiR Base accession:
MI0000269, MI0000289, MI0000223, and MI0000697); for driving
differentiation of a hematopoietic progenitor cell toward a
lymphoid progenitor cell: miR-181 (see, e.g., MiR Base accession:
MI0000269, MI0000270, MI0000271, MI0000289, MI0000683, MI0003139,
MI0000223, MI0000723, MI0000697, MI0000724, MI0000823, and
MI0005450); for blocking differentiation of a hematopoietic
progenitor cell toward a lymphoid progenitor cell: miR-146 (see,
e.g., MiR Base accession: MI0000477, MI0003129, MI0003782,
MI0000170, and MI0004665); for blocking differentiation of a
hematopoietic progenitor cell toward a myeloid progenitor cell:
miR-155, miR-24a, and/or miR-17 (see, e.g., MiR Base accession:
MI0000071 and MI0000687); for driving differentiation of a lymphoid
progenitor cell toward a T cell fate: miR-150 (see, e.g., MiR Base
accession: MI0000479 and MI0000172); for blocking differentiation
of a myeloid progenitor cell toward a granulocyte fate: miR-223
(see, e.g., MiR Base accession: MI0000300 and MI0000703); for
blocking differentiation of a myeloid progenitor cell toward a
monocyte fate: miR-17-5p (see, e.g., MiR Base accession:
MIMAT0000070 and MIMAT0000649), miR-20a (see, e.g., MiR Base
accession: MI0000076 and MI0000568), and/or miR-106a (see, e.g.,
MiR Base accession: MI0000113 and MI0000406); for blocking
differentiation of a myeloid progenitor cell toward a red blood
cell fate: miR-150 (see, e.g., MiR Base accession: MI0000479 and
MI0000172), miR-155, miR-221 (see, e.g., MiR Base accession:
MI0000298 and MI0000709), and/or miR-222 (see, e.g., MiR Base
accession: MI0000299 and MI0000710); and for driving
differentiation of a myeloid progenitor cell toward a red blood
cell fate: miR-451 (see, e.g., MiR Base accession: MI0001729,
MI0017360, MI0001730, and MI0021960) and/or miR-16 (see, e.g., MiR
Base accession: MI0000070, MI0000115, MI0000565, and
MI0000566).
[0226] For examples of signaling proteins (e.g., extracellular
signaling proteins) that can be delivered (e.g., as protein or as
DNA or RNA encoding the protein) in combination with a gene editing
tool, see FIG. 18B. The same proteins can be used as part of the
outer shell of a subject nanoparticle in a similar manner as a
targeting ligand, e.g., for the purpose of biasing differentiation
in target cells that receive the nanoparticle. For example, the
following signaling proteins (e.g., extracellular signaling
proteins) can be used for the following purposes: for driving
differentiation of a hematopoietic stem cell toward a common
lymphoid progenitor cell lineage: IL-7 (see, e.g., NCBI Gene ID
3574); for driving differentiation of a hematopoietic stem cell
toward a common myeloid progenitor cell lineage: IL-3 (see, e.g.,
NCBI Gene ID 3562), GM-CSF (see, e.g., NCBI Gene ID 1437), and/or
M-CSF (see, e.g., NCBI Gene ID 1435); for driving differentiation
of a common lymphoid progenitor cell toward a B-cell fate: IL-3,
IL-4 (see, e.g., NCBI Gene ID: 3565), and/or IL-7; for driving
differentiation of a common lymphoid progenitor cell toward a
Natural Killer Cell fate: IL-15 (see, e.g., NCBI Gene ID 3600); for
driving differentiation of a common lymphoid progenitor cell toward
a T-cell fate: IL-2 (see, e.g., NCBI Gene ID 3558), IL-7, and/or
Notch (see, e.g., NCBI Gene IDs 4851, 4853, 4854, 4855); for
driving differentiation of a common lymphoid progenitor cell toward
a dendritic cell fate: Flt-3 ligand (see, e.g., NCBI Gene ID 2323);
for driving differentiation of a common myeloid progenitor cell
toward a dendritic cell fate: Flt-3 ligand, GM-CSF, and/or
TNF-alpha (see, e.g., NCBI Gene ID 7124); for driving
differentiation of a common myeloid progenitor cell toward a
granulocyte-macrophage progenitor cell lineage: GM-CSF; for driving
differentiation of a common myeloid progenitor cell toward a
megakaryocyte-erythroid progenitor cell lineage: IL-3, SCF (see,
e.g., NCBI Gene ID 4254), and/or Tpo (see, e.g., NCBI Gene ID
7173); for driving differentiation of a megakaryocyte-erythroid
progenitor cell toward a megakaryocyte fate: IL-3, IL-6 (see, e.g.,
NCBI Gene ID 3569), SCF, and/or Tpo; for driving differentiation of
a megakaryocyte-erythroid progenitor cell toward a erythrocyte
fate: erythropoietin (see, e.g., NCBI Gene ID 2056); for driving
differentiation of a megakaryocyte toward a platelet fate: IL-11
(see, e.g., NCBI Gene ID 3589) and/or Tpo; for driving
differentiation of a granulocyte-macrophage progenitor cell toward
a monocyte lineage: GM-CSF and/or M-CSF; for driving
differentiation of a granulocyte-macrophage progenitor cell toward
a myeloblast lineage: GM-CSF; for driving differentiation of a
monocyte toward a monocyte-derived dendritic cell fate: Flt-3
ligand, GM-CSF, IFN-alpha (see, e.g., NCBI Gene ID 3439), and/or
IL-4; for driving differentiation of a monocyte toward a macrophage
fate: IFN-gamma, IL-6, IL-10 (see, e.g., NCBI Gene ID 3586), and/or
M-CSF; for driving differentiation of a myeloblast toward a
neutrophil fate: G-CSF (see, e.g., NCBI Gene ID 1440), GM-CSF,
IL-6, and/or SCF; for driving differentiation of a myeloblast
toward a eosinophil fate: GM-CSF, IL-3, and/or IL-5 (see, e.g.,
NCBI Gene ID 3567); and for driving differentiation of a myeloblast
toward a basophil fate: G-CSF, GM-CSF, and/or IL-3.
[0227] Examples of proteins that can be delivered (e.g., as protein
and/or a nucleic acid such as DNA or RNA encoding the protein) in
combination with a gene editing tool include but are not limited
to: SOX17, HEX, OSKM (Oct4/Sox2/Klf4/c-myc), and/or bFGF (e.g., to
drive differentiation toward hepatic stem cell lineage); HNF4a
(e.g., to drive differentiation toward hepatocyte fate); Poly
(1:0), BMP-4, bFGF, and/or 8-Br-cAMP (e.g., to drive
differentiation toward endothelial stem cell/progenitor lineage);
VEGF (e.g., to drive differentiation toward arterial endothelium
fate); Sox-2, Brn4, Mytl1, Neurod2, Ascl1 (e.g., to drive
differentiation toward neural stem cell/progenitor lineage); and
BDNF, FCS, Forskolin, and/or SHH (e.g., to drive differentiation
neuron, astrocyte, and/or oligodendrocyte fate).
[0228] Examples of signaling proteins (e.g., extracellular
signaling proteins) that can be delivered (e.g., as protein and/or
a nucleic acid such as DNA or RNA encoding the protein) in
combination with a gene editing tool include but are not limited
to: cytokines (e.g., IL-2 and/or IL-15, e.g., for activating CD8+
T-cells); ligands and or signaling proteins that modulate one or
more of the Notch, Wnt, and/or Smad signaling pathways; SCF; stem
cell differentiating factors (e.g. Sox2, Oct3/4, Nanog, Klf4,
c-Myc, and the like); and temporary surface marker "tags" and/or
fluorescent reporters for subsequent
isolation/purification/concentration. For example, a fibroblast may
be converted into a neural stem cell via delivery of Sox2, while it
will turn into a cardiomyocyte in the presence of Oct3/4 and small
molecule "epigenetic resetting factors." In a patient with
Huntington's disease or a CXCR4 mutation, these fibroblasts may
respectively encode diseased phenotypic traits associated with
neurons and cardiac cells. By delivering gene editing corrections
and these factors in a single package, the risk of deleterious
effects due to one or more, but not all of the factors/payloads
being introduced can be significantly reduced.
[0229] Because the timing and/or location of payload release can be
controlled (described in more detail elsewhere in this disclosure),
the packaging of multiple payloads in the same package (e.g., same
nanoparticle) does not preclude one from achieving different
release times and/or locations for different payloads. For example
the release of the above proteins (and/or a DNAs or mRNAs encoding
same) and/or non-coding RNAs can be controlled separately from the
release of the one or more gene editing tools that are part of the
same package. For example, proteins and/or nucleic acids (e.g.,
DNAs, mRNAs, non-coding RNAs, miRNAs) that control cell
proliferation and/or differentiation, or that control bias toward
NHEJ or HDR, can be released earlier than the one or more gene
editing tools or can be released later than the one or more gene
editing tools. This can be achieved, e.g., by using more than one
sheddable layer and/or by using more than one core (e.g., where one
core has a different release profile than the other, e.g., uses a
different D- to L-isomer ratio, uses a different ESP:ENP:EPP
profile, and the like).
ii. Sheddable Layer (Sheddable Coat)
[0230] In some embodiments, a subject nanoparticle includes a
sheddable layer (also referred to herein as a "transient
stabilizing layer") that surrounds (encapsulates) the core. In some
cases a subject sheddable layer can protect the payload before and
during initial cellular uptake. For example, without a sheddable
layer, much of the payload can be lost during cellular
internalization. Once in the cellular environment, a sheddable
layer `sheds` (e.g., the layer can be pH- and/or or
glutathione-sensitive), exposing the components of the core.
[0231] In some cases a subject sheddable layer includes silica. In
some cases, when a subject nanoparticle includes a sheddable layer
(e.g., of silica), greater intracellular delivery efficiency can be
observed despite decreased probability of cellular uptake. Without
wishing to be bound by any particular theory, coating a
nanoparticle core with a sheddable layer (e.g., silica coating) can
seal the core, stabilizing it until shedding of the layer, which
leads to release of the payload (e.g., upon processing in the
intended subcellular compartment). Following cellular entry through
receptor-mediated endocytosis, the nanoparticle sheds its outermost
layer, the sheddable layer degrades in the acidifying environment
of the endosome or reductive environment of the cytosol, and
exposes the core, which in some cases exposes localization signals
such as nuclear localization signals (NLSs) and/or mitochondrial
localization signals. Moreover, nanoparticle cores encapsulated by
a sheddable layer can be stable in serum and can be suitable for
administration in vivo.
[0232] Any desired sheddable layer can be used, and one of ordinary
skill in the art can take into account where in the target cell
(e.g., under what conditions, such as low pH) they desire the
payload to be released (e.g., endosome, cytosol, nucleus, lysosome,
and the like). Different sheddable layers may be more desirable
depending on when, where, and/or under what conditions it would be
desirable for the sheddable coat to shed (and therefore release the
payload). For example, a sheddable layer can be acid labile. In
some cases the sheddable layer is an anionic sheddable layer (an
anionic coat). In some cases the sheddable layer comprises silica,
a peptoid, a polycysteine, and/or a ceramic (e.g., a bioceramic).
In some cases the sheddable includes one or more of: calcium,
manganese, magnesium, iron (e.g., the sheddable layer can be
magnetic, e.g., Fe.sub.3MnO.sub.2), and lithium. Each of these can
include phosphate or sulfate. As such, in some cases the sheddable
includes one or more of: calcium phosphate, calcium sulfate,
manganese phosphate, manganese sulfate, magnesium phosphate,
magnesium sulfate, iron phosphate, iron sulfate, lithium phosphate,
and lithium sulfate; each of which can have a particular effect on
how and/or under which conditions the sheddable layer will `shed.`
Thus, in some cases the sheddable layer includes one or more of:
silica, a peptoid, a polycysteine, a ceramic (e.g., a bioceramic),
calcium, calcium phosphate, calcium sulfate, manganese, manganese
phosphate, manganese sulfate, magnesium, magnesium phosphate,
magnesium sulfate, iron, iron phosphate, iron sulfate, lithium,
lithium phosphate, and lithium sulfate (in any combination thereof)
(e.g., the sheddable layer can be a coating of silica, peptoid,
polycysteine, a ceramic (e.g., a bioceramic), calcium phosphate,
calcium sulfate, manganese phosphate, manganese sulfate, magnesium
phosphate, magnesium sulfate, iron phosphate, iron sulfate, lithium
phosphate, lithium sulfate, or a combination thereof). In some
cases the sheddable layer includes silica (e.g., the sheddable
layer can be a silica coat). In some cases the sheddable layer
includes an alginate gel.
[0233] In some cases different release times for different payloads
are desirable. For example, in some cases it is desirable to
release a payload early (e.g., within 0.5-7 days of contacting a
target cell) and in some cases it is desirable to release a payload
late (e.g., within 6 days-30 days of contacting a target cell). For
example, in some cases it may be desirable to release a payload
(e.g., a gene editing tool such as a CRISPR/Cas guide RNA, a DNA
molecule encoding said CRISPR/Cas guide RNA, a CRISPR/Cas
RNA-guided polypeptide, and/or a nucleic acid molecule encoding
said CRISPR/Cas RNA-guided polypeptide) within 0.5-7 days of
contacting a target cell (e.g., within 0.5-5 days, 0.5-3 days, 1-7
days, 1-5 days, or 1-3 days of contacting a target cell). In some
cases it may be desirable to release a payload (e.g., a DNA donor
template, e.g., for homology directed repair--HDR) within 6-40 days
of contacting a target cell (e.g., within 6-30, 6-20, 6-15, 7-40,
7-30, 7-20, 7-15, 9-40, 9-30, 9-20, or 9-15 days of contacting a
target cell). In some cases release times can be controlled by
delivering nanoparticles having different payloads at different
times. In some cases release times can be controlled by delivering
nanoparticles at the same time (as part of different formulations
or as part of the same formulation), where the components of the
nanoparticle are designed to achieve the desired release times. For
example, one may use a sheddable layer that degrades faster or
slower, core components that are more or less resistant to
degradation, core components that are more or less susceptible to
de-condensation, etc.--and any or all of the components can be
selected in any convenient combination to achieve the desired
timing.
[0234] In some cases it is desirable to delay the release of a
payload (e.g., a DNA donor template) relative to another payload
(e.g., one or more gene editing tools). As an example, in some
cases a first nanoparticle includes a donor DNA template as a
payload is designed such that the payload is released within 6-40
days of contacting a target cell (e.g., within 6-30, 6-20, 6-15,
7-40, 7-30, 7-20, 7-15, 9-40, 9-30, 9-20, or 9-15 days of
contacting a target cell), while a second nanoparticle that
includes one or more gene editing tools (e.g., a ZFP or nucleic
acid encoding the ZFP, a TALE or a nucleic acid encoding the TALE,
a ZFN or nucleic acid encoding the ZFN, a TALEN or a nucleic acid
encoding the TALEN, a CRISPR/Cas guide RNA or DNA molecule encoding
the CRISPR/Cas guide RNA, a CRISPR/Cas RNA-guided polypeptide or a
nucleic acid molecule encoding the CRISPR/Cas RNA-guided
polypeptide, and the like) as a payload is designed such that the
payload is released within 0.5-7 days of contacting a target cell
(e.g., within 0.5-5 days, 0.5-3 days, 1-7 days, 1-5 days, or 1-3
days of contacting a target cell). The second nanoparticle can be
part of the same or part of a different formulation as the first
nanoparticle.
[0235] In some cases, a nanoparticle includes more than one
payload, where it is desirable for the payloads to be released at
different times. This can be achieved in a number of different
ways. For example, a nanoparticle can have more than one core,
where one core is made with components that can release the payload
early (e.g., within 0.5-7 days of contacting a target cell, e.g.,
within 0.5-5 days, 0.5-3 days, 1-7 days, 1-5 days, or 1-3 days of
contacting a target cell) (e.g., an siRNA, an mRNA, and/or a genome
editing tool such as a ZFP or nucleic acid encoding the ZFP, a TALE
or a nucleic acid encoding the TALE, a ZFN or nucleic acid encoding
the ZFN, a TALEN or a nucleic acid encoding the TALEN, a CRISPR/Cas
guide RNA or DNA molecule encoding the CRISPR/Cas guide RNA, a
CRISPR/Cas RNA-guided polypeptide or a nucleic acid molecule
encoding the CRISPR/Cas RNA-guided polypeptide, and the like) and
the other is made with components that can release the payload
(e.g., a DNA donor template) later (e.g., within 6-40 days of
contacting a target cell, e.g., within 6-30, 6-20, 6-15, 7-40,
7-30, 7-20, 7-15, 9-40, 9-30, 9-20, or 9-15 days of contacting a
target cell).
[0236] As another example, a nanoparticle can include more than one
sheddable layer, where the outer sheddable layer is shed (releasing
a payload) prior to an inner sheddable layer being shed (releasing
another payload). In some cases, the inner payload is a DNA donor
template (e.g., for homology directed repair--HDR) and the outer
payload is one or more gene editing tools (e.g., a ZFP or nucleic
acid encoding the ZFP, a TALE or a nucleic acid encoding the TALE,
a ZFN or nucleic acid encoding the ZFN, a TALEN or a nucleic acid
encoding the TALEN, a CRISPR/Cas guide RNA or DNA molecule encoding
the CRISPR/Cas guide RNA, a CRISPR/Cas RNA-guided polypeptide or a
nucleic acid molecule encoding the CRISPR/Cas RNA-guided
polypeptide, and the like). The inner and outer payloads can be any
desired payload and either or both can include, for example, one or
more siRNAs and/or one or more mRNAs. As such, in some cases a
nanoparticle can have more than one sheddable layer and can be
designed to release one payload early (e.g., within 0.5-7 days of
contacting a target cell, e.g., within 0.5-5 days, 0.5-3 days, 1-7
days, 1-5 days, or 1-3 days of contacting a target cell) (e.g., an
siRNA, an mRNA, a genome editing tool such as a ZFP or nucleic acid
encoding the ZFP, a TALE or a nucleic acid encoding the TALE, a ZFN
or nucleic acid encoding the ZFN, a TALEN or a nucleic acid
encoding the TALEN, a CRISPR/Cas guide RNA or DNA molecule encoding
the CRISPR/Cas guide RNA, a CRISPR/Cas RNA-guided polypeptide or a
nucleic acid molecule encoding the CRISPR/Cas RNA-guided
polypeptide, and the like), and another payload (e.g., an siRNA, an
mRNA, a DNA donor template) later (e.g., within 6-40 days of
contacting a target cell, e.g., within 6-30, 6-20, 6-15, 7-40,
7-30, 7-20, 7-15, 9-40, 9-30, 9-20, or 9-15 days of contacting a
target cell).
[0237] In some embodiments (e.g., in embodiments described above),
time of altered gene expression can be used as a proxy for the time
of payload release. As an illustrative example, if one desires to
determine if a payload has been released by day 12, one can assay
for the desired result of nanoparticle delivery on day 12. For
example, if the desired result was to reduce the expression of a
target gene of the target cell, e.g., by delivering an siRNA, then
the expression of the target gene can be assayed/monitored to
determine if the siRNA has been released. As another example, if
the desired result was to express a protein of interest, e.g., by
delivering a DNA or mRNA encoding the protein of interest, then the
expression of the protein of interest can be assayed/monitored to
determine if the payload has been released. As yet another example,
if the desired result was to alter the genome of the target cell,
e.g., via cleaving genomic DNA and/or inserting a sequence of a
donor DNA template, the expression from the targeted locus and/or
the presence of genomic alterations can be assayed/monitored to
determine if the payload has been released.
[0238] As such, in some cases a sheddable layer provides for a
staged release of nanoparticle components. For example, in some
cases, a nanoparticle has more than one (e.g., two, three, or four)
sheddable layers. For example, for a nanoparticle with two
sheddable layers, such a nanoparticle can have, from inner-most to
outer-most: a core, e.g., with a first payload; a first sheddable
layer, an intermediate layer e.g., with a second payload; and a
second sheddable layer surrounding the intermediate layer (see,
e.g., FIG. 9). Such a configuration (multiple sheddable layers)
facilitates staged release of various desired payloads. As a
further illustrative example, a nanoparticle with two sheddable
layers (as described above) can include one or more desired gene
editing tools in the core (e.g., one or more of: a DNA donor
template, a CRISPR/Cas guide RNA, a DNA encoding a CRISPR/Cas guide
RNA, and the like), and another desired gene editing tool in the
intermediate layer (e.g., one or more of: a programmable gene
editing protein such as a CRISPR/Cas protein, a ZFP, a ZFN, a TALE,
a TALEN, etc.; a DNA or RNA encoding a programmable gene editing
protein; a CRISPR/Cas guide RNA; a DNA encoding a CRISPR/Cas guide
RNA; and the like)--in any desired combination.
[0239] An example of adding a sheddable layer (e.g., two solutions
passed through a microfluidic mixing chip with the appropriate
residence time (flowrate)) can be found in the Examples
section.
Alternative Packaging (e.g., Lipid Formulations)
[0240] In some embodiments, a subject core (e.g., including any
combination of components and/or configurations described above) is
part of a lipid-based delivery system, e.g., a cationic lipid
delivery system (see, e.g., Chesnoy and Huang, Annu Rev Biophys
Biomol Struct. 2000, 29:27-47; Hirko et al., Curr Med Chem. 2003
Jul. 10(14):1185-93; and Liu et al., Curr Med Chem. 2003 Jul.
10(14):1307-15). In some cases a subject core (e.g., including any
combination of components and/or configurations described above) is
not surrounded by a sheddable layer. As noted above a core can
include an anionic polymer composition (e.g., poly(glutamic acid)),
a cationic polymer composition (e.g., poly(arginine), a cationic
polypeptide composition (e.g., a histone tail peptide), and a
payload (e.g., nucleic acid and/or protein payload).
[0241] In some cases in which the core is part of a lipid-based
delivery system, the core was designed with timed and/or positional
release in mind. For example, in some cases the core includes ESPs,
ENPs, and/or EPPs, and in some such cases these components are
present at ratios such that payload release is delayed until a
desired condition (e.g., cellular location, cellular condition such
as pH, presence of a particular enzyme, and the like) is
encountered by the core (e.g., described above). In some such
embodiments the core includes polymers of D-isomers of an anionic
amino acid and polymers of L-isomers of an anionic amino acid, and
in some cases the polymers of D- and L- isomers are present,
relative to one another, within a particular range of ratios (e.g.,
described above). In some cases the core includes polymers of
D-isomers of a cationic amino acid and polymers of L-isomers of a
cationic amino acid, and in some cases the polymers of D- and L-
isomers are present, relative to one another, within a particular
range of ratios (e.g., described above). In some cases the core
includes polymers of D-isomers of an anionic amino acid and
polymers of L-isomers of a cationic amino acid, and in some cases
the polymers of D- and L-isomers are present, relative to one
another, within a particular range of ratios (e.g., described
above). In some cases the core includes polymers of L-isomers of an
anionic amino acid and polymers of D-isomers of a cationic amino
acid, and in some cases the polymers of D- and L-isomers are
present, relative to one another, within a particular range of
ratios (e.g., described above). In some cases the core includes a
protein that includes an NLS (e.g., described above). In some cases
the core includes an HTP (e.g., described above).
[0242] Cationic lipids are nonviral vectors that can be used for
gene delivery and have the ability to condense plasmid DNA. After
synthesis of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium
chloride for lipofection, improving molecular structures of
cationic lipids has been an active area, including head group,
linker, and hydrophobic domain modifications. Modifications have
included the use of multivalent polyamines, which can improve DNA
binding and delivery via enhanced surface charge density, and the
use of sterol-based hydrophobic groups such as
3B--[N--(N',N'-dimethylaminoethane)-carbamoyl] cholesterol, which
can limit toxicity. Helper lipids such as dioleoyl
phosphatidylethanolamine (DOPE) can be used to improve transgene
expression via enhanced liposomal hydrophobicity and hexagonal
inverted-phase transition to facilitate endosomal escape. In some
cases a lipid formulation includes one or more of: DLin-DMA,
DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, C12-200, a
cholesterol a PEG-lipid, a lipiopolyamine, dexamethasone-spermine
(DS), and disubstituted spermine (D.sub.2S) (e.g., resulting from
the conjugation of dexamethasone to polyamine spermine). DLin-DMA,
DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 and DLin-MC3-DMA can be
synthesized by methods outlined in the art (see, e.g., Heyes et.
al, J. Control Release, 2005, 107, 276-287; Semple et. al, Nature
Biotechnology, 2010, 28, 172-176; Akinc et. al, Nature
Biotechnology, 2008, 26, 561-569; Love et. al, PNAS, 2010, 107,
1864-1869; international patent application publication
WO2010054401; all of which are hereby incorporated by reference in
their entirety.
[0243] Examples of various lipid-based delivery systems include,
but are not limited to those described in the following
publications: international patent publication No. WO2016081029;
U.S. patent application publication Nos. US20160263047 and
US20160237455; and U.S. Pat. Nos. 9,533,047; 9,504,747; 9,504,651;
9,486,538; 9,393,200; 9,326,940; 9,315,828; and 9,308,267; all of
which are hereby incorporated by reference in their entirety.
[0244] As such, in some cases a subject core is surrounded by a
lipid (e.g., a cationic lipid such as a LIPOFECTAMINE transfection
reagent). In some cases a subject core is present in a lipid
formulation (e.g., a lipid nanoparticle formulation). A lipid
formulation can include a liposome and/or a lipoplex. A lipid
formulation can include a Spontaneous Vesicle Formation by Ethanol
Dilution (SNALP) liposome (e.g., one that includes cationic lipids
together with neutral helper lipids which can be coated with
polyethylene glycol (PEG) and/or protamine).
[0245] A lipid formulation can be a lipidoid-based formulation. The
synthesis of lipidoids has been extensively described and
formulations containing these compounds can be included in a
subject lipid formulation (see, e.g., Mahon et al., Bioconjug Chem.
2010 21:1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21;
Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al., Proc
Natl Acad Sci USA. 2010 107:1864-1869; and Siegwart et al., Proc
Natl Acad Sci USA. 2011 108:12996-3001; all of which are
incorporated herein by reference in their entirety). In some cases
a subject lipid formulation can include one or more of (in any
desired combination): 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine
(DOPC); 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE);
N-[1-(2,3-Dioleyloxy)prophyl]N,N,N-trimethylammonium chloride
(DOTMA); 1,2-Dioleoyloxy-3-trimethylammonium-propane (DOTAP);
Dioctadecylamidoglycylspermine (DOGS);
N-(3-Aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1 (GAP-DLRIE);
propanaminium bromide; cetyltrimethylammonium bromide (CTAB);
6-Lauroxyhexyl ornithinate (LHON);
Dioleoyloxypropyl)-2,4,6-trimethylpyridinium (20c);
2,3-Dioleyloxy-N-[2(sperminecarboxamido-ethyl]-N,N-dimethyl-1
(DOSPA); propanaminium trifluoroacetate;
1,2-Dioleyl-3-trimethylammonium-propane (DOPA);
N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1 (MDRIE);
propanaminium bromide; dimyristooxypropyl dimethyl hydroxyethyl
ammonium bromide (DMRI);
3.beta.-[N--(N',N'-Dimethylaminoethane)-carbamoyl]cholesterol
DC-Chol; bis-guanidium-tren-cholesterol (BGTC);
1,3-Diodeoxy-2-(6-carboxy-spermyl)-propylamide (DOSPER);
Dimethyloctadecylammonium bromide (DDAB);
Dioctadecylamidoglicylspermidin (DSL);
rac-[(2,3-Dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium
(CLIP-1); chloride rac-[2(2,3-Dihexadecyloxypropyl (CLIP-6);
oxymethyloxy)ethyl]trimethylammonium bromide;
ethyldimyristoylphosphatidylcholine (EDMPC);
1,2-Distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA);
1,2-Dimyristoyl-trimethylammonium propane (DMTAP);
O,O'-Dimyristyl-N-lysyl aspartate (DMKE);
1,2-Distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC);
N-Palmitoyl D-erythro-sphingosyl carbamoyl-spermine (CCS);
N-t-Butyl-N0-tetradecyl-3-tetradecylaminopropionamidine;
diC14-amidine; octadecenolyoxy[ethyl-2-heptadecenyl-3 hydroxyethyl]
imidazolinium (DOTIM); chloride
N1-Cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN);
2-[3-[bis(3-aminopropyl)amino]propylamino]-N-[2-[di(tetradecyl)am-
ino]-2-oxoethyl]acetamide (RPR209120);
ditetradecylcarbamoylme-ethyl-acetamide;
1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA);
2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane; DLin-KC2-DMA;
dilinoleyl-methyl-4-dimethylaminobutyrate; DLin-MC3-DMA;
DLin-K-DMA; 98N12-5; C12-200; a cholesterol; a PEG-lipid; a
lipiopolyamine; dexamethasone-spermine (DS); and disubstituted
spermine (D.sub.2S).
iii. Surface Coat (Outer Shell)
[0246] In some cases, the sheddable layer (the coat), is itself
coated by an additional layer, referred to herein as an "outer
shell," "outer coat," or "surface coat." A surface coat can serve
multiple different functions. For example, a surface coat can
increase delivery efficiency and/or can target a subject
nanoparticle to a particular cell type. The surface coat can
include a peptide, a polymer, or a ligand-polymer conjugate. The
surface coat can include a targeting ligand. For example, an
aqueous solution of one or more targeting ligands (with or without
linker domains) can be added to a coated nanoparticle suspension
(suspension of nanoparticles coated with a sheddable layer). For
example, in some cases the final concentration of protonated
anchoring residues (of an anchoring domain) is between 25 and 300
.mu.M. In some cases, the process of adding the surface coat yields
a monodispersed suspension of particles with a mean particle size
between 50 and 150 nm and a zeta potential between 0 and -10
mV.
[0247] In some cases, the surface coat interacts electrostatically
with the outermost sheddable layer. For example, in some cases, a
nanoparticle has two sheddable layers (e.g., from inner-most to
outer-most: a core, e.g., with a first payload; a first sheddable
layer, an intermediate layer e.g., with a second payload; and a
second sheddable layer surrounding the intermediate layer), and the
outer shell (surface coat) can interact with (e.g.,
electrostatically) the second sheddable layer. In some cases, a
nanoparticle has only one sheddable layer (e.g., an anionic silica
layer), and the outer shell can in some cases electrostatically
interact with the sheddable layer.
[0248] Thus, in cases where the sheddable layer (e.g., outermost
sheddable layer) is anionic (e.g., in some cases where the
sheddable layer is a silica coat), the surface coat can interact
electrostatically with the sheddable layer if the surface coat
includes a cationic component. For example, in some cases the
surface coat includes a delivery molecule in which a targeting
ligand is conjugated to a cationic anchoring domain. The cationic
anchoring domain interacts electrostatically with the sheddable
layer and anchors the delivery molecule to the nanoparticle.
Likewise, in cases where the sheddable layer (e.g., outermost
sheddable layer) is cationic, the surface coat can interact
electrostatically with the sheddable layer if the surface coat
includes an anionic component.
[0249] In some embodiments, the surface coat includes a cell
penetrating peptide (CPP). In some cases, a polymer of a cationic
amino acid can function as a CPP (also referred to as a `protein
transduction domain`--PTD), which is a term used to refer to a
polypeptide, polynucleotide, carbohydrate, or organic or inorganic
compound that facilitates traversing a lipid bilayer, micelle, cell
membrane, organelle membrane, or vesicle membrane. A PTD attached
to another molecule (e.g., embedded in and/or interacting with a
sheddable layer of a subject nanoparticle), which can range from a
small polar molecule to a large macromolecule and/or a
nanoparticle, facilitates the molecule traversing a membrane, for
example going from extracellular space to intracellular space, or
cytosol to within an organelle (e.g., the nucleus).
[0250] Examples of CPPs include but are not limited to a minimal
undecapeptide protein transduction domain (corresponding to
residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR (SEQ ID NO:
160); a polyarginine sequence comprising a number of arginines
sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9,
10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer
Gene Ther. 9(6):489-96); an Drosophila Antennapedia protein
transduction domain (Noguchi et al. (2003) Diabetes
52(7):1732-1737); a truncated human calcitonin peptide (Trehin et
al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al.
(2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR
(SEQ ID NO: 161); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID
NO: 162); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 163); and
RQIKIWFQNRRMKWKK (SEQ ID NO: 164). Example CPPs include but are not
limited to: YGRKKRRQRRR (SEQ ID NO: 160), RKKRRQRRR (SEQ ID NO:
165), an arginine homopolymer of from 3 arginine residues to 50
arginine residues, RKKRRQRR (SEQ ID NO: 166), YARAAARQARA (SEQ ID
NO: 167), THRLPRRRRRR (SEQ ID NO: 168), and GGRRARRRRRR (SEQ ID NO:
169). In some embodiments, the CPP is an activatable CPP (ACPP)
(Aguilera et al. (2009) lntegr Biol (Camb) June; 1(5-6): 371-381).
ACPPs comprise a polycationic CPP (e.g., Arg9 or "R9") connected
via a cleavable linker to a matching polyanion (e.g., Glu9 or
"E9"), which reduces the net charge to nearly zero and thereby
inhibits adhesion and uptake into cells. Upon cleavage of the
linker, the polyanion is released, locally unmasking the
polyarginine and its inherent adhesiveness, thus "activating" the
ACPP to traverse the membrane
[0251] In some cases a CPP can be added to the nanoparticle by
contacting a coated core (a core that is surrounded by a sheddable
layer) with a composition (e.g., solution) that includes the CPP.
The CPP can then interact with the sheddable layer (e.g.,
electrostatically).
[0252] In some cases, the surface coat includes a polymer of a
cationic amino acid (e.g., a poly(arginine) such as
poly(L-arginine) and/or poly(D-arginine), a poly(lysine) such as
poly(L-lysine) and/or poly(D-lysine), a poly(histidine) such as
poly(L- histidine) and/or poly(D-histidine), a poly(ornithine) such
as poly(L-ornithine) and/or poly(D-ornithine), poly(citrulline)
such as poly(L-citrulline) and/or poly(D-citrulline), and the
like). As such, in some cases the surface coat includes
poly(arginine), e.g., poly(L-arginine).
[0253] In some embodiments, the surface coat includes a
heptapeptide such as selank (TKPRPGP--SEQ ID NO: 147) (e.g.,
N-acetyl selank) and/or semax (MEHFPGP--SEQ ID NO: 148) (e.g.,
N-acetyl semax). As such, in some cases the surface coat includes
selank (e.g., N-acetyl selank). In some cases the surface coat
includes semax (e.g., N-acetyl semax).
[0254] In some embodiments the surface coat includes a delivery
molecule. A delivery molecule includes a targeting ligand and in
some cases the targeting ligand is conjugated to an anchoring
domain (e.g. a cationic anchoring domain). In some case a targeting
ligand is conjugated to an anchoring domain (e.g. a cationic
anchoring domain) via an intervening linker.
Targeting Ligand
[0255] A variety of targeting ligands (e.g., as part of a subject
delivery molecule) can be used as part of a surface coat, and
numerous different targeting ligands are envisioned. In some
embodiments the targeting ligand is a fragment (e.g., a binding
domain) of a wild type protein. For example, in some cases the
peptide targeting ligand of a subject delivery molecule can have a
length of from 4-50 amino acids (e.g., from 4-40, 4-35, 4-30, 4-25,
4-20, 4-15, 5-50, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 7-50, 7-40,
7-35, 7-30, 7-25, 7-20, 7-15, 8-50, 8-40, 8-35, 8-30, 8-25, 8-20,
or 8-15 amino acids). The targeting ligand can be a fragment of a
wild type protein, but in some cases has a mutation (e.g.,
insertion, deletion, substitution) relative to the wild type amino
acid sequence (i.e., a mutation relative to a corresponding wild
type protein sequence). For example, a targeting ligand can include
a mutation that increases or decreases binding affinity with a
target cell surface protein.
[0256] In some cases the targeting ligand is an antigen-binding
region of an antibody (e.g., an ScFv). "Fv" is the minimum antibody
fragment which contains a complete antigen-recognition and -binding
site. In a two-chain Fv species, this region consists of a dimer of
one heavy- and one light-chain variable domain in tight,
non-covalent association. In a single-chain Fv species (scFv), one
heavy- and one light-chain variable domain can be covalently linked
by a flexible peptide linker such that the light and heavy chains
can associate in a "dimeric" structure analogous to that in a
two-chain Fv species. For a review of scFv see Pluckthun, in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
[0257] In some cases a targeting ligand includes a viral
glycoprotein, which in some cases binds to ubiqituous surface
markers such as heparin sulfate proteoglycans, and may induce
micropinocytosis in some cell populations through membrane ruffling
associated processes. Poly(L-arginine) is another example targeting
ligand that can also be used for binding to surface markers such as
heparin sulfate proteoglycans.
[0258] In some cases, a targeting ligand can include a mutation
that adds a cysteine residue, which can facilitate conjugation to a
linker and/or an anchoring domain (e.g., cationic anchoring
domain). For example, cysteine can be used for crosslinking
(conjugation) via sulfhydryl chemistry (e.g., a disulfide bond)
and/or amine-reactive chemistry.
[0259] In some cases, a targeting ligand includes an internal
cysteine residue. In some cases, a targeting ligand includes a
cysteine residue at the N- and/or C-terminus. In some cases, in
order to include a cysteine residue, a targeting ligand is mutated
(e.g., insertion or substitution), e.g., relative to a
corresponding wild type sequence. As such, any of the targeting
ligands described herein can be modified by inserting and/or
substituting in a cysteine residue (e.g., internal, N-terminal,
C-terminal insertion of or substitution with a cysteine
residue).
[0260] By "corresponding" wild type sequence is meant a wild type
sequence from which the subject sequence was or could have been
derived (e.g., a wild type protein sequence having high sequence
identity to the sequence of interest). For example, for a targeting
ligand that has one or more mutations (e.g., substitution,
insertion) but is otherwise highly similar to a wild type sequence,
the amino acid sequence to which it is most similar may be
considered to be a corresponding wild type amino acid sequence.
[0261] A corresponding wild type protein/sequence does not have to
be 100% identical (e.g., can be 85% or more identical, 90% or more
identical, 95% or more identical, 98% or more identical, 99% or
more identical, etc.) (outside of the position(s) that is
modified), but the targeting ligand and corresponding wild type
protein (e.g., fragment of a wild protein) can bind to the intended
cell surface protein, and retain enough sequence identity (outside
of the region that is modified) that they can be considered
homologous. The amino acid sequence of a "corresponding" wild type
protein sequence can be identified/evaluated using any convenient
method (e.g., using any convenient sequence comparison/alignment
software such as BLAST, MUSCLE, T-COFFEE, etc.).
[0262] Examples of targeting ligands that can be used as part of a
surface coat (e.g., as part of a delivery molecule of a surface
coat) include, but are not limited to, those listed in Table 1.
Examples of targeting ligands that can be used as part of a subject
delivery molecule include, but are not limited to, those listed in
Table 3 (many of the sequences listed in Table 3 include the
targeting ligand (e.g., SNRWLDVK for row 2) conjugated to a
cationic polypeptide domain, e.g., 9R, 6R, etc., via a linker
(e.g., GGGGSGGGGS). Examples of amino acid sequences that can be
included in a targeting ligand include, but are not limited to:
NPKLTRMLTFKFY (SEQ ID NO: xx) (IL2), TSVGKYPNTGYYGD (SEQ ID NO: xx)
(CD3), SNRWLDVK (Siglec), EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF);
EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF), EKFILKVRPAFKAV (SEQ ID NO:
xx) (SCF), SNYSIIDKLVNIVDDLVECVKENS (SEQ ID NO: xx) (cKit), and
Ac-SNYSAibADKAibANAibADDAibAEAibAKENS (SEQ ID NO: xx) (cKit). Thus
in some cases a targeting ligand includes an amino acid sequence
that has 85% or more (e.g., 90% or more, 95% or more, 98% or more,
99% or more, or 100%) sequence identity with NPKLTRMLTFKFY (SEQ ID
NO: xx) (IL2), TSVGKYPNTGYYGD (SEQ ID NO: xx) (CD3), SNRWLDVK
(Siglec), EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF); EKFILKVRPAFKAV (SEQ
ID NO: xx) (SCF), EKFILKVRPAFKAV (SEQ ID NO: xx) (SCF), or
SNYSIIDKLVNIVDDLVECVKENS (SEQ ID NO: xx) (cKit).
TABLE-US-00004 TABLE 1 Examples of Targeting ligands Cell Surface
SEQ Protein Targeting Ligand Sequence ID NO: Family B GPCR Exendin
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS 1 Exendin (S11C)
HGEGTFTSDLCKQMEEEAVRLFIEWLKNGGPSSGAPPPS 2 FGF receptor FGF fragment
KRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEE 3
RGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERL ESNNYNTY FGF fragment
KNGGFFLRIHPDGRVDGVREKS 4 FGF fragment HFKDPK 5 FGF fragment
LESNNYNT 6 E-selectin MIASQFLSALTLVLLIKESGA 7 L-selectin
MVFPWRCEGTYWGSRNILKLWVWTLLCCDFLIHHGTHC 8
MIFPWKCQSTQRDLWNIFKLWGWTMLCCDFLAHHGTDC 9
MIFPWKCQSTQRDLWNIFKLWGWTMLCC 10 P-selectin PSGL-1
MAVGASGLEGDKMAGAMPLQLLLLLILLGPGNSLQLWDT 271 (SELPLG)
WADEAEKALGPLLARDRRQATEYEYLDYDFLPETEPPEM
LRNSTDTTPLTGPGTPESTTVEPAARRSTGLDAGGAVTE
LTTELANMGNLSTDSAAMEIQTTQPAATEAQTTQPVPTE
AQTTPLAATEAQTTRLTATEAQTTPLAATEAQTTPPAAT
EAQTTQPTGLEAQTTAPAAMEAQTTAPAAMEAQTTPPAA
MEAQTTQTTAMEAQTTAPEATEAQTTQPTATEAQTTPLA
AMEALSTEPSATEALSMEPTTKRGLFIPFSVSSVTHMGI
PMAASNLSVNYPVGAPDHISVKQCLLAILILALVATIFF
VCTVVLAVRLSRKGHMYPVRNYSPTEMVCISSLLPDGGE
GPSATANGGLSKAKSPGLTPEPREDREGDDLTLHSFLP E-selectin ESL-1
MAACGRVRRMFRLSAALHLLLLFAAGAEKLPGQGVHSQG 272 (GLG1)
QGPGANFVSFVGQAGGGGPAGQQLPQLPQSSQLQQQQQQ
QQQQQQPQPPQPPFPAGGPPARRGGAGAGGGWKLAEEES
CREDVTRVCPKHTWSNNLAVLECLQDVREPENEISSDCN
HLLWNYKLNLTTDPKFESVAREVCKSTITEIKECADEPV
GKGYMVSCLVDHRGNITEYQCHQYITKMTAIIFSDYRLI
CGFMDDCKNDINILKCGSIRLGEKDAHSQGEVVSCLEKG
LVKEAEEREPKIQVSELCKKAILRVAELSSDDFHLDRHL
YFACRDDRERFCENTQAGEGRVYKCLFNHKFEESMSEKC
REALTTRQKLIAQDYKVSYSLAKSCKSDLKKYRCNVENL
PRSREARLSYLLMCLESAVHRGRQVSSECQGEMLDYRRM
LMEDFSLSPEIILSCRGEIEHHCSGLHRKGRTLHCLMKV
VRGEKGNLGMNCQQALQTLIQETDPGADYRIDRALNEAC
ESVIQTACKHIRSGDPMILSCLMEHLYTEKMVEDCEHRL
LELQYFISRDWKLDPVLYRKCQGDASRLCHTHGWNETSE
FMPQGAVFSCLYRHAYRTEEQGRRLSRECRAEVQRILHQ
RAMDVKLDPALQDKCLIDLGKWCSEKTETGQELECLQDH
LDDLVVECRDIVGNLTELESEDIQIEALLMRACEPIIQN
FCHDVADNQIDSGDLMECLIQNKHQKDMNEKCAIGVTHF
QLVQMKDFRFSYKFKMACKEDVLKLCPNIKKKVDVVICL
STTVRNDTLQEAKEHRVSLKCRRQLRVEELEMTEDIRLE
PDLYEACKSDIKNFCSAVQYGNAQIIECLKENKKQLSTR
CHQKVFKLQETEMMDPELDYTLMRVCKQMIKRFCPEADS
KTMLQCLKQNKNSELMDPKCKQMITKRQITQNTDYRLNP
MLRKACKADIPKFCHGILTKAKDDSELEGQVISCLKLRY
ADQRLSSDCEDQIRIIIQESALDYRLDPQLQLHCSDEIS
SLCAEEAAAQEQTGQVEECLKVNLLKIKTELCKKEVLNM
LKESKADIFVDPVLHTACALDIKHHCAAITPGRGRQMSC
LMEALEDKRVRLQPECKKRLNDRIEMWSYAAKVAPADGF
SDLAMQVMTSPSKNYILSVISGSICILFLIGLMCGRITK
RVTRELKDRLQYRSETMAYKGLVWSQDVTGSPA PSGL-1 See above 271 (SELPLG)
CD44 MDKFWWHAAWGLCLVPLSLAQIDLNITCRFAGVFHVEKN 273
GRYSISRTEAADLCKAFNSTLPTMAQMEKALSIGFETCR
YGFIEGHVVIPRIHPNSICAANNTGVYILTSNTSQYDTY
CFNASAPPEEDCTSVTDLPNAFDGPITITIVNRDGTRYV
QKGEYRTNPEDIYPSNPTDDDVSSGSSSERSSTSGGYIF
YTFSTVHPIPDEDSPWITDSTDRIPATTLMSTSATATET
ATKRQETWDWFSWLFLPSESKNHLHTTTQMAGTSSNTIS
AGWEPNEENEDERDRHLSFSGSGIDDDEDFISSTISTTP
RAFDHTKQNQDWTQWNPSHSNPEVLLQTTTRMTDVDRNG
TTAYEGNWNPEAHPPLIHHEHHEEEETPHSTSTIQATPS
STTEETATQKEQWFGNRWHEGYRQTPKEDSHSTTGTAAA
SHATSHPMQGRTTPSPSDSSWNTDFFNPISHPMGRGHQA
GRRMDMDSSHSITLQPTANPNTGLVEDLDRTGPLSMTTQ
QSNSQSFSTSHEGLEEDKDHPTTSTLTSSNRNDVTGGRR
DPNHSEGSTTLLEGYTSHYPHTKESRTFIPVTSAKGTGS
FGVTAVTVGDSNSNVNRSLSGDQDTFHPSGGSHTTHGSE
SDGHSHGSQEGGANTTSGPIRTPQIPEWLIILASLLALA
LILAVCIAVNSRRRCGQKKKLVINSGNGAVEDRKPSGLN
GEASKSQEMVHLVNKESSETPDQFMTADETRNLQNVDMK IGV DR3
MEQRPRGCAAVAAALLLVLLGARAQGGTRSPRCDCAGDF 274 (TNFRSF25)
HKKIGLFCCRGCPAGHYLKAPCTEPCGNSTCLVCPQDTF
LAWENHHNSECARCQACDEQASQVALENCSAVADTRCGC
KPGWFVECQVSQCVSSSPFYCQPCLDCGALHRHTRLLCS
RRDTDCGTCLPGFYEHGDGCVSCPTPPPSLAGAPWGAVQ
SAVPLSVAGGRVGVFWVQVLLAGLVVPLLLGATLTYTYR
HCWPHKPLVTADEAGMEALTPPPATHLSPLDSAHTLLAP
PDSSEKICTVQLVGNSWTPGYPETQEALCPQVTWSWDQL
PSRALGPAAAPTLSPESPAGSPAMMLQPGPQLYDVMDAV
PARRWKEFVRTLGLREAEIEAVEVEIGRFRDQQYEMLKR
WRQQQPAGLGAVYAALERMGLDGCVEDLRSRLQRGP LAMP1
MAAPGSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGT 275
ACIMANFSAAFSVNYDTKSGPKNMTFDLPSDATVVLNRS
SCGKENTSDPSLVIAFGRGHTLTLNFTRNATRYSVQLMS
FVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCV
SGTQVHMNNVTVTLHDATIQAYLSNSSFSRGETRCEQDR
PSPTTAPPAPPSPSPSPVPKSPSVDKYNVSGTNGTCLLA
SMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHL
VTLELHSEGTTVLLFQFGMNASSSRFFLQGIQLNTILPD
ARDPAFKAANGSLRALQATVGNSYKCNAEEHVRVTKAFS
VNIFKVWVQAFKVEGGQFGSVEECLLDENSMLIPIAVGG ALAGLVLIVLIAYLVGRKRSHAGYQTI
LAMP2 MVCFRLFPVPGSGLVLVCLVLGAVRSYALELNLTDSENA 276
TCLYAKWQMNFTVRYETTNKTYKTVTISDHGTVTYNGSI
CGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFS
YNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCNSL
STLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTS
TVAPTIHTTVPSPTTTPTPKEKPEAGTYSVNNGNDTCLL
ATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLR
LNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFS
IANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLR
VQPFNVTQGKYSTAQDCSADDDNFLVPIAVGAALAGVLI LVLLAYFIGLKHHHAGYEQF
Mac2-BP MTPPRLFWVWLLVAGTQGVNDGDMRLADGGATMQGRVEI 277 (galectin 3
binding FYRGQWGTVCDLNWDLTDASVVCRALGFENATQALGRAA protein)
FGQGSGPIMLDEVQCTGTEASLADCKSLGWLKSNCRHER (LGALS3BP)
DAGVVCTNETRSTHTLDLSRELSEALGQIFDSQRGCDLS
ISVNVQGEDALGFCGHTVILTANLEAQALWKEPGSNVTM
SVDAECVPMVRDLLRYFYSRRIDITLSSVKCFHKLASAY
GARQLQGYCASLFAILLPQDPSFQMPLDLYAYAVATGDA
LLEKLCLQFLAWNFEALTQAEAWPSVPTDLLQLLLPRSD
LAVPSELALLKAVDTWSWGERASHEEVEGLVEKIRFPMM
LPEELFELQFNLSLYWSHEALFQKKTLQALEFHTVPFQL
LARYKGLNLTEDTYKPRIYTSPTWSAFVTDSSWSARKSQ
LVYQSRRGPLVKYSSDYFQAPSDYRYYPYQSFQTPQHPS
FLFQDKRVSWSLVYLPTIQSCWNYGFSCSSDELPVLGLT
KSGGSDRTIAYENKALMLCEGLFVADVTDFEGWKAAIPS
ALDTNSSKSTSSFPCPAGHFNGFRTVIRPFYLTNSSGVD Transferrin Transferrin
ligand THRPPMWSPVWP 11 receptor .alpha.5.beta.1 integrin
.alpha.5.beta.1 ligand RRETAWA 12 RGD RGDGW 181 integrin Integrin
binding (Ac)-GCGYGRGDSPG(NH2) 188 peptide GCGYGRGDSPG 182
.alpha.5.beta.3 integrin .alpha.5.beta.3 ligand DGARYCRGDCFDG 187
rabies virus YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG 183 glycoprotein
(RVG) c-Kit stem cell factor
EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVL 184 receptor (SCF)
PSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDK (CD117)
LVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFR
IFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTK PFMLPPVA CD27 CD70
PEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQR 185
FAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGP
ALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSST
TASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQR
LTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP CD150 SH2 domain-
SSGLVPRGSHMDAVAVYHGKISRETGEKLLLATGLDGSY 186 containing protein
LLRDSESVPGVYCLCVLYHGYIYTYRVSQTETSWWSAET 1A (SH2D1A)
APGVHKRYFRKIKNLISAFQKPDQGIVIPLQYPVEKKSS ARSTQGTTGIREDPDVCLKAP
[0263] A targeting ligand (e.g., of a delivery molecule) can
include the amino acid sequence RGD and/or an amino acid sequence
having 85% or more sequence identity (e.g., 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100%
sequence identity) with the amino acid sequence set forth in any
one of SEQ ID NOs: 1-12. In some cases, a targeting ligand includes
the amino acid sequence RGD and/or the amino acid sequence set
forth in any one of SEQ ID NOs: 1-12. In some embodiments, a
targeting ligand can include a cysteine (internal, C-terminal, or
N-terminal), and can also include the amino acid sequence RGD
and/or an amino acid sequence having 85% or more sequence identity
(e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, 99.5% or more, or 100% sequence identity) with the amino acid
sequence set forth in any one of SEQ ID NOs: 1-12.
[0264] A targeting ligand (e.g., of a delivery molecule) can
include the amino acid sequence RGD and/or an amino acid sequence
having 85% or more sequence identity (e.g., 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100%
sequence identity) with the amino acid sequence set forth in any
one of SEQ ID NOs: 1-12 and 181-187. In some cases, a targeting
ligand includes the amino acid sequence RGD and/or the amino acid
sequence set forth in any one of SEQ ID NOs: 1-12 and 181-187. In
some embodiments, a targeting ligand can include a cysteine
(internal, C-terminal, or N-terminal), and can also include the
amino acid sequence RGD and/or an amino acid sequence having 85% or
more sequence identity (e.g., 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, 99.5% or more, or 100% sequence
identity) with the amino acid sequence set forth in any one of SEQ
ID NOs: 1-12 and 181-187.
[0265] A targeting ligand (e.g., of a delivery molecule) can
include the amino acid sequence RGD and/or an amino acid sequence
having 85% or more sequence identity (e.g., 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100%
sequence identity) with the amino acid sequence set forth in any
one of SEQ ID NOs: 1-12, 181-187, and 271-277. In some cases, a
targeting ligand includes the amino acid sequence RGD and/or the
amino acid sequence set forth in any one of SEQ ID NOs: 1-12,
181-187, and 271-277. In some embodiments, a targeting ligand can
include a cysteine (internal, C-terminal, or N-terminal), and can
also include the amino acid sequence RGD and/or an amino acid
sequence having 85% or more sequence identity (e.g., 90% or more,
95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more,
or 100% sequence identity) with the amino acid sequence set forth
in any one of SEQ ID NOs: 1-12, 181-187, and 271-277.
[0266] In some cases, a targeting ligand (e.g., of a delivery
molecule) can include an amino acid sequence having 85% or more
sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98%
or more, 99% or more, 99.5% or more, or 100% sequence identity)
with the amino acid sequence set forth in any one of SEQ ID NOs:
181-187, and 271-277. In some cases, a targeting ligand includes
the amino acid sequence set forth in any one of SEQ ID NOs:
181-187, and 271-277. In some embodiments, a targeting ligand can
include a cysteine (internal, C-terminal, or N-terminal), and can
also include an amino acid sequence having 85% or more sequence
identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more,
99% or more, 99.5% or more, or 100% sequence identity) with the
amino acid sequence set forth in any one of SEQ ID NOs: 181-187,
and 271-277.
[0267] In some cases, a targeting ligand (e.g., of a delivery
molecule) can include an amino acid sequence having 85% or more
sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98%
or more, 99% or more, 99.5% or more, or 100% sequence identity)
with the amino acid sequence set forth in any one of SEQ ID NOs:
181-187. In some cases, a targeting ligand includes the amino acid
sequence set forth in any one of SEQ ID NOs: 181-187. In some
embodiments, a targeting ligand can include a cysteine (internal,
C-terminal, or N-terminal), and can also include an amino acid
sequence having 85% or more sequence identity (e.g., 90% or more,
95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more,
or 100% sequence identity) with the amino acid sequence set forth
in any one of SEQ ID NOs: 181-187.
[0268] In some cases, a targeting ligand (e.g., of a delivery
molecule) can include an amino acid sequence having 85% or more
sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98%
or more, 99% or more, 99.5% or more, or 100% sequence identity)
with the amino acid sequence set forth in any one of SEQ ID NOs:
271-277. In some cases, a targeting ligand includes the amino acid
sequence set forth in any one of SEQ ID NOs: 271-277. In some
embodiments, a targeting ligand can include a cysteine (internal,
C-terminal, or N-terminal), and can also include an amino acid
sequence having 85% or more sequence identity (e.g., 90% or more,
95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more,
or 100% sequence identity) with the amino acid sequence set forth
in any one of SEQ ID NOs: 271-277.
[0269] The terms "targets" and "targeted binding" are used herein
to refer to specific binding. The terms "specific binding,"
"specifically binds," and the like, refer to non-covalent or
covalent preferential binding to a molecule relative to other
molecules or moieties in a solution or reaction mixture (e.g., an
antibody specifically binds to a particular polypeptide or epitope
relative to other available polypeptides, a ligand specifically
binds to a particular receptor relative to other available
receptors). In some embodiments, the affinity of one molecule for
another molecule to which it specifically binds is characterized by
a K.sub.d (dissociation constant) of 10.sup.-5 M or less (e.g.,
10.sup.-6 M or less, 10.sup.-7 M or less, 10.sup.-8 M or less,
10.sup.-9 M or less, 10.sup.-10 M or less, 10.sup.11 M or less,
10.sup.-12 M or less, 10.sup.-13 M or less, 10.sup.-14 M or less,
10.sup.-15 M or less, or 10.sup.-16 M or less). "Affinity" refers
to the strength of binding, increased binding affinity correlates
with a lower K.sub.d.
[0270] In some cases, the targeting ligand provides for targeted
binding to a cell surface protein selected from a family B
G-protein coupled receptor (GPCR), a receptor tyrosine kinase
(RTK), a cell surface glycoprotein, and a cell-cell adhesion
molecule. Consideration of a ligand's spatial arrangement upon
receptor docking can be used to accomplish a desired functional
selectivity and endosomal sorting biases, e.g., so that the
structure function relationship between the ligand and the target
is not disrupted due to the conjugation of the targeting ligand to
the payload or anchoring domain (e.g., cationic anchoring domain).
For example, conjugation to a nucleic acid, protein,
ribonucleoprotein, or anchoring domain (e.g., cationic anchoring
domain) could potentially interfere with the binding cleft(s).
[0271] Thus, in some cases, where a crystal structure of a desired
target (cell surface protein) bound to its ligand is available (or
where such a structure is available for a related protein), one can
use 3D structure modeling and sequence threading to visualize sites
of interaction between the ligand and the target. This can
facilitate, e.g., selection of internal sites for placement of
substitutions and/or insertions (e.g., of a cysteine residue).
[0272] As an example, in some cases, the targeting ligand provides
for binding to a family B G protein coupled receptor (GPCR) (also
known as the `secretin-family`). In some cases, the targeting
ligand provides for binding to both an allosteric-affinity domain
and an orthosteric domain of the family B GPCR to provide for the
targeted binding and the engagement of long endosomal recycling
pathways, respectively (see e.g., the examples section below as
well as FIG. 11 and FIG. 12).
[0273] G-protein-coupled receptors (GPCRs) share a common molecular
architecture (with seven putative transmembrane segments) and a
common signaling mechanism, in that they interact with G proteins
(heterotrimeric GTPases) to regulate the synthesis of intracellular
second messengers such as cyclic AMP, inositol phosphates,
diacylglycerol and calcium ions. Family B (the secretin-receptor
family or `family 2`) of the GPCRs is a small but structurally and
functionally diverse group of proteins that includes receptors for
polypeptide hormones and molecules thought to mediate intercellular
interactions at the plasma membrane (see e.g., Harmar et al.,
Genome Biol. 2001; 2(12):REVIEWS3013). There have been important
advances in structural biology as relates to members of the
secretin-receptor family, including the publication of several
crystal structures of their N-termini, with or without bound
ligands, which work has expanded the understanding of ligand
binding and provides a useful platform for structure-based ligand
design (see e.g., Poyner et al., Br J Pharmacol. 2012 May;
166(1):1-3).
[0274] For example, one may desire to use a subject delivery
molecule to target the pancreatic cell surface protein GLP1R (e.g.,
to target .beta.-islets) using the Exendin-4 ligand, or a
derivative thereof (e.g., a cysteine substituted Exendin-4
targeting ligand such as that presented as SEQ ID NO: 2). Because
GLP1R is abundant within the brain and pancreas, a targeting ligand
that provides for targeting binding to GLP1R can be used to target
the brain and pancreas. Thus, targeting GLP1R facilitates methods
(e.g., treatment methods) focused on treating diseases (e.g., via
delivery of one or more gene editing tools) such as Huntington's
disease (CAG repeat expansion mutations), Parkinson's disease
(LRRK2 mutations), ALS (SOD1 mutations), and other CNS diseases.
Targeting GLP1R also facilitates methods (e.g., treatment methods)
focused on delivering a payload to pancreatic .beta.-islets for the
treatment of diseases such as diabetes mellitus type I, diabetes
mellitus type II, and pancreatic cancer (e.g., via delivery of one
or more gene editing tools).
[0275] When targeting GLP1R using a modified version of exendin-4,
an amino acid for cysteine substitution and/or insertion (e.g., for
conjugation to a nucleic acid payload) can be identified by
aligning the Exendin-4 amino acid sequence, which is
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO. 1), to crystal
structures of glucagon-GCGR (4ERS) and GLP1-GLP1R-ECD complex (PDB:
3IOL), using PDB 3 dimensional renderings, which may be rotated in
3D space in order to anticipate the direction that a cross-linked
complex must face in order not to disrupt the two binding clefts
(see e.g., the examples section below as well as FIG. 11 and FIG.
12). When a desirable cross-linking site (e.g., site for
substitution/insertion of a cysteine residue) of a targeting ligand
(that targets a family B GPCR) is sufficiently orthogonal to the
two binding clefts of the corresponding receptor, high-affinity
binding may occur as well as concomitant long endosomal recycling
pathway sequestration (e.g., for optimal payload release). The
cysteine substitution at amino acid positions 10, 11, and/or 12 of
SEQ ID NO: 1 confers bimodal binding and specific initiation of a
Gs-biased signaling cascade, engagement of beta arrestin, and
receptor dissociation from the actin cytoskeleton. In some cases,
this targeting ligand triggers internalization of the nanoparticle
via receptor-mediated endocytosis, a mechanism that is not engaged
via mere binding to the GPCR's N-terminal domain without
concomitant orthosteric site engagement (as is the case with mere
binding of the affinity strand, Exendin-4 [31-39]).
[0276] In some cases, a subject targeting ligand includes an amino
acid sequence having 85% or more (e.g., 90% or more, 95% or more,
98% or more, 99% or more, or 100%) identity to the exendin-4 amino
acid sequence (SEQ ID NO: 1). In some such cases, the targeting
ligand includes a cysteine substitution or insertion at one or more
of positions corresponding to L10, S11, and K12 of the amino acid
sequence set forth in SEQ ID NO: 1. In some cases, the targeting
ligand includes a cysteine substitution or insertion at a position
corresponding to S11 of the amino acid sequence set forth in SEQ ID
NO: 1. In some cases, a subject targeting ligand includes an amino
acid sequence having the exendin-4 amino acid sequence (SEQ ID NO:
1). In some cases, the targeting ligand is conjugated (with or
without a linker) to an anchoring domain (e.g., a cationic
anchoring domain).
[0277] As another example, in some cases a targeting ligand
according to the present disclosure provides for binding to a
receptor tyrosine kinase (RTK) such as fibroblast growth factor
(FGF) receptor (FGFR). Thus in some cases the targeting ligand is a
fragment of an FGF (i.e., comprises an amino acid sequence of an
FGF). In some cases, the targeting ligand binds to a segment of the
RTK that is occupied during orthosteric binding (e.g., see the
examples section below). In some cases, the targeting ligand binds
to a heparin-affinity domain of the RTK. In some cases, the
targeting ligand provides for targeted binding to an FGF receptor
and comprises an amino acid sequence having 85% or more sequence
identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more,
99% or more, 99.5% or more, or 100% sequence identity) with the
amino acid sequence KNGGFFLRIHPDGRVDGVREKS (SEQ ID NO: 4). In some
cases, the targeting ligand provides for targeted binding to an FGF
receptor and comprises the amino acid sequence set forth as SEQ ID
NO: 4.
[0278] In some cases, small domains (e.g., 5-40 amino acids in
length) that occupy the orthosteric site of the RTK may be used to
engage endocytotic pathways relating to nuclear sorting of the RTK
(e.g., FGFR) without engagement of cell-proliferative and
proto-oncogenic signaling cascades, which can be endemic to the
natural growth factor ligands. For example, the truncated bFGF
(tbFGF) peptide (a.a.30-115), contains a bFGF receptor binding site
and a part of a heparin-binding site, and this peptide can
effectively bind to FGFRs on a cell surface, without stimulating
cell proliferation. The sequences of tbFGF are
KRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLAS
KCVTDECFFFERLESNNYNTY (SEQ ID NO: 13) (see, e.g., Cai et al., Int J
Pharm. 2011 Apr. 15; 408(1-2):173-82).
[0279] In some cases, the targeting ligand provides for targeted
binding to an FGF receptor and comprises the amino acid sequence
HFKDPK (SEQ ID NO: 5) (see, e.g., the examples section below). In
some cases, the targeting ligand provides for targeted binding to
an FGF receptor, and comprises the amino acid sequence LESNNYNT
(SEQ ID NO: 6) (see, e.g., the examples section below).
[0280] In some cases, a targeting ligand according to the present
disclosure provides for targeted binding to a cell surface
glycoprotein. In some cases, the targeting ligand provides for
targeted binding to a cell-cell adhesion molecule. For example, in
some cases, the targeting ligand provides for targeted binding to
CD34, which is a cell surface glycoprotein that functions as a
cell-cell adhesion factor, and which is protein found on
hematopoietic stem cells (e.g., of the bone marrow). In some cases,
the targeting ligand is a fragment of a selectin such as
E-selectin, L-selectin, or P-selectin (e.g., a signal peptide found
in the first 40 amino acids of a selectin). In some cases a subject
targeting ligand includes sushi domains of a selectin (e.g.,
E-selectin, L-selectin, P-selectin).
[0281] In some cases, the targeting ligand comprises an amino acid
sequence having 85% or more sequence identity (e.g., 90% or more,
95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more,
or 100% sequence identity) with the amino acid sequence
MIASQFLSALTLVLLIKESGA (SEQ ID NO: 7). In some cases, the targeting
ligand comprises the amino acid sequence set forth as SEQ ID NO: 7.
In some cases, the targeting ligand comprises an amino acid
sequence having 85% or more sequence identity (e.g., 90% or more,
95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more,
or 100% sequence identity) with the amino acid sequence
MVFPWRCEGTYWGSRNILKLWVWTLLCCDFLIHHGTHC (SEQ ID NO: 8). In some
cases, the targeting ligand comprises the amino acid sequence set
forth as SEQ ID NO: 8. In some cases, targeting ligand comprises an
amino acid sequence having 85% or more sequence identity (e.g., 90%
or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5%
or more, or 100% sequence identity) with the amino acid sequence
MIFPWKCQSTQRDLWNIFKLWGWTMLCCDFLAHHGTDC (SEQ ID NO: 9). In some
cases, targeting ligand comprises the amino acid sequence set forth
as SEQ ID NO: 9. In some cases, targeting ligand comprises an amino
acid sequence having 85% or more sequence identity (e.g., 90% or
more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or
more, or 100% sequence identity) with the amino acid sequence
MIFPWKCQSTQRDLWNIFKLWGWTMLCC (SEQ ID NO: 10). In some cases,
targeting ligand comprises the amino acid sequence set forth as SEQ
ID NO: 10.
[0282] Fragments of selectins that can be used as a subject
targeting ligand (e.g., a signal peptide found in the first 40
amino acids of a selectin) can in some cases attain strong binding
to specifically-modified sialomucins, e.g., various Sialyl
Lewis.sup.x modifications/O-sialylation of extracellular CD34 can
lead to differential affinity for P-selectin, L-selectin and
E-selectin to bone marrow, lymph, spleen and tonsillar
compartments. Conversely, in some cases a targeting ligand can be
an extracellular portion of CD34. In some such cases, modifications
of sialylation of the ligand can be utilized to differentially
target the targeting ligand to various selectins.
[0283] In some cases, a targeting ligand according to the present
disclosure provides for targeted binding to E-selectin. E-selectin
can mediate the adhesion of tumor cells to endothelial cells and
ligands for E-selectin can play a role in cancer metastasis. As an
example, P-selectin glycoprotein-1 (PSGL-1) (e.g., derived from
human neutrophils) can function as a high-efficiency ligand for
E-selectin (e.g., expressed by the endothelium), and a subject
targeting ligand can therefore in some cases include the PSGL-1
amino acid sequence (or a fragment thereof the binds to
E-selectin). As another example, E-selectin ligand-1 (ESL-1) can
bind E-selectin and a subject targeting ligand can therefore in
some cases include the ESL-1 amino acid sequence (or a fragment
thereof the binds to E-selectin). In some cases, a targeting ligand
with the PSGL-1 and/or ESL-1 amino acid sequence (or a fragment
thereof the binds to E-selectin) bears one or more sialyl Lewis
modifications in order to bind E-selectin. As another example, in
some cases CD44, death receptor-3 (DR3), LAMP1, LAMP2, and Mac2-BP
can bind E-selectin and a subject targeting ligand can therefore in
some cases include the amino acid sequence (or a fragment thereof
the binds to E-selectin) of any one of: CD44, death receptor-3
(DR3), LAMP1, LAMP2, and Mac2-BP.
[0284] In some cases, a targeting ligand according to the present
disclosure provides for targeted binding to P-selectin. In some
cases PSGL-1 can provide for such targeted binding. In some cases a
subject targeting ligand can therefore in some cases include the
PSGL-1 amino acid sequence (or a fragment thereof the binds to
P-selectin). In some cases, a targeting ligand with the PSGL-1
amino acid sequence (or a fragment thereof the binds to P-selectin)
bears one or more sialyl Lewis modifications in order to bind
P-selectin.
[0285] In some cases, a targeting ligand according to the present
disclosure provides for targeted binding to a transferrin receptor.
In some such cases, the targeting ligand comprises an amino acid
sequence having 85% or more sequence identity (e.g., 90% or more,
95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more,
or 100% sequence identity) with the amino acid sequence
THRPPMWSPVWP (SEQ ID NO: 11). In some cases, targeting ligand
comprises the amino acid sequence set forth as SEQ ID NO: 11.
[0286] In some cases, a targeting ligand according to the present
disclosure provides for targeted binding to an integrin (e.g.,
.alpha.5.beta.1 integrin). In some such cases, the targeting ligand
comprises an amino acid sequence having 85% or more sequence
identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more,
99% or more, 99.5% or more, or 100% sequence identity) with the
amino acid sequence RRETAWA (SEQ ID NO: 12). In some cases,
targeting ligand comprises the amino acid sequence set forth as SEQ
ID NO: 12. In some cases, the targeting ligand comprises an amino
acid sequence having 85% or more sequence identity (e.g., 90% or
more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or
more, or 100% sequence identity) with the amino acid sequence RGDGW
(SEQ ID NO: 181). In some cases, targeting ligand comprises the
amino acid sequence set forth as SEQ ID NO: 181. In some cases, the
targeting ligand comprises the amino acid sequence RGD.
[0287] In some cases, a targeting ligand according to the present
disclosure provides for targeted binding to an integrin. In some
such cases, the targeting ligand comprises an amino acid sequence
having 85% or more sequence identity (e.g., 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100%
sequence identity) with the amino acid sequence GCGYGRGDSPG (SEQ ID
NO: 182). In some cases, the targeting ligand comprises the amino
acid sequence set forth as SEQ ID NO: 182. In some cases such a
targeting ligand is acetylated on the N-terminus and/or amidated
(NH2) on the C-terminus.
[0288] In some cases, a targeting ligand according to the present
disclosure provides for targeted binding to an integrin (e.g.,
.alpha.5.beta.3 integrin). In some such cases, the targeting ligand
comprises an amino acid sequence having 85% or more sequence
identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more,
99% or more, 99.5% or more, or 100% sequence identity) with the
amino acid sequence DGARYCRGDCFDG (SEQ ID NO: 187). In some cases,
the targeting ligand comprises the amino acid sequence set forth as
SEQ ID NO: 187.
[0289] In some embodiments, a targeting ligand used to target the
brain includes an amino acid sequence from rabies virus
glycoprotein (RVG) (e.g., YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG (SEQ ID
NO: 183)). In some such cases, the targeting ligand comprises an
amino acid sequence having 85% or more sequence identity (e.g., 90%
or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5%
or more, or 100% sequence identity) with the amino acid sequence
set forth as SEQ ID NO: 183. As for any of targeting ligand (as
described elsewhere herein), RVG can be conjugated and/or fused to
an anchoring domain (e.g., 9R peptide sequence). For example, a
subject delivery molecule used as part of a surface coat of a
subject nanoparticle can include the sequence
YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGRRRRRRRRR (SEQ ID NO: 180).
[0290] In some cases, a targeting ligand according to the present
disclosure provides for targeted binding to c-Kit receptor. In some
such cases, the targeting ligand comprises an amino acid sequence
having 85% or more sequence identity (e.g., 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100%
sequence identity) with the amino acid sequence set forth as SEQ ID
NO: 184. In some cases, the targeting ligand comprises the amino
acid sequence set forth as SEQ ID NO: 184.
[0291] In some cases, a targeting ligand according to the present
disclosure provides for targeted binding to CD27. In some such
cases, the targeting ligand comprises an amino acid sequence having
85% or more sequence identity (e.g., 90% or more, 95% or more, 97%
or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence
identity) with the amino acid sequence set forth as SEQ ID NO: 185.
In some cases, the targeting ligand comprises the amino acid
sequence set forth as SEQ ID NO: 185.
[0292] In some cases, a targeting ligand according to the present
disclosure provides for targeted binding to CD150. In some such
cases, the targeting ligand comprises an amino acid sequence having
85% or more sequence identity (e.g., 90% or more, 95% or more, 97%
or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence
identity) with the amino acid sequence set forth as SEQ ID NO: 186.
In some cases, the targeting ligand comprises the amino acid
sequence set forth as SEQ ID NO: 186.
[0293] In some embodiments, a targeting ligand provides for
targeted binding to KLS CD27+/IL-7Ra-/CD150+/CD34- hematopoietic
stem and progenitor cells (HSPCs). For example, a gene editing
tool(s) (described elsewhere herein) can be introduced in order to
disrupt expression of a BCL11a transcription factor and
consequently generate fetal hemoglobin. As another example, the
beta-globin (HBB) gene may be targeted directly to correct the
altered E7V substitution with a corresponding homology-directed
repair donor template. As one illustrative example, a CRISPR/Cas
RNA-guided polypeptide (e.g., Cas9, CasX, CasY, Cpf1) can be
delivered with an appropriate guide RNA such that it will bind to
loci in the HBB gene and create double-stranded or single-stranded
breaks in the genome, initiating genomic repair. In some cases, a
DNA donor template (single stranded or double stranded) is
introduced (as part of a payload) and is release for 14-30 days
while a guide RNA/CRISPR/Cas protein complex (a ribonucleoprotein
complex) can be released over the course of from 1-7 days. In some
cases, a payload can include an siRNA for ku70 or ku80, e.g., which
can be used to promote homologous directed repair (HDR) and limit
indel formation. In some cases, an mRNA for SIRT6 is released over
14-30 d to promote HDR-driven insertion of a donor strand following
nuclease-mediated site-specific cleavage.
[0294] In some embodiments, a targeting ligand provides for
targeted binding to CD4+ or CD8+ T-cells, hematopoietic stem and
progenitor cells (HSPCs), or peripheral blood mononuclear cells
(PBMCs), in order to modify the T-cell receptor. For example, a
gene editing tool(s) (described elsewhere herein) can be introduced
in order to modify the T-cell receptor. The T-cell receptor may be
targeted directly and substituted with a corresponding
homology-directed repair donor template for a novel T-cell
receptor. As one example, a CRISPR/Cas RNA-guided polypeptide
(e.g., Cas9, CasX, CasY, Cpf1) can be delivered with an appropriate
guide RNA such that it will bind to loci in the TCR gene and create
double-stranded or single-stranded breaks in the genome, initiating
genomic repair. In some cases, a DNA donor template (single
stranded or double stranded) is introduced (as part of a payload)
for HDR. It would be evident to skilled artisans that other CRISPR
guide RNA and HDR donor sequences, targeting beta-globin, CCR5, the
T-cell receptor, or any other gene of interest, and/or other
expression vectors may be employed in accordance with the present
disclosure.
[0295] Also provided are delivery molecules with two different
peptide sequences that together constitute a targeting ligand. For
example, in some cases a targeting ligand is bivalent (e.g.,
heterobivalent). In some cases, cell-penetrating peptides and/or
heparin sulfate proteoglycan binding ligands are used as
heterobivalent endocytotic triggers along with any of the targeting
ligands of this disclosure. A heterobivalent targeting ligand can
include an affinity sequence from one of targeting ligand and an
orthosteric binding sequence (e.g., one known to engage a desired
endocytic trafficking pathway) from a different targeting
ligand.
Anchoring Domain
[0296] In some embodiments, the surface coat includes a delivery
molecule that includes a targeting ligand conjugated to an
anchoring domain (e.g., cationic anchoring domain) (see e.g., FIG.
10, panels A-B). In some cases a targeting ligand is conjugate to
an anchoring domain (or to a linker) distal to the active region of
the targeting ligand, e.g., in order to preserve activity.
Anchoring domains (e.g., cationic anchoring domains) can include
repeating cationic residues (e.g., arginine, lysine, histidine). In
some cases, a cationic anchoring domain has a length in a range of
from 3 to 30 amino acids (e.g., from 3-28, 3-25, 3-24, 3-20, 4-30,
4-28, 4-25, 4-24, or 4-20 amino acids). In some cases, a cationic
anchoring domain has a length in a range of from 4 to 24 amino
acids. Suitable examples of an anchoring domain (e.g., cationic
anchoring domain) include, but are not limited to: RRRRRRRRR
(9R)(SEQ ID NO: 15) and HHHHHH (6H)(SEQ ID NO: 16).
[0297] In some cases, an anchoring domain (e.g., cationic anchoring
domain) of a subject delivery molecule is used as an anchor to coat
the surface of a nanoparticle with the delivery molecule, e.g., so
that the targeting ligand is used to target the nanoparticle to a
desired cell/cell surface protein (see e.g., FIG. 8, FIG. 9, and
FIG. 10). Thus, in some cases, the anchoring domain (e.g., cationic
anchoring domain) interacts electrostatically with a charged
sheddable layer of a nanoparticle. In some cases, the stabilization
layer has a negative charge and a positively anchoring domain
(e.g., cationic anchoring domain) can therefore interact with the
stabilization layer, effectively anchoring the delivery molecule to
the nanoparticle and coating the nanoparticle surface with a
subject targeting ligand (e.g., see FIG. 8, FIG. 9, and FIG.
10).
[0298] Conjugation of a targeting ligand to an anchoring domain can
be accomplished by any convenient technique and many different
conjugation chemistries will be known to one of ordinary skill in
the art. In some cases the conjugation is via sulfhydryl chemistry
(e.g., a disulfide bond). In some cases the conjugation is
accomplished using amine-reactive chemistry (e.g., an amine present
on a side chain from an amino acid residue in the anchoring
domain). As noted above, the targeting ligand can include a
cysteine residue, which can facilitate conjugation. Likewise, an
anchoring domain (e.g., a cationic anchoring domain) can include a
cysteine residue, which can facilitate conjugation. In some cases,
the targeting ligand and the anchoring domain (e.g., cationic
anchoring domain) are conjugated by virtue of being part of the
same polypeptide.
Linker
[0299] In some embodiments a targeting ligand according to the
present disclosure is conjugated to an anchoring domain (e.g., a
cationic anchoring domain) via an intervening linker (e.g., see
FIG. 10). The linker can be a protein linker or non-protein linker.
A linker can in some cases aid in stability, prevent complement
activation, and/or provide flexibility to the ligand relative to
the anchoring domain.
[0300] Conjugation of a targeting ligand to a linker or a linker to
an anchoring domain can be accomplished in a number of different
ways. In some cases the conjugation is via sulfhydryl chemistry
(e.g., a disulfide bond, e.g., between two cysteine residues, e.g.,
see FIG. 10). In some cases the conjugation is accomplished using
amine-reactive chemistry. In some cases, a targeting ligand
includes a cysteine residue and is conjugated to the linker via the
cysteine residue; and/or an anchoring domain includes a cysteine
residue and is conjugated to the linker via the cysteine residue.
In some cases, the linker is a peptide linker and includes a
cysteine residue. In some cases, the targeting ligand and a peptide
linker are conjugated by virtue of being part of the same
polypeptide; and/or the anchoring domain and a peptide linker are
conjugated by virtue of being part of the same polypeptide.
[0301] In some cases, a subject linker is a polypeptide and can be
referred to as a polypeptide linker. It is to be understood that
while polypeptide linkers are contemplated, non-polypeptide linkers
(chemical linkers) are used in some cases. For example, in some
embodiments the linker is a polyethylene glycol (PEG) linker.
Suitable protein linkers include polypeptides of between 4 amino
acids and 60 amino acids in length (e.g., 4-50, 4-40, 4-30, 4-25,
4-20, 4-15, 4-10, 6-60, 6-50, 6-40, 6-30, 6-25, 6-20, 6-15, 6-10,
8-60, 8-50, 8-40, 8-30, 8-25, 8-20, or 8-15 amino acids in
length).
[0302] In some embodiments, a subject linker is rigid (e.g., a
linker that include one or more proline residues). One non-limiting
example of a rigid linker is GAPGAPGAP (SEQ ID NO: 17). In some
cases, a polypeptide linker includes a C residue at the N- or
C-terminal end. Thus, in some case a rigid linker is selected from:
GAPGAPGAPC (SEQ ID NO: 18) and CGAPGAPGAP (SEQ ID NO: 19).
[0303] Peptide linkers with a degree of flexibility can be used.
Thus, in some cases, a subject linker is flexible. The linking
peptides may have virtually any amino acid sequence, bearing in
mind that flexible linkers will have a sequence that results in a
generally flexible peptide. The use of small amino acids, such as
glycine and alanine, are of use in creating a flexible peptide. The
creation of such sequences is routine to those of skill in the art.
A variety of different linkers are commercially available and are
considered suitable for use. Example linker polypeptides include
glycine polymers (G).sub.n, glycine-serine polymers (including, for
example, (GS).sub.n, GSGGS.sub.n (SEQ ID NO: 20), GGSGGS.sub.n (SEQ
ID NO: 21), and GGGS.sub.n (SEQ ID NO: 22), where n is an integer
of at least one), glycine-alanine polymers, alanine-serine
polymers. Example linkers can comprise amino acid sequences
including, but not limited to, GGSG (SEQ ID NO: 23), GGSGG (SEQ ID
NO: 24), GSGSG (SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ
ID NO: 27), GSSSG (SEQ ID NO: 28), and the like. The ordinarily
skilled artisan will recognize that design of a peptide conjugated
to any elements described above can include linkers that are all or
partially flexible, such that the linker can include a flexible
linker as well as one or more portions that confer less flexible
structure. Additional examples of flexible linkers include, but are
not limited to: GGGGGSGGGGG (SEQ ID NO: 29) and GGGGGSGGGGS (SEQ ID
NO: 30). As noted above, in some cases, a polypeptide linker
includes a C residue at the N- or C-terminal end. Thus, in some
cases a flexible linker includes an amino acid sequence selected
from: GGGGGSGGGGGC (SEQ ID NO: 31), CGGGGGSGGGGG (SEQ ID NO: 32),
GGGGGSGGGGSC (SEQ ID NO: 33), and CGGGGGSGGGGS (SEQ ID NO: 34).
[0304] In some cases, a subject polypeptide linker is
endosomolytic. Endosomolytic polypeptide linkers include but are
not limited to: KALA (SEQ ID NO: 35) and GALA (SEQ ID NO: 36). As
noted above, in some cases, a polypeptide linker includes a C
residue at the N- or C-terminal end. Thus, in some cases a subject
linker includes an amino acid sequence selected from: CKALA (SEQ ID
NO: 37), KALAC (SEQ ID NO: 38), CGALA (SEQ ID NO: 39), and GALAC
(SEQ ID NO: 40).
Illustrative Examples of Sulfhydryl Coupling Reactions
[0305] (e.g., for Conjugation Via Sulfhydryl Chemistry, e.g., Using
a Cysteine Residue)
[0306] (e.g., for Conjugating a Targeting Ligand to a Linker,
Conjugating a Targeting Ligand to an Anchoring Domain (e.g.,
Cationic Anchoring Domain), Conjugating a Linker to an Anchoring
Domain (e.g., Cationic Anchoring Domain), and the Like)
[0307] Disulfide Bond
[0308] Cysteine residues in the reduced state, containing free
sulfhydryl groups, readily form disulfide bonds with protected
thiols in a typical disulfide exchange reaction.
##STR00001##
[0309] Thioether/Thioester Bond
[0310] Sulfhydryl groups of cysteine react with maleimide and acyl
halide groups, forming stable thioether and thioester bonds
respectively.
##STR00002##
[0311] Azide--Alkyne Cycloaddition
[0312] This conjugation is facilitated by chemical modification of
the cysteine residue to contain an alkyne bond, or by the use of
L-propargyl cysteine (pictured below) in synthetic peptide
preparation. Coupling is then achieved by means of Cu promoted
click chemistry.
##STR00003##
Examples of Targeting Ligands
[0313] Examples of targeting ligands include, but are not limited,
to those that include to the following amino acid sequences:
TABLE-US-00005 SCF (targets/binds to c-Kit receptor) (SEQ ID NO:
184) EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVV
QLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKK
SFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDS RVSVTKPFMLPPVA;
CD70 (targets/binds to CD27) (SEQ ID NO: 185)
PEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLE
SLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRD
GIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGC
TIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQVVVRP; and (SEQ ID NO: 186)
SH2 domain-containing protein 1A (SH2D1A) (targets/binds to CD150)
SSGLVPRGSHMDAVAVYHGKISRETGEKLLLATGLDGSYLLRDSESVPGV
YCLCVLYHGYIYTYRVSQTETGSWSAETAPGVHKRYFRKIKNLISAFQKP
DQGIVIPLQYPVEKKSSARSTQGTTGIREDPDVCLKAP
Thus, non-limiting examples of targeting ligands (which can be used
alone or in combination with other targeting ligands) include:
TABLE-US-00006 9R-SCF (SEQ ID NO: 189)
RRRRRRRRRMEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLP
SHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECV
KENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVV
SSTLSPEKDSRVSVTKPFMLPPVA 9R-CD70 (SEQ ID NO: 190)
RRRRRRRRRPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFA
QAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELD
KGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISL
LRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVR P CD70-9R (SEQ
ID NO: 191) PEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLE
SLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRD
GIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGC
TIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRPRRRRRRRR R 6H-SH2D1A (SEQ
ID NO: 192) MGSSHHHHHHSSGLVPRGSHMDAVAVYHGKISRETGEKLLLATGLDGSYL
LRDSESVPGVYCLCVLYHGYIYTYRVSQTETGSWSAETAPGVHKRYFRKI
KNLISAFQKPDQGIVIPLQYPVEKKSSARSTQGTTGIREDPDVCLKAP 6H-SH2D1A (SEQ ID
NO: 193) RRRRRRRRRSSGLVPRGSHMDAVAVYHGKISRETGEKLLLATGLDGSYLL
RDSESVPGVYCLCVLYHGYIYTYRVSQTETGSWSAETAPGVHKRYFRKIK
NLISAFQKPDQGIVIPLQYPVEKKSSARSTQGTTGIREDPDVCLKAP Illustrative
examples of delivery molecules and components (0a) Cysteine
conjugation anchor 1 (CCA1) [anchoring domain (e.g., cationic
anchoring domain)-linker (GAPGAPGAP)-cysteine] (SEQ ID NO: 41)
RRRRRRRRR GAPGAPGAP C
(0b) Cysteine Conjugation Anchor 2 (CCA2)
[0314] [cysteine--linker (GAPGAPGAP)--anchoring domain (e.g.,
cationic anchoring domain)] C GAPGAPGAP RRRRRRRRR (SEQ ID NO:
42)
(1a) .alpha.5.beta.1 Ligand
[0314] [0315] [anchoring domain (e.g., cationic anchoring
domain)--linker (GAPGAPGAP)--Targeting ligand] RRRRRRRRR GAPGAPGAP
RRETAWA (SEQ ID NO: 45)
(1b) .alpha.5.beta.1 Ligand
[0315] [0316] [Targeting ligand--linker (GAPGAPGAP)--anchoring
domain (e.g., cationic anchoring domain)] RRETAWA GAPGAPGAP
RRRRRRRRR (SEQ ID NO: 46)
(1c) .alpha.5.beta.1 Ligand--Cys Left
[0317] CGAPGAPGAP (SEQ ID NO: 19)
Note: This can be conjugated to CCA1 (see above) either via
sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive
chemistry.
(1d) .alpha.5.beta.1 Ligand--Cys Right
[0318] GAPGAPGAPC (SEQ ID NO: 18)
Note: This can be conjugated to CCA2 (see above) either via
sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive
chemistry.
(2a) RGD .alpha.5.beta.1 Ligand
[0319] [anchoring domain (e.g., cationic anchoring domain)--linker
(GAPGAPGAP)--Targeting ligand] RRRRRRRRR GAPGAPGAP RGD (SEQ ID NO:
47)
(2b) RGD .alpha.5b1 Ligand
[0319] [0320] [Targeting ligand--linker (GAPGAPGAP)--anchoring
domain (e.g., cationic anchoring domain)] RGD GAPGAPGAP RRRRRRRRR
(SEQ ID NO: 48) [0321] (2c) RGD Ligand--Cys Left
[0322] CRGD (SEQ ID NO: 49)
Note: This can be conjugated to CCA1 (see above) either via
sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive
chemistry.
(2d) RGD Ligand--Cys Right
[0323] RGDC (SEQ ID NO: 50)
Note: This can be conjugated to CCA2 (see above) either via
sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive
chemistry.
(3a) Transferrin Ligand
[0324] [anchoring domain (e.g., cationic anchoring domain)--linker
(GAPGAPGAP)--Targeting ligand] RRRRRRRRR GAPGAPGAP THRPPMWSPVWP
(SEQ ID NO: 51)
(3b) Transferrin Ligand
[0324] [0325] [Targeting ligand--linker (GAPGAPGAP)--anchoring
domain (e.g., cationic anchoring domain)] THRPPMWSPVWP GAPGAPGAP
RRRRRRRRR (SEQ ID NO: 52)
(3c) Transferrin Ligand--Cys Left
[0326] CTHRPPMWSPVWP (SEQ ID NO: 53)
[0327] CPTHRPPMWSPVWP (SEQ ID NO: 54)
Note: This can be conjugated to CCA1 (see above) either via
sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive
chemistry.
(3d) Transferrin Ligand--Cys Right
[0328] THRPPMWSPVWPC (SEQ ID NO: 55)
Note: This can be conjugated to CCA2 (see above) either via
sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive
chemistry.
(4a) E-Selectin Ligand [1-21]
[0329] [anchoring domain (e.g., cationic anchoring domain)--linker
(GAPGAPGAP)--Targeting ligand] RRRRRRRRR GAPGAPGAP
MIASQFLSALTLVLLIKESGA (SEQ ID NO: 56) (4b) E-selectin ligand [1-21]
[0330] [Targeting ligand--linker (GAPGAPGAP)--anchoring domain
(e.g., cationic anchoring domain)] MIASQFLSALTLVLLIKESGA GAPGAPGAP
RRRRRRRRR (SEQ ID NO: 57)
(4c) E-Selectin Ligand [1-21]--Cys Left
[0331] CMIASQFLSALTLVLLIKESGA (SEQ ID NO: 58)
Note: This can be conjugated to CCA1 (see above) either via
sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive
chemistry.
(4d) E-Selectin Ligand [1-21]--Cys Right
[0332] MIASQFLSALTLVLLIKESGAC (SEQ ID NO: 59)
Note: This can be conjugated to CCA2 (see above) either via
sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive
chemistry.
(5a) FGF Fragment [26-47]
[0333] [anchoring domain (e.g., cationic anchoring domain)--linker
(GAPGAPGAP)--Targeting ligand] RRRRRRRRR GAPGAPGAP
KNGGFFLRIHPDGRVDGVREKS (SEQ ID NO: 60) Note: This can be conjugated
to CCA1 (see above) either via sulfhydryl chemistry (e.g., a
disulfide bond) or amine-reactive chemistry.
(5b) FGF Fragment [26-47]
[0334] [Targeting ligand--linker (GAPGAPGAP)--anchoring domain
(e.g., cationic anchoring domain)]
[0335] KNGGFFLRIHPDGRVDGVREKS GAPGAPGAP RRRRRRRRR (SEQ ID NO:
61)
Note: This can be conjugated to CCA1 (see above) either via
sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive
chemistry.
(5c) FGF Fragment [25-47]--Cys on Left is Native
[0336] CKNGGFFLRIHPDGRVDGVREKS (SEQ ID NO: 43)
Note: This can be conjugated to CCA1 (see above) either via
sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive
chemistry.
(5d) FGF Fragment [26-47]--Cys Right
[0337] KNGGFFLRIHPDGRVDGVREKSC (SEQ ID NO: 44)
Note: This can be conjugated to CCA2 (see above) either via
sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive
chemistry.
(6a) Exendin (S11C) [1-39]
[0338] HGEGTFTSDLCKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 2)
Note: This can be conjugated to CCA1 (see above) either via
sulfhydryl chemistry (e.g., a disulfide bond) or amine-reactive
chemistry.
Multivalent Surface Coat
[0339] In some cases the surface coat includes any one or more of
(in any desired combination): (i) one or more of the above
described polymers, (ii) one or more targeting ligands, one or more
CPPs, and one or more heptapeptides. For example, in some cases a
surface coat can include one or more (e.g., two or more, three or
more) targeting ligands, but can also include one or more of the
above described cationic polymers. In some cases a surface coat can
include one or more (e.g., two or more, three or more) targeting
ligands, but can also include one or more CPPs.
[0340] In some cases, a surface coat includes a combination of
targeting ligands that provides for targeted binding to CD34 and
heparin sulfate proteoglycans. For example, poly(L-arginine) can be
used as part of a surface coat to provide for targeted binding to
heparin sulfate proteoglycans. As such, in some cases, after
surface coating a nanoparticle with a cationic polymer (e.g.,
poly(L-arginine)), the coated nanoparticle is incubated with
hyaluronic acid, thereby forming a zwitterionic and multivalent
surface.
[0341] In some embodiments, the surface coat is multivalent. A
multivalent surface coat is one that includes two or more targeting
ligands (e.g., two or more delivery molecules that include
different ligands). An example of a multimeric (in this case
trimeric) surface coat (outer shell) is one that includes the
targeting ligands stem cell factor (SCF) (which targets c-Kit
receptor, also known as CD117), CD70 (which targets CD27), and SH2
domain-containing protein 1A (SH2D1A) (which targets CD150). For
example, in some cases, to target hematopoietic stem cells (HSCs)
[KLS (c-Kit.sup.+Lin.sup.-Sca-1.sup.+) and
CD27.sup.+/IL-7Ra.sup.-/CD150.sup.+/CD34.sup.-], a subject
nanoparticle includes a surface coat that includes a combination of
the targeting ligands SCF, CD70, and SH2 domain-containing protein
1A (SH2D1A), which target c-Kit, CD27, and CD150, respectively
(see, e.g., Table 1). In some cases, such a surface coat can
selectively target HSPCs and long-term HSCs
(c-Kit+/Lin-/Sca-1+/CD27+/IL-7Ra-/CD150+/CD34-) over other lymphoid
and myeloid progenitors.
[0342] In some example embodiments, all three targeting ligands
(SCF, CD70, and SH2D1A) are anchored to the nanoparticle via fusion
to a cationic anchoring domain (e.g., a poly-histidine such as 6H,
a poly-arginine such as 9R, and the like). For example, (1) the
targeting polypeptide SCF (which targets c-Kit receptor) can
include
XMEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFS
NISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVAS
ETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAX (SEQ ID NO: 194), where the X is a
cationic anchoring domain (e.g., a poly-histidine such as 6H, a
poly-arginine such as 9R, and the like), e.g., which can in some
cases be present at the N- and/or C-terminal end, or can be
embedded within the polypeptide sequence; (2) the targeting
polypeptide CD70 (which targets CD27) can include
XPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGWDVAELQLNHT
GPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAV
GICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRPX
(SEQ ID NO: 195), where the X is a cationic anchoring domain (e.g.,
a poly-histidine such as 6H, a poly-arginine such as 9R, and the
like), e.g., which can in some cases be present at the N- and/or
C-terminal end, or can be embedded within the polypeptide sequence;
and (3) the targeting polypeptide SH2D1A (which targets CD150) can
include
XSSGLVPRGSHMDAVAVYHGKISRETGEKLLLATGLDGSYLLRDSESVPGVYCLCVLYHGYIYTYR
VSQTETGSWSAETAPGVHKRYFRKIKNLISAFQKPDQGIVIPLQYPVEKKSSARSTQGTTGIREDP
DVCLKAP (SEQ ID NO: 196), where the X is a cationic anchoring
domain (e.g., a poly-histidine such as 6H, a poly-arginine such as
9R, and the like), e.g., which can in some cases be present at the
N- and/or C-terminal end, or can be embedded within the polypeptide
sequence (e.g., such as
MGSSXSSGLVPRGSHMDAVAVYHGKISRETGEKLLLATGLDGSYLLRDSESVPGVYCLCVLYHGY
IYTYRVSQTETGSWSAETAPGVHKRYFRKIKNLISAFQKPDQGIVIPLQYPVEKKSSARSTQGTTGI
REDPDVCLKAP (SEQ ID NO: 197)).
[0343] As noted above, nanoparticles of the disclosure can include
multiple targeting ligands (as part of a surface coat) in order to
target a desired cell type, or in order to target a desired
combination of cell types. Examples of cells of interest within the
mouse and human hematopoietic cell lineages are depicted in FIG. 17
(panels A-B), along with markers that have been identified for
those cells. For example, various combinations of cell surface
markers of interest include, but are not limited to: [Mouse] (i)
CD150; (ii) Sca1, cKit, CD150; (iii) CD150 and CD49b; (iv) Sca1,
cKit, CD150, and CD49b; (v) CD150 and Flt3; (vi) Sca1, cKit, CD150,
and Flt3; (vii) Flt3 and CD34; (viii) Flt3, CD34, Sca1, and cKit;
(ix) Flt3 and CD127; (x) Sca1, cKit, Flt3, and CD127; (xi) CD34;
(xii) cKit and CD34; (xiii) CD16/32 and CD34; (xiv) cKit, CD16/32,
and CD34; and (xv) cKit; and [Human] (i) CD90 and CD49f; (ii) CD34,
CD90, and CD49f; (iii) CD34; (iv) CD45RA and CD10; (v) CD34,
CD45RA, and CD10; (vi) CD45RA and CD135; (vii) CD34, CD38, CD45RA,
and CD135; (viii) CD135; (ix) CD34, CD38, and CD135; and (x) CD34
and CD38. Thus, in some cases a surface coat includes one or more
targeting ligands that provide targeted binding to a surface
protein or combination of surface proteins selected from: [Mouse]
(i) CD150; (ii) Sca1, cKit, CD150; (iii) CD150 and CD49b; (iv)
Sca1, cKit, CD150, and CD49b; (v) CD150 and Flt3; (vi) Sca1, cKit,
CD150, and Flt3; (vii) Flt3 and CD34; (viii) Flt3, CD34, Sca1, and
cKit; (ix) Flt3 and CD127; (x) Sca1, cKit, Flt3, and CD127; (xi)
CD34; (xii) cKit and CD34; (xiii) CD16/32 and CD34; (xiv) cKit,
CD16/32, and CD34; and (xv) cKit; and [Human] (i) CD90 and CD49f;
(ii) CD34, CD90, and CD49f; (iii) CD34; (iv) CD45RA and CD10; (v)
CD34, CD45RA, and CD10; (vi) CD45RA and CD135; (vii) CD34, CD38,
CD45RA, and CD135; (viii) CD135; (ix) CD34, CD38, and CD135; and
(x) CD34 and CD38. Because a subject nanoparticle can include more
than one targeting ligand, and because some cells include
overlapping markers, multiple different cell types can be targeted
using combinations of surface coats, e.g., in some cases a surface
coat may target one specific cell type while in other cases a
surface coat may target more than one specific cell type (e.g., 2
or more, 3 or more, 4 or more cell types). For example, any
combination of cells within the hematopoietic lineage can be
targeted. As an illustrative example, targeting CD34 (using a
targeting ligand that provides for targeted binding to CD34) can
lead to nanoparticle delivery of a payload to several different
cells within the hematopoietic lineage (see, e.g., FIG. 17, panels
A and B).
iv. Delivery
[0344] Provided are methods of delivering a nucleic acid, protein,
or ribonucleoprotein payload to a cell. As noted above, in some
cases the payload includes a gene editing tool. Thus, in some cases
a subject method is used to perform site-specific genome editing,
which in some cases, e.g., when performed in the presence of a
donor DNA template, leads to and homology-directed repair.
[0345] Such methods include a step of contacting a cell with a
subject nanoparticle (or subject viral or non-viral delivery
vehicle). A subject nanoparticle (or subject viral or non-viral
delivery vehicle) can be used to deliver a payload to any desired
eukaryotic target cell. In some cases, the target cell is a
mammalian cell (e.g., a cell of a rodent, a mouse, a rat, an
ungulate, a cow, a sheep, a pig, a horse, a camel, a rabbit, a
canine (dog), a feline (cat), a primate, a non-human primate, or a
human). Any cell type can be targeted, and in some cases specific
targeting of particular cells depends on the presence of targeting
ligands, e.g., as part of the surface coat, where the targeting
ligands provide for targeting binding to a particular cell type.
For example, cells that can be targeted include but are not limited
to bone marrow cells, hematopoietic stem cells (HSCs), long-term
HSCs, short-term HSCs, hematopoietic stem and progenitor cells
(HSPCs), peripheral blood mononuclear cells (PBMCs), myeloid
progenitor cells, lymphoid progenitor cells, T-cells, B-cells, NKT
cells, NK cells, dendritic cells, monocytes, granulocytes,
erythrocytes, megakaryocytes, mast cells, basophils, eosinophils,
neutrophils, macrophages, erythroid progenitor cells (e.g., HUDEP
cells), megakaryocyte-erythroid progenitor cells (MEPs), common
myeloid progenitor cells (CMPs), multipotent progenitor cells
(MPPs), hematopoietic stem cells (HSCs), short term HSCs (ST-HSCs),
IT-HSCs, long term HSCs (LT-HSCs), endothelial cells, neurons,
astrocytes, pancreatic cells, pancreatic .beta.-islet cells, muscle
cells, skeletal muscle cells, cardiac muscle cells, hepatic cells,
fat cells, intestinal cells, cells of the colon, and cells of the
stomach.
[0346] Examples of various applications (e.g., for targeting
neurons, cells of the pancreas, hematopoietic stem cells and
multipotent progenitors, etc.) are discussed above, e.g., in the
context of targeting ligands. For example, Hematopoietic stem cells
and multipotent progenitors can be targeted for gene editing (e.g.,
insertion) in vivo. Even editing 1% of bone marrow cells in vivo
(approximately 15 billion cells) would target more cells than an ex
vivo therapy (approximately 10 billion cells). As another example,
pancreatic cells (e.g., .beta. islet cells) can be targeted, e.g.,
to treat pancreatic cancer, to treat diabetes, etc. As another
example, somatic cells in the brain such as neurons can be targeted
(e.g., to treat indications such as Huntington's disease,
Parkinson's (e.g., LRRK2 mutations), and ALS (e.g., SOD1
mutations)). In some cases this can be achieved through direct
intracranial injections.
[0347] As another example, endothelial cells and cells of the
hematopoietic system (e.g., megakaryocytes and/or any progenitor
cell upstream of a megakaryocyte such as a megakaryocyte-erythroid
progenitor cell (MEP), a common myeloid progenitor cell (CMP), a
multipotent progenitor cell (MPP), a hematopoietic stem cells
(HSC), a short term HSC (ST-HSC), an IT-HSC, a long term HSC
(LT-HSC)--see, e.g., FIG. 17) can be targeted with a subject
nanoparticle (or subject viral or non-viral delivery vehicle) to
treat Von Willebrand's disease. For example, a cell (e.g., an
endothelial cell, a megakaryocyte and/or any progenitor cell
upstream of a megakaryocyte such as an MEP, a CMP, an MPP, an HSC
such as an ST-HSC, an IT-HSC, and/or an LT-HSC) harboring a
mutation in the gene encoding von Willebrand factor (VWF) can be
targeted (in vitro, ex vivo, in vivo) in order to introduce an
active protein (e.g., via delivery of a functional VWF protein
and/or a nucleic acid encoding a functional VWF protein) and/or in
order to edit the mutated gene, e.g., by introducing a replacement
sequence (e.g., via delivery of a gene editing tool and delivery of
a DNA donor template). In some of the above cases (e.g., in cases
related to treating Von Willebrand's disease, in cases related to
targeting a cell harboring a mutation in the gene encoding VWF), a
subject targeting ligand provides for targeted binding to
E-selectin.
[0348] As another example, a cell of a stem cell lineage (e.g., a
stem and/or progenitor cell of the hematopoietic lineage, e.g., a
GMP, MEP, CMP, MLP, MPP, and/or an HSC) can be targeted with a
subject nanoparticle (or subject viral or non-viral delivery
vehicle) in order to increase expression of stem cell factor (SCF)
in the cell, which can therefore drive proliferation of the
targeted cell. For example, a subject nanoparticle (or subject
viral or non-viral delivery vehicle) can be used to deliver SCF
and/or a nucleic acid (DNA or mRNA) encoding SCF to the targeted
cell.
[0349] Methods and compositions of this disclosure can be used to
treat any number of diseases, including any disease that is linked
to a known causative mutation, e.g., a mutation in the genome. For
example, methods and compositions of this disclosure can be used to
treat sickle cell disease, thalassemia, HIV, myelodysplastic
syndromes, JAK2-mediated polycythemia vera, JAK2-mediated primary
myelofibrosis, JAK2-mediated leukemia, and various hematological
disorders. As additional non-limiting examples, the methods and
compositions of this disclosure can also be used for B-cell
antibody generation, immunotherapies (e.g., delivery of a
checkpoint blocking reagent), and stem cell differentiation
applications.
[0350] As noted above, in some embodiments, a targeting ligand
provides for targeted binding to KLS CD27+/IL-7Ra-/CD150+/CD34-
hematopoietic stem and progenitor cells (HSPCs). For example, a
gene editing tool(s) (described elsewhere herein) can be introduced
in order to disrupt expression of a BCL11a transcription factor and
consequently generate fetal hemoglobin. As another example, the
beta-globin (HBB) gene may be targeted directly to correct the
altered E7V substitution with a corresponding homology-directed
repair donor template. As one illustrative example, a CRISPR/Cas
RNA-guided polypeptide (e.g., Cas9, CasX, CasY, Cpf1) can be
delivered with an appropriate guide RNA such that it will bind to
loci in the HBB gene and create double-stranded or single-stranded
breaks in the genome, initiating genomic repair. In some cases, a
DNA donor template (single stranded or double stranded) is
introduced (as part of a payload) and is release for 14-30 days
while a guide RNA/CRISPR/Cas protein complex (a ribonucleoprotein
complex) can be released over the course of from 1-7 days. In some
cases, a payload can include an siRNA for ku70 or ku80, e.g., which
can be used to promote homologous directed repair (HDR) and limit
indel formation. In some cases, an mRNA for SIRT6 is released over
14-30 d to promote HDR-driven insertion of a donor strand following
nuclease-mediated site-specific cleavage.
[0351] In some embodiments, a targeting ligand provides for
targeted binding to CD4+ or CD8+ T-cells, hematopoietic stem and
progenitor cells (HSPCs), or peripheral blood mononuclear cells
(PBMCs), in order to modify the T-cell receptor. For example, a
gene editing tool(s) (described elsewhere herein) can be introduced
in order to modify the T-cell receptor. The T-cell receptor may be
targeted directly and substituted with a corresponding
homology-directed repair donor template for a novel T-cell
receptor. As one example, a CRISPR/Cas RNA-guided polypeptide
(e.g., Cas9, CasX, CasY, Cpf1) can be delivered with an appropriate
guide RNA such that it will bind to loci in the TCR gene and create
double-stranded or single-stranded breaks in the genome, initiating
genomic repair. In some cases, a DNA donor template (single
stranded or double stranded) is introduced (as part of a payload)
for HDR. It would be evident to skilled artisans that other CRISPR
guide RNA and HDR donor sequences, targeting beta-globin, CCR5, the
T-cell receptor, or any other gene of interest, and/or other
expression vectors may be employed in accordance with the present
disclosure.
[0352] In some cases, when contacting a cell with a subject
nanoparticle (or subject viral or non-viral delivery vehicle), the
contacting is in vitro (e.g., the cell is in culture), e.g., the
cell can be a cell of an established tissue culture cell line. In
some cases, the contacting is ex vivo (e.g., the cell is a primary
cell (or a recent descendant) isolated from an individual, e.g. a
patient). In some cases, the cell is in vivo and is therefore
inside of (part of) an organism. As an example of in vivo contact,
in some cases the contacting step includes administration of a
subject nanoparticle (or subject viral or non-viral delivery
vehicle) to an individual.
[0353] A subject nanoparticle (or subject viral or non-viral
delivery vehicle) may be introduced to the subject (i.e.,
administered to an individual) via any of the following routes:
systemic, local, parenteral, subcutaneous (s.c.), intravenous
(i.v.), intracranial (i.c.), intraspinal, intraocular, intradermal
(i.d.), intramuscular (i.m.), intralymphatic (LI.), or into spinal
fluid. A subject nanoparticle (or subject viral or non-viral
delivery vehicle) may be introduced by injection (e.g., systemic
injection, direct local injection, local injection into or near a
tumor and/or a site of tumor resection, etc.), catheter, or the
like. Examples of methods for local delivery (e.g., delivery to a
tumor and/or cancer site) include, e.g., by bolus injection, e.g.
by a syringe, e.g. into a joint, tumor, or organ, or near a joint,
tumor, or organ; e.g., by continuous infusion, e.g. by cannulation,
e.g. with convection (see e.g. US Application No. 20070254842,
incorporated here by reference).
[0354] The number of administrations of treatment to a subject may
vary. Introducing a subject nanoparticle (or subject viral or
non-viral delivery vehicle) into an individual may be a one-time
event; but in certain situations, such treatment may elicit
improvement for a limited period of time and require an on-going
series of repeated treatments. In other situations, multiple
administrations of a subject nanoparticle (or subject viral or
non-viral delivery vehicle) may be required before an effect is
observed. As will be readily understood by one of ordinary skill in
the art, the exact protocols depend upon the disease or condition,
the stage of the disease and parameters of the individual being
treated.
[0355] A "therapeutically effective dose" or "therapeutic dose" is
an amount sufficient to effect desired clinical results (i.e.,
achieve therapeutic efficacy). A therapeutically effective dose can
be administered in one or more administrations. For purposes of
this disclosure, a therapeutically effective dose of a subject
nanoparticle (or subject viral or non-viral delivery vehicle) is an
amount that is sufficient, when administered to the individual, to
palliate, ameliorate, stabilize, reverse, prevent, slow or delay
the progression of a disease state/ailment.
[0356] An example therapeutic intervention is one that creates
resistance to HIV infection in addition to ablating any retroviral
DNA that has been integrated into the host genome. T-cells are
directly affected by HIV and thus a hybrid blood targeting strategy
for CD34+ and CD45+ cells may be explored for delivering dual
guided nucleases. By simultaneously targeting HSCs and T-cells and
delivering an ablation to the CCR5-.DELTA.32 and gag/rev/pol genes
through multiple guided nucleases (e.g., within a single particle),
a universal HIV cure can be created with persistence through the
patient's life.
v. Co-Delivery (not Necessarily a Nanoparticle of the
Disclosure)
[0357] As noted above, one advantage of delivering multiple
payloads as part of the same package is that the efficiency of each
payload is not diluted. As such, in some embodiments, one or more
gene editing tools (e.g., as described above) is delivered in
combination with (e.g., as part of the same package/delivery
vehicle, where the delivery vehicle does not need to be a
nanoparticle of the disclosure) a protein (and/or a DNA or mRNA
encoding same) and/or a non-coding RNA that increases genomic
editing efficiency. In some embodiments, one or more gene editing
tools is delivered in combination with (e.g., as part of the same
package/delivery vehicle, where the delivery vehicle does not need
to be a nanoparticle of the disclosure) a protein (and/or a DNA or
mRNA encoding same) and/or a non-coding RNA that controls cell
division and/or differentiation. For example, in some cases one or
more gene editing tools is delivered in combination with (e.g., as
part of the same package/delivery vehicle, where the delivery
vehicle does not need to be a nanoparticle of the disclosure) a
protein (and/or a DNA or mRNA encoding same) and/or a non-coding
RNA that controls cell division. In some cases one or more gene
editing tools is delivered in combination with (e.g., as part of
the same package/delivery vehicle, where the delivery vehicle does
not need to be a nanoparticle of the disclosure) a protein (and/or
a DNA or mRNA encoding same) and/or a non-coding RNA that controls
differentiation. In some cases, one or more gene editing tools is
delivered in combination with (e.g., as part of the same
package/delivery vehicle, where the delivery vehicle does not need
to be a nanoparticle of the disclosure) a protein (and/or a DNA or
mRNA encoding same) and/or a non-coding RNA that biases the cell
DNA repair machinery toward non-homologous end joining (NHEJ) or
homology directed repair (HDR).
[0358] As noted above, in some cases the delivery vehicle does not
need to be a nanoparticle of the disclosure. For example, in some
cases the delivery vehicle is viral and in some cases the delivery
vehicle is non-viral. Examples of non-viral delivery systems
include materials that can be used to co-condense multiple nucleic
acid payloads, or combinations of protein and nucleic acid
payloads. Examples include, but are not limited to: (1) lipid based
particles such as zwitterionic or cationic lipids, and exosome or
exosome-derived vesicles; (2) inorganic/hybrid composite particles
such as those that include ionic complexes co-condensed with
nucleic acids and/or protein payloads, and complexes that can be
condensed from cationic ionic states of Ca, Mg, Si, Fe and
physiological anions such as O.sup.2-, OH, PO.sub.4.sup.3-,
SO.sub.4.sup.2-; (3) carbohydrate Delivery vehicles such as
cyclodextrin and/or alginate; (4) polymeric and/or co-polymeric
complexes such as poly(amino-acid) based electrostatic complexes,
poly(Amido-Amine), and cationic poly(B-Amino Ester); and (5) virus
like particles (e.g., protein and nucleic acid based) such as
Li2016 artificial viruses. Examples of viral delivery systems
include but are not limited to: AAV, adenoviral, retroviral, and
lentiviral.
[0359] Examples of Payloads for Co-Delivery
[0360] In some embodiments one or more gene editing tools can be
delivered in combination with (e.g., as part of the same
package/delivery vehicle, where the delivery vehicle does not need
to be a nanoparticle of the disclosure) one or more of: SCF (and/or
a DNA or mRNA encoding SCF), HoxB4 (and/or a DNA or mRNA encoding
HoxB4), BCL-XL (and/or a DNA or mRNA encoding BCL-XL), SIRT6
(and/or a DNA or mRNA encoding SIRT6), a nucleic acid molecule
(e.g., an siRNA and/or an LNA) that suppresses miR-155, a nucleic
acid molecule (e.g., an siRNA, an shRNA, a microRNA) that reduces
ku70 expression, and a nucleic acid molecule (e.g., an siRNA, an
shRNA, a microRNA) that reduces ku80 expression.
[0361] For examples of microRNAs (delivered as RNAs or as DNA
encoding the RNAs) that can be delivered together, see FIG. 18A.
For example, the following microRNAs can be used for the following
purposes: for blocking differentiation of a pluripotent stem cell
toward ectoderm lineage: miR-430/427/302; for blocking
differentiation of a pluripotent stem cell toward endoderm lineage:
miR-109 and/or miR-24; for driving differentiation of a pluripotent
stem cell toward endoderm lineage: miR-122 and/or miR-192; for
driving differentiation of an ectoderm progenitor cell toward a
keratinocyte fate: miR-203; for driving differentiation of a neural
crest stem cell toward a smooth muscle fate: miR-145; for driving
differentiation of a neural stem cell toward a glial cell fate
and/or toward a neuron fate: miR-9 and/or miR-124a; for blocking
differentiation of a mesoderm progenitor cell toward a chondrocyte
fate: miR-199a; for driving differentiation of a mesoderm
progenitor cell toward an osteoblast fate: miR-296 and/or miR-2861;
for driving differentiation of a mesoderm progenitor cell toward a
cardiac muscle fate: miR-1; for blocking differentiation of a
mesoderm progenitor cell toward a cardiac muscle fate: miR-133; for
driving differentiation of a mesoderm progenitor cell toward a
skeletal muscle fate: miR-214, miR-206, miR-1 and/or miR-26a; for
blocking differentiation of a mesoderm progenitor cell toward a
skeletal muscle fate: miR-133, miR-221, and/or miR-222; for driving
differentiation of a hematopoietic progenitor cell toward
differentiation: miR-223; for blocking differentiation of a
hematopoietic progenitor cell toward differentiation: miR-128a
and/or miR-181a; for driving differentiation of a hematopoietic
progenitor cell toward a lymphoid progenitor cell: miR-181; for
blocking differentiation of a hematopoietic progenitor cell toward
a lymphoid progenitor cell: miR-146; for blocking differentiation
of a hematopoietic progenitor cell toward a myeloid progenitor
cell: miR-155, miR-24a, and/or miR-17; for driving differentiation
of a lymphoid progenitor cell toward a T cell fate: miR-150; for
blocking differentiation of a myeloid progenitor cell toward a
granulocyte fate: miR-223; for blocking differentiation of a
myeloid progenitor cell toward a monocyte fate: miR-17-5p, miR-20a,
and/or miR-106a; for blocking differentiation of a myeloid
progenitor cell toward a red blood cell fate: miR-150, miR-155,
miR-221, and/or miR-222; and for driving differentiation of a
myeloid progenitor cell toward a red blood cell fate: miR-451
and/or miR-16.
For examples of signaling proteins (e.g., extracellular signaling
proteins) that can be delivered together with one or more gene
editing tools (e.g., as described elsewhere herein), see FIG. 18B.
For example, the following signaling proteins (e.g., extracellular
signaling proteins) (e.g., delivered as protein or as a nucleic
acid such as DNA or RNA encoding the protein) can be used for the
following purposes: for driving differentiation of a hematopoietic
stem cell toward a common lymphoid progenitor cell lineage: IL-7;
for driving differentiation of a hematopoietic stem cell toward a
common myeloid progenitor cell lineage: IL-3, GM-CSF, and/or M-CSF;
for driving differentiation of a common lymphoid progenitor cell
toward a B-cell fate: IL-3, IL-4, and/or IL-7; for driving
differentiation of a common lymphoid progenitor cell toward a
Natural Killer Cell fate: IL-15; for driving differentiation of a
common lymphoid progenitor cell toward a T-cell fate: IL-2, IL-7,
and/or Notch; for driving differentiation of a common lymphoid
progenitor cell toward a dendritic cell fate: Flt-3 ligand; for
driving differentiation of a common myeloid progenitor cell toward
a dendritic cell fate: Flt-3 ligand, GM-CSF, and/or TNF-alpha; for
driving differentiation of a common myeloid progenitor cell toward
a granulocyte-macrophage progenitor cell lineage: GM-CSF; for
driving differentiation of a common myeloid progenitor cell toward
a megakaryocyte-erythroid progenitor cell lineage: IL-3, SCF,
and/or Tpo; for driving differentiation of a
megakaryocyte-erythroid progenitor cell toward a megakaryocyte
fate: IL-3, IL-6, SCF, and/or Tpo; for driving differentiation of a
megakaryocyte-erythroid progenitor cell toward a erythrocyte fate:
erythropoietin; for driving differentiation of a megakaryocyte
toward a platelet fate: IL-11 and/or Tpo; for driving
differentiation of a granulocyte-macrophage progenitor cell toward
a monocyte lineage: GM-CSF and/or M-CSF; for driving
differentiation of a granulocyte-macrophage progenitor cell toward
a myeloblast lineage: GM-CSF; for driving differentiation of a
monocyte toward a monocyte-derived dendritic cell fate: Flt-3
ligand, GM-CSF, IFN-alpha, and/or IL-4; for driving differentiation
of a monocyte toward a macrophage fate: IFN-gamma, IL-6, IL-10,
and/or M-CSF; for driving differentiation of a myeloblast toward a
neutrophil fate: G-CSF, GM-CSF, IL-6, and/or SCF; for driving
differentiation of a myeloblast toward a eosinophil fate: GM-CSF,
IL-3, and/or IL-5; and for driving differentiation of a myeloblast
toward a basophil fate: G-CSF, GM-CSF, and/or IL-3.
[0362] Examples of proteins that can be delivered (e.g., as protein
and/or a nucleic acid such as DNA or RNA encoding the protein)
together with one or more gene editing tools (e.g., as described
elsewhere herein) include but are not limited to: SOX17, HEX, OSKM
(Oct4/Sox2/Klf4/c-myc), and/or bFGF (e.g., to drive differentiation
toward hepatic stem cell lineage); HNF4a (e.g., to drive
differentiation toward hepatocyte fate); Poly (I:C), BMP-4, bFGF,
and/or 8-Br-cAMP (e.g., to drive differentiation toward endothelial
stem cell/progenitor lineage); VEGF (e.g., to drive differentiation
toward arterial endothelium fate); Sox-2, Brn4, Myt11, Neurod2,
Ascl1 (e.g., to drive differentiation toward neural stem
cell/progenitor lineage); and BDNF, FCS, Forskolin, and/or SHH
(e.g., to drive differentiation neuron, astrocyte, and/or
oligodendrocyte fate).
[0363] Examples of signaling proteins (e.g., extracellular
signaling proteins) that can be delivered (e.g., as protein and/or
a nucleic acid such as DNA or RNA encoding the protein) together
with one or more gene editing tools (e.g., as described elsewhere
herein) include but are not limited to: cytokines (e.g., IL-2
and/or IL-15, e.g., for activating CD8+ T-cells); ligands and or
signaling proteins that modulate one or more of the Notch, Wnt,
and/or Smad signaling pathways; SCF; stem cell differentiating
factors (e.g. Sox2, Oct3/4, Nanog, Klf4, c-Myc, and the like); and
temporary surface marker "tags" and/or fluorescent reporters for
subsequent isolation/purification/concentration. For example, a
fibroblast may be converted into a neural stem cell via delivery of
Sox2, while it will turn into a cardiomyocyte in the presence of
Oct3/4 and small molecule "epigenetic resetting factors." In a
patient with Huntington's disease or a CXCR4 mutation, these
fibroblasts may respectively encode diseased phenotypic traits
associated with neurons and cardiac cells. By delivering gene
editing corrections and these factors in a single package, the risk
of deleterious effects due to one or more, but not all of the
factors/payloads being introduced can be significantly reduced.
[0364] Applications include in vivo approaches wherein a cell death
cue may be conditional upon a gene edit not being successful, and
cell differentiation/proliferation/activation is tied to a
tissue/organ-specific promoter and/or exogenous factor. A diseased
cell receiving a gene edit may activate and proliferate, but due to
the presence of another promoter-driven expression cassette (e.g.
one tied to the absence of tumor suppressor such as p21 or p53),
those cells will subsequently be eliminated. The cells expressing
desired characteristics, on the other hand, may be triggered to
further differentiate into the desired downstream lineages.
vi. Kits
[0365] Also within the scope of the disclosure are kits. For
example, in some cases a subject kit can include one or more of (in
any combination): (i) a targeting ligand, (ii) a linker, (iii) a
targeting ligand conjugated to a linker, (iv) a targeting ligand
conjugated to an anchoring domain (e.g., with or without a linker),
(v) an agent for use as a sheddable layer (e.g., silica), (vi) a
payload, e.g., an siRNA or a transcription template for an siRNA or
shRNA; a gene editing tool, and the like, (vii) a polymer that can
be used as a cationic polymer, (viii) a polymer that can be used as
an anionic polymer, (ix) a polypeptide that can be used as a
cationic polypeptide, e.g., one or more HTPs, and (x) a subject
viral or non-viral delivery vehicle. In some cases, a subject kit
can include instructions for use. Kits typically include a label
indicating the intended use of the contents of the kit. The term
label includes any writing, or recorded material supplied on or
with the kit, or which otherwise accompanies the kit.
Exemplary Non-Limiting Aspects of the Disclosure
[0366] Aspects, including embodiments, of the present subject
matter described above may be beneficial alone or in combination,
with one or more other aspects or embodiments. Without limiting the
foregoing description, certain non-limiting aspects of the
disclosure are provided below in Set A (numbered 1-74), Set B
(numbered 1-33), and Set C (numbered 1-11). As will be apparent to
those of ordinary skill in the art upon reading this disclosure,
each of the individually numbered aspects may be used or combined
with any of the preceding or following individually numbered
aspects. This is intended to provide support for all such
combinations of aspects and is not limited to combinations of
aspects explicitly provided below:
Aspects (Set A)
[0367] 1. A nanoparticle, comprising a core and a sheddable layer
encapsulating the core, wherein the core comprises:
[0368] (i) an anionic polymer composition;
[0369] (ii) a cationic polymer composition;
[0370] (iii) a cationic polypeptide composition; and
[0371] (iv) a nucleic acid and/or protein payload,
[0372] wherein (a) said anionic polymer composition comprises
polymers of D-isomers of an anionic amino acid and polymers of
L-isomers of an anionic amino acid; and/or (b) said cationic
polymer composition comprises polymers of D-isomers of a cationic
amino acid and polymers of L-isomers of a cationic amino acid.
2. The nanoparticle of 1, wherein said anionic polymer composition
comprises a first anionic polymer selected from poly(D-glutamic
acid) (PDEA) and poly(D-aspartic acid) (PDDA); and comprises a
second anionic polymer selected from poly(L-glutamic acid) (PLEA)
and poly(L-aspartic acid) (PLDA). 3. The nanoparticle of 1 or 2,
wherein said cationic polymer composition comprises a first
cationic polymer selected from poly(D-arginine), poly(D-lysine),
poly(D-histidine), poly(D-ornithine), and poly(D-citrulline); and
comprises a second cationic polymer selected from poly(L-arginine),
poly(L-lysine), poly(L-histidine), poly(L-ornithine), and
poly(L-citrulline). 4. The nanoparticle of any one of 1-3, wherein
said polymers of D-isomers of an anionic amino acid are present at
a ratio, relative to said polymers of L-isomers of an anionic amino
acid, in a range of from 10:1 to 1:10. 5. The nanoparticle of any
one of 1-4, wherein said polymers of D-isomers of a cationic amino
acid are present at a ratio, relative to said polymers of L-isomers
of a cationic amino acid, in a range of from 10:1 to 1:10. 6. A
nanoparticle, comprising a core and a sheddable layer encapsulating
the core, wherein the core comprises:
[0373] (a) an anionic polymer composition;
[0374] (b) a cationic polymer composition;
[0375] (c) a cationic polypeptide composition; and
[0376] (d) a nucleic acid and/or protein payload,
[0377] wherein one of (a) and (b) comprises a D-isomer polymer of
an amino acid, and the other of (a) and (b) comprises an L-isomer
polymer of an amino acid, and wherein the ratio of the D-isomer
polymer to the L-isomer polymer is in a range of from 10:1 to
1.5:1, or from 1:1.5 to 1:10.
7. The nanoparticle of 6, wherein said anionic polymer composition
comprises an anionic polymer selected from poly(D-glutamic acid)
(PDEA) and poly(D-aspartic acid) (PDDA). 8. The nanoparticle of 7,
wherein said cationic polymer composition comprises a cationic
polymer selected from poly(L-arginine), poly(L-lysine),
poly(L-histidine), poly(L-ornithine), and poly(L-citrulline). 9.
The nanoparticle of 6, wherein said cationic polymer composition
comprises a cationic polymer selected from poly(D-arginine),
poly(D-lysine), poly(D-histidine), poly(D-ornithine), and
poly(D-citrulline). 10. The nanoparticle of 9, wherein said anionic
polymer composition comprises an anionic polymer selected from
poly(L-glutamic acid) (PLEA) and poly(L-aspartic acid) (PLDA). 11.
The nanoparticle of any one of 1-10, wherein the sheddable layer is
an anionic coat. 12. The nanoparticle of any one of 1-11, wherein
the sheddable layer is pH and/or glutathione sensitive. 13. The
nanoparticle of any one of 1-12, wherein the sheddable layer
comprises one or more of: silica, a peptoid, a polycysteine,
calcium, calcium phosphate, calcium sulfate, manganese, manganese
phosphate, manganese sulfate, magnesium, magnesium phosphate,
magnesium sulfate, iron, iron phosphate, iron sulfate, lithium,
lithium phosphate, and lithium sulfate. 14. The nanoparticle of 8,
wherein the sheddable layer is a silica coat. 15. The nanoparticle
of any one of 1-14, further comprising a surface coat surrounding
the sheddable layer. 16. The nanoparticle of 15, wherein the
surface coat comprises a cationic component that interacts
electrostatically with the sheddable layer. 17. The nanoparticle of
15 or 16, wherein the surface coat comprises one or more of: a
polymer of a cationic amino acid, a poly(arginine), an anchoring
domain, a cationic anchoring domain, a cell penetrating peptide, a
viral glycoprotein, a heparin sulfate proteoglycan, and a targeting
ligand. 18. The nanoparticle of any one of 15-17, wherein the
surface coat is zwitterionic and multivalent. 19. The nanoparticle
of any one of 15-18, wherein the surface coat comprises one or more
targeting ligands. 20. The nanoparticle of 19, wherein at least one
of the one or more targeting ligands is conjugated to an anchoring
domain that interacts with the sheddable layer. 21. The
nanoparticle of 21, wherein the anchoring domain is a cationic
anchoring domain selected from RRRRRRRRR (SEQ ID NO: 15) and HHHHHH
(SEQ ID NO: 16). 22. The nanoparticle of 21 or 21, wherein the
anchoring domain is conjugated to the at least one of the one or
more targeting ligands via a linker. 23. The nanoparticle of 22,
wherein the linker is not a polypeptide. 24. The nanoparticle of
22, wherein the linker is a polypeptide. 25. The nanoparticle of
any one of 22-24, wherein the linker is conjugated to the targeting
ligand via sulfhydryl or amine-reactive chemistry, and/or the
linker is conjugated to the anchoring domain via sulfhydryl or
amine-reactive chemistry. 26. The nanoparticle of any one of 22-25,
wherein said at least one of the one or more targeting ligands
comprises a cysteine residue and is conjugated to the linker via
the cysteine residue. 27. The nanoparticle of any one of 19-26,
wherein said one or more targeting ligands provides for targeted
binding to a family B G-protein coupled receptor (GPCR). 28. The
nanoparticle of 27, wherein said targeting ligand comprises a
cysteine substitution, at one or more internal amino acid
positions, relative to a corresponding wild type amino acid
sequence. 29. The nanoparticle of 27 or 28, wherein said targeting
ligand comprises an amino acid sequence having 85% or more identity
to the amino acid sequence HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS
(SEQ ID NO: 1). 30. The nanoparticle of 29, wherein said targeting
ligand comprises a cysteine substitution at one or more of
positions L10, S11, and K12 of the amino acid sequence set forth in
SEQ ID NO: 1). 31. The nanoparticle of 30, wherein said targeting
ligand comprises the amino acid sequence
HGEGTFTSDLCKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 2). 32. The
nanoparticle of any one of 19-31, wherein the surface coat
comprises one or more targeting ligands that provides for targeted
binding to a cell surface protein selected from c-Kit, CD27, and
CD150. 33. The nanoparticle of any one of 19-32, wherein the
surface coat comprises one or more targeting ligands selected from
the group consisting of: rabies virus glycoprotein (RVG) fragment,
ApoE-transferrin, lactoferrin, melanoferritin, ovotransferritin,
L-selectin, E-selectin, P-selectin, PSGL-1, ESL-1, CD44, death
receptor-3 (DR3), LAMP1, LAMP2, Mac2-BP, stem cell factor (SCF),
CD70, SH2 domain-containing protein 1A (SH2D1A), a exendin-4, GLP1,
a targeting ligand that targets .alpha.5.beta.1, RGD, a Transferrin
ligand, an FGF fragment, succinic acid, a bisphosphonate, CD90,
CD45f, CD34, a hematopoietic stem cell chemotactic lipid,
sphingosine, ceramide, sphingosine-1-phosphate,
ceramide-1-phosphate, and an active targeting fragment of any of
the above. 34. The nanoparticle of any one of 19-33, wherein the
surface coat comprises stem cell factor (SCF) or a targeting
fragment thereof, CD70 or a targeting fragment thereof, and SH2
domain-containing protein 1A (SH2D1A) or a targeting fragment
thereof. 35. The nanoparticle of any one of 19-34, wherein the
surface coat comprises one or more targeting ligands that provides
for targeted binding to target cells selected from: bone marrow
cells, hematopoietic stem cells (HSCs), hematopoietic stem and
progenitor cells (HSPCs), peripheral blood mononuclear cells
(PBMCs), myeloid progenitor cells, lymphoid progenitor cells,
T-cells, B-cells, NKT cells, NK cells, dendritic cells, monocytes,
granulocytes, erythrocytes, megakaryocytes, mast cells, basophils,
eosinophils, neutrophils, macrophages, erythroid progenitor cells,
megakaryocyte-erythroid progenitor cells (MEPs), common myeloid
progenitor cells (CMPs), multipotent progenitor cells (MPPs),
hematopoietic stem cells (HSCs), short term HSCs (ST-HSCs),
IT-HSCs, long term HSCs (LT-HSCs), endothelial cells, neurons,
astrocytes, pancreatic cells, pancreatic .beta.-islet cells, liver
cells, muscle cells, skeletal muscle cells, cardiac muscle cells,
hepatic cells, fat cells, intestinal cells, cells of the colon, and
cells of the stomach. 36. The nanoparticle of any one of 19-34,
wherein the surface coat comprises two or more targeting ligands,
the combination of which provides for targeted binding to cells
selected from: bone marrow cells, hematopoietic stem cells (HSCs),
hematopoietic stem and progenitor cells (HSPCs), peripheral blood
mononuclear cells (PBMCs), myeloid progenitor cells, lymphoid
progenitor cells, T-cells, B-cells, NKT cells, NK cells, dendritic
cells, monocytes, granulocytes, erythrocytes, megakaryocytes, mast
cells, basophils, eosinophils, neutrophils, macrophages, erythroid
progenitor cells (e.g., HUDEP cells), megakaryocyte-erythroid
progenitor cells (MEPs), common myeloid progenitor cells (CMPs),
multipotent progenitor cells (MPPs), hematopoietic stem cells
(HSCs), short term HSCs (ST-HSCs), IT-HSCs, long term HSCs
(LT-HSCs), endothelial cells, neurons, astrocytes, pancreatic
cells, pancreatic .beta.-islet cells, liver cells, muscle cells,
skeletal muscle cells, cardiac muscle cells, hepatic cells, fat
cells, intestinal cells, cells of the colon, and cells of the
stomach. 37. The nanoparticle of any one of 1-36, wherein the
cationic polypeptide composition comprises a polypeptide that
comprises a nuclear localization signal (NLS). 38. The nanoparticle
of 37, wherein the NLS comprises the amino acid sequence set forth
in any one of SEQ ID NOs: 151-157 and 201-264. 39. The nanoparticle
of any one of 1-38, wherein the cationic polypeptide composition
comprises a histone tail peptide (HTP). 40. The nanoparticle of 39,
wherein the HTP is conjugated to a cationic amino acid polymer. 41.
The nanoparticle of 40, wherein the HTP is conjugated to a cationic
amino acid polymer via a cysteine residue. 42. The nanoparticle of
40 or 41, wherein the cationic amino acid polymer comprises
poly(lysine). 43. The nanoparticle of any one of 38-42, wherein
said cationic polypeptide composition comprises histone peptides
having a branched structure. 44. The nanoparticle of any one of
1-43, wherein the payload comprises one or more of: (i) a
CRISPR/Cas guide RNA, (ii) a DNA molecule encoding a CRISPR/Cas
guide RNA, (iii) a nucleic acid molecule encoding a CRISPR/Cas
RNA-guided polypeptide, (iv) a CRISPR/Cas RNA-guided polypeptide,
(v) a CRISPR/Cas guide RNA complexed with a CRISPR/Cas RNA-guided
polypeptide, (vi) a nucleic acid molecule encoding a zinc finger
protein (ZFP), (vii) a ZFP, (viii) a nucleic acid molecule encoding
a transcription activator-like effector (TALE) protein, (ix) a TALE
protein, and (x) a DNA donor template. 45. The nanoparticle of any
one of 1-44, wherein the payload comprises (i) a CRISPR/Cas guide
RNA and/or a DNA molecule encoding said CRISPR/Cas guide RNA; and
(ii) a CRISPR/Cas RNA-guided polypeptide and/or a nucleic acid
molecule encoding said CRISPR/Cas RNA-guided polypeptide. 46. The
nanoparticle of 45, wherein the payload further comprises a DNA
donor template. 47. The nanoparticle of any one of 1-46, further
comprising one or more of: SCF, a nucleic acid encoding SCF, HoxB4,
a nucleic acid encoding HoxB4, BCL-XL, a nucleic acid encoding
BCL-XL, SIRT6, a nucleic acid encoding SIRT6, a nucleic acid
molecule (e.g., an siRNA, an LNA) that suppresses miR-155, a
nucleic acid molecule (e.g., an siRNA, an shRNA, a microRNA) that
in reduces ku70 expression, and a nucleic acid molecule (e.g., an
siRNA, an shRNA, a microRNA) that in reduces ku80 expression. 48. A
nanoparticle formulation, comprising:
[0378] (a) a first nanoparticle according to any one of 1-47,
wherein the payload comprises one or more of: (i) a CRISPR/Cas
guide RNA, (ii) a DNA molecule encoding a CRISPR/Cas guide RNA,
(iii) a nucleic acid molecule encoding a CRISPR/Cas RNA-guided
polypeptide, (iv) a CRISPR/Cas RNA-guided polypeptide, (v) a
CRISPR/Cas guide RNA complexed with a CRISPR/Cas RNA-guided
polypeptide, (vi) a nucleic acid molecule encoding a zinc finger
protein (ZFP), (vii) a ZFP, (viii) a nucleic acid molecule encoding
a transcription activator-like effector (TALE) protein, and (ix) a
TALE protein; and
[0379] (b) a second nanoparticle comprising a nucleic acid payload
that comprises a DNA donor template.
49. A multi-layered nanoparticle, comprising:
[0380] (a) an inner core comprising a payload comprising a DNA
donor template;
[0381] (b) a first sheddable layer surrounding the inner core;
[0382] (c) an intermediate core surrounding the first sheddable
layer, wherein the intermediate core comprises one or more of: (i)
a CRISPR/Cas guide RNA, (ii) a DNA molecule encoding a CRISPR/Cas
guide RNA, (iii) a nucleic acid molecule encoding a CRISPR/Cas
RNA-guided polypeptide, (iv) a CRISPR/Cas RNA-guided polypeptide,
(v) a CRISPR/Cas guide RNA complexed with a CRISPR/Cas RNA-guided
polypeptide, (vi) a zinc finger protein (ZFP), (vii) a DNA molecule
encoding a ZFP, (viii) a transcription activator-like effector
(TALE) protein, and (ix) a DNA molecule encoding a TALE protein;
and
[0383] (d) a second sheddable layer surrounding the intermediate
core.
50. The multi-layered nanoparticle of 49, wherein the first and/or
second sheddable layer comprises one or more of: silica, a peptoid,
a polycysteine, calcium, calcium phosphate, calcium sulfate,
manganese, manganese phosphate, manganese sulfate, magnesium,
magnesium phosphate, magnesium sulfate, iron, iron phosphate, iron
sulfate, lithium, lithium phosphate, and lithium sulfate. 51. The
multi-layered nanoparticle of 49 or 50, comprising a surface coat
surrounding the second sheddable layer. 52. The multi-layered
nanoparticle of 51, wherein the surface coat comprises a cationic
component that interacts electrostatically with the second
sheddable layer. 53. The multi-layered nanoparticle of 51 or 52,
wherein the surface coat comprises one or more of: a polymer of a
cationic amino acid, a poly(arginine), a cell penetrating peptide,
a viral glycoprotein, a heparin sulfate proteoglycan, and a
targeting ligand. 54. The multi-layered nanoparticle of any one of
49-53, wherein the surface coat is zwitterionic and multivalent.
55. The multi-layered nanoparticle of any one of 49-54, wherein the
surface coat comprises one or more targeting ligands. 56. A method
of delivering a nucleic acid and/or protein payload to a target
cell, the method comprising: contacting a eukaryotic target cell
with the nanoparticle of any one of 1-47, the nanoparticle
formulation of 48, and/or the multi-layered nanoparticle of any one
of 49-55. 57. The method of 56, wherein the payload includes a gene
editing tool. 58. The method of 56 or 57, wherein the payload
includes one or more of: a CRISPR/Cas guide RNA, a DNA molecule
encoding a CRISPR/Cas guide RNA, a CRISPR/Cas RNA-guided
polypeptide, a nucleic acid molecule encoding a CRISPR/Cas
RNA-guided polypeptide, a zinc finger nuclease, a nucleic acid
molecule encoding a zinc finger nuclease, a TALE or TALEN, a
nucleic acid molecule encoding a TALE or TALEN, DNA donor template,
a nucleic acid molecule encoding a site-specific recombinase (e.g.,
Cre recombinase, Dre recombinase, Flp recombinase, KD recombinase,
B2 recombinase, B3 recombinase, R recombinase, Hin recombinase, Tre
recombinase, PhiC31 integrase, Bxb1 integrase, R4 integrase, lambda
integrase, HK022 integrase, HP1 integrase), a site-specific
recombinase, a nucleic acid molecule encoding a resolvase and/or
invertase (e.g., Gin, Hin, .gamma..delta.3, Tn3, Sin, Beta), a
resolvase and/or invertase (e.g., Gin, Hin, .gamma..delta.3, Tn3,
Sin, Beta), a transposon and/or a DNA derived from a transposon
(e.g., a bacterial transposon such as Tn3, Tn5, Tn7, Tn9, Tn10,
Tn903, Tn1681; a eukaryotic transposon such as a Tc1/mariner super
family transposon, a PiggyBac superfamily transposon, an hAT
superfamily transposon, PiggyBac, Sleeping Beauty, Frog Prince,
Minos, Himar1, mariner), and a transposase. 59. The method of any
one of 56-58, wherein the target cell is a mammalian cell 60. The
method of any one of 56-59, wherein the target cell is a human cell
61. The method of any one of 56-60, wherein the target cell is in
culture in vitro. 62. The method of any one of 56-60, wherein the
target cell is in vivo. 63. The method of 62, wherein said
contacting includes a step of administering the nanoparticle to an
individual 64. The method of 63, wherein the individual has
Huntington's disease, ALS, Parkinson's disease, pancreatic cancer,
diabetes, or von Willebrand's disease. 65. The method of any one of
56-64, wherein the nanoparticle includes a surface coat comprising
a targeting ligand. 66. The method of 65, wherein the targeting
ligand provides for target binding to cells selected from: bone
marrow cells, hematopoietic stem cells (HSCs), hematopoietic stem
and progenitor cells (HSPCs), peripheral blood mononuclear cells
(PBMCs), myeloid progenitor cells, lymphoid progenitor cells,
T-cells, B-cells, NKT cells, NK cells, dendritic cells, monocytes,
granulocytes, erythrocytes, megakaryocytes, mast cells, basophils,
eosinophils, neutrophils, macrophages, erythroid progenitor cells
(e.g., HUDEP cells), megakaryocyte-erythroid progenitor cells
(MEPs), common myeloid progenitor cells (CMPs), multipotent
progenitor cells (MPPs), hematopoietic stem cells (HSCs), short
term HSCs (ST-HSCs), IT-HSCs, long term HSCs (LT-HSCs), endothelial
cells, neurons, astrocytes, pancreatic cells, pancreatic
.beta.-islet cells, liver cells, muscle cells, skeletal muscle
cells, cardiac muscle cells, hepatic cells, fat cells, intestinal
cells, cells of the colon, and cells of the stomach. 67. The method
of any one of 56-66, wherein the target cell is selected from: a
bone marrow cell, a hematopoietic stem cell (HSC), a hematopoietic
stem and progenitor cell (HSPC), a peripheral blood mononuclear
cell (PBMC), a myeloid progenitor cell, a lymphoid progenitor cell,
a T-cell, a B-cell, a NKT cell, a NK cell, a dendritic cell, a
monocyte, a granulocyte, an erythrocyte, a megakaryocyte, a mast
cell, a basophil, an eosinophil, a neutrophil, a macrophage, an
erythroid progenitor cell, a megakaryocyte-erythroid progenitor
cell (MEP), a common myeloid progenitor cell (CMP), a multipotent
progenitor cell (MPP), a hematopoietic stem cell (HSC), a short
term HSC (ST-HSC), an IT-HSC, a long term HSC (LT-HSC), an
endothelial cell, a neuron, an astrocyte, a pancreatic cell, a
pancreatic .beta.-islet cell, a liver cell, a muscle cell, a
skeletal muscle cell, a cardiac muscle cell, a hepatic cell, a fat
cell, an intestinal cell, a cell of the colon, and a cell of the
stomach. 68. The method of any one of 56-67, wherein the target
cell is a stem and/or progenitor cell and the payload comprises
stem cell factor (SCF) and/or a nucleic acid encoding SCF. 69. The
method of any one of 56-67, wherein (i) the target cell is from an
individual with von Willebrand's disease and/or the target cell
includes a genomic mutation in the gene encoding VWF such that the
cell produces sub-normal levels of functional VWF; (ii) the target
cell is any one of: a megakaryocyte, an endothelial cell, an MEP, a
CMP, an MPP, an HSC, a ST-HSC, and a LT-HSC; and (iii) the payload
includes a functional VWF protein and/or a nucleic acid encoding a
functional VWF. 70. A branched histone molecule, comprising: one or
more histone tail peptides (HTPs) conjugated to side chains of a
cationic polymer. 71. The branched histone molecule of 70, wherein
the cationic polymer comprises poly(arginine) or poly(lysine). 72.
The branched histone molecule of 70 or 71, wherein up to 40% of the
side chains of the cationic polymer are conjugated to said one or
more HTPs. 73. A branched histone molecule, comprising: one or more
histone tail peptides (HTPs) conjugated to one another such that
the branched histone molecule forms a structure selected from: a
brush polymer, a web (e.g., spider web structure), a graft polymer,
a star-shaped polymer, a comb polymer, a polymer network, and a
dendrimer. 74. The branched histone molecule of 73, wherein the
branched histone molecule forms a web structure.
Aspects (Set B)
[0384] 1. A lipid formulation for delivering a protein and/or
nucleic acid payload, the lipid formulation comprising: a lipid and
a core, wherein the core comprises:
[0385] (i) an anionic polymer composition;
[0386] (ii) a cationic polymer composition;
[0387] (iii) a cationic polypeptide composition; and
[0388] (iv) a nucleic acid and/or protein payload,
[0389] wherein (a) said anionic polymer composition comprises
polymers of D-isomers of an anionic amino acid and polymers of
L-isomers of an anionic amino acid; and/or (b) said cationic
polymer composition comprises polymers of D-isomers of a cationic
amino acid and polymers of L-isomers of a cationic amino acid.
2. The lipid formulation of 1, wherein said anionic polymer
composition comprises a first anionic polymer selected from
poly(D-glutamic acid) (PDEA) and poly(D-aspartic acid) (PDDA); and
comprises a second anionic polymer selected from poly(L-glutamic
acid) (PLEA) and poly(L-aspartic acid) (PLDA). 3. The lipid
formulation of 1 or 2, wherein said cationic polymer composition
comprises a first cationic polymer selected from poly(D-arginine),
poly(D-lysine), poly(D-histidine), poly(D-ornithine), and
poly(D-citrulline); and comprises a second cationic polymer
selected from poly(L-arginine), poly(L-lysine), poly(L-histidine),
poly(L-ornithine), and poly(L-citrulline). 4. The lipid formulation
of any one of 1-3, wherein said polymers of D-isomers of an anionic
amino acid are present at a ratio, relative to said polymers of
L-isomers of an anionic amino acid, in a range of from 10:1 to
1:10. 5. The lipid formulation of any one of 1-4, wherein said
polymers of D-isomers of a cationic amino acid are present at a
ratio, relative to said polymers of L-isomers of a cationic amino
acid, in a range of from 10:1 to 1:10. 6. A lipid formulation for
delivering a protein and/or nucleic acid payload, the lipid
formulation comprising: a lipid and a core, wherein the core
comprises:
[0390] (a) an anionic polymer composition;
[0391] (b) a cationic polymer composition;
[0392] (c) a cationic polypeptide composition; and
[0393] (d) a nucleic acid and/or protein payload,
[0394] wherein one of (a) and (b) comprises a D-isomer polymer of
an amino acid, and the other of (a) and (b) comprises an L-isomer
polymer of an amino acid.
7. The lipid formulation of 6, wherein the ratio of the D-isomer
polymer to the L-isomer polymer is in a range of from 10:1 to
1.5:1, or from 1:1.5 to 1:10. 8. The lipid formulation of 7,
wherein said anionic polymer composition comprises an anionic
polymer selected from poly(D-glutamic acid) (PDEA) and
poly(D-aspartic acid) (PDDA). 9. The lipid formulation of 8,
wherein said cationic polymer composition comprises a cationic
polymer selected from poly(L-arginine), poly(L-lysine),
poly(L-histidine), poly(L-ornithine), and poly(L-citrulline). 10.
The lipid formulation of 7, wherein said cationic polymer
composition comprises a cationic polymer selected from
poly(D-arginine), poly(D-lysine), poly(D-histidine),
poly(D-ornithine), and poly(D-citrulline). 11. The lipid
formulation of 10, wherein said anionic polymer composition
comprises an anionic polymer selected from poly(L-glutamic acid)
(PLEA) and poly(L-aspartic acid) (PLDA). 12. The lipid formulation
of any one of 1-11, wherein the cationic polypeptide composition
comprises a polypeptide that comprises a nuclear localization
signal (NLS). 13. The lipid formulation of 12, wherein the NLS
comprises the amino acid sequence set forth in any one of SEQ ID
NOs: 151-157 and 201-264. 14. The lipid formulation of any one of
1-13, wherein the cationic polypeptide composition comprises a
histone tail peptide (HTP). 15. The lipid formulation of 14,
wherein the HTP is conjugated to a cationic amino acid polymer. 16.
The lipid formulation of 15, wherein the HTP is conjugated to a
cationic amino acid polymer via a cysteine residue. 17. The lipid
formulation of 14 or 15, wherein the cationic amino acid polymer
comprises poly(lysine). 18. The lipid formulation of any one of
1-17, wherein said cationic polypeptide composition comprises
histone peptides having a branched structure. 19. The lipid
formulation of any one of 1-18, wherein the payload comprises one
or more of: (i) a CRISPR/Cas guide RNA, (ii) a DNA molecule
encoding a CRISPR/Cas guide RNA, (iii) a nucleic acid molecule
encoding a CRISPR/Cas RNA-guided polypeptide, (iv) a CRISPR/Cas
RNA-guided polypeptide, (v) a CRISPR/Cas guide RNA complexed with a
CRISPR/Cas RNA-guided polypeptide, (vi) a nucleic acid molecule
encoding a zinc finger protein (ZFP), (vii) a ZFP, (viii) a nucleic
acid molecule encoding a transcription activator-like effector
(TALE) protein, (ix) a TALE protein, (x) a DNA donor template, (xi)
a nucleic acid molecule encoding a site-specific recombinase (e.g.,
Cre recombinase, Dre recombinase, Flp recombinase, KD recombinase,
B2 recombinase, B3 recombinase, R recombinase, Hin recombinase, Tre
recombinase, PhiC31 integrase, Bxb1 integrase, R4 integrase, lambda
integrase, HK022 integrase, HP1 integrase), (xii) a site-specific
recombinase, (xiii) a nucleic acid molecule encoding a resolvase
and/or invertase (e.g., Gin, Hin, .gamma..delta.3, Tn3, Sin, Beta),
(xiv) a resolvase and/or invertase (e.g., Gin, Hin,
.gamma..delta.3, Tn3, Sin, Beta), (xv) a transposon and/or a DNA
derived from a transposon (e.g., a bacterial transposon such as
Tn3, Tn5, Tn7, Tn9, Tn10, Tn903, Tn1681; a eukaryotic transposon
such as a Tc1/mariner super family transposon, a PiggyBac
superfamily transposon, an hAT superfamily transposon, PiggyBac,
Sleeping Beauty, Frog Prince, Minos, Himar1, mariner), and (xvi) a
transposase. 20. The lipid formulation of any one of 1-19, wherein
the payload comprises (i) a CRISPR/Cas guide RNA and/or a DNA
molecule encoding said CRISPR/Cas guide RNA; and (ii) a CRISPR/Cas
RNA-guided polypeptide and/or a nucleic acid molecule encoding said
CRISPR/Cas RNA-guided polypeptide. 21. The lipid formulation of 20,
wherein the payload further comprises a DNA donor template. 22. A
method of delivering a nucleic acid and/or protein payload to a
target cell, the method comprising: contacting a eukaryotic target
cell with the lipid formulation of any one of 1-21. 23. The method
of 22, wherein the payload includes a gene editing tool. 24. The
method of 22 or 23, wherein the payload includes one or more of: a
CRISPR/Cas guide RNA, a DNA molecule encoding a CRISPR/Cas guide
RNA, a CRISPR/Cas RNA-guided polypeptide, a nucleic acid molecule
encoding a CRISPR/Cas RNA-guided polypeptide, a zinc finger
nuclease, a nucleic acid molecule encoding a zinc finger nuclease,
a TALE or TALEN, a nucleic acid molecule encoding a TALE or TALEN,
DNA donor template, a nucleic acid molecule encoding a
site-specific recombinase (e.g., Cre recombinase, Dre recombinase,
Flp recombinase, KD recombinase, B2 recombinase, B3 recombinase, R
recombinase, Hin recombinase, Tre recombinase, PhiC31 integrase,
Bxb1 integrase, R4 integrase, lambda integrase, HK022 integrase,
HP1 integrase), a site-specific recombinase, a nucleic acid
molecule encoding a resolvase and/or invertase (e.g., Gin, Hin,
.gamma..delta.3, Tn3, Sin, Beta), a resolvase and/or invertase
(e.g., Gin, Hin, .gamma..delta.3, Tn3, Sin, Beta), a transposon
and/or a DNA derived from a transposon (e.g., a bacterial
transposon such as Tn3, Tn5, Tn7, Tn9, Tn10, Tn903, Tn1681; a
eukaryotic transposon such as a Tc1/mariner super family
transposon, a PiggyBac superfamily transposon, an hAT superfamily
transposon, PiggyBac, Sleeping Beauty, Frog Prince, Minos, Himar1,
mariner), and a transposase. 25. The method of any one of 22-24,
wherein the target cell is a mammalian cell 26. The method of any
one of 22-25, wherein the target cell is a human cell 27. The
method of any one of 22-26, wherein the target cell is in culture
in vitro. 28. The method of any one of 22-26, wherein the target
cell is in vivo. 29. The method of 28, wherein said contacting
includes a step of administering the lipid formulation to an
individual 30. The method of 29, wherein the individual has
Huntington's disease, ALS, Parkinson's disease, pancreatic cancer,
diabetes, or von Willebrand's disease. 31. The method of any one of
22-30, wherein the target cell is selected from: a bone marrow
cell, a hematopoietic stem cell (HSC), a hematopoietic stem and
progenitor cell (HSPC), a peripheral blood mononuclear cell (PBMC),
a myeloid progenitor cell, a lymphoid progenitor cell, a T-cell, a
B-cell, a NKT cell, a NK cell, a dendritic cell, a monocyte, a
granulocyte, an erythrocyte, a megakaryocyte, a mast cell, a
basophil, an eosinophil, a neutrophil, a macrophage, an erythroid
progenitor cell, a megakaryocyte-erythroid progenitor cell (MEP), a
common myeloid progenitor cell (CMP), a multipotent progenitor cell
(MPP), a hematopoietic stem cell (HSC), a short term HSC (ST-HSC),
an IT-HSC, a long term HSC (LT-HSC), an endothelial cell, a neuron,
an astrocyte, a pancreatic cell, a pancreatic .beta.-islet cell, a
liver cell, a muscle cell, a skeletal muscle cell, a cardiac muscle
cell, a hepatic cell, a fat cell, an intestinal cell, a cell of the
colon, and a cell of the stomach. 32. The method of any one of
22-31, wherein the target cell is a stem and/or progenitor cell and
the payload comprises stem cell factor (SCF) and/or a nucleic acid
encoding SCF. 33. The method of any one of 22-31, wherein (i) the
target cell is a cell of an individual with von Willebrand's
disease and/or the target cell includes a genomic mutation in the
gene encoding VWF such that the cell produces sub-normal levels of
functional VWF; (ii) the target cell is any one of: a
megakaryocyte, an endothelial cell, an MEP, a CMP, an MPP, an HSC,
a ST-HSC, and a LT-HSC; and (iii) the payload includes a functional
VWF protein and/or a nucleic acid encoding a functional VWF.
Aspects (Set C)
[0395] 1. A method of delivering a nucleic acid and/or protein
payload to a target cell, the method comprising: contacting a
eukaryotic target cell with a viral or non-viral delivery vehicle
comprising:
[0396] (a) a gene editing tool; and
[0397] (b) a nucleic acid or protein agent that induces
proliferation of and/or biases differentiation of the target
cell.
2. The method of 1, wherein (a) comprises one or more of: (i) a
CRISPR/Cas guide RNA, (ii) a DNA molecule encoding a CRISPR/Cas
guide RNA, (iii) a nucleic acid molecule encoding a CRISPR/Cas
RNA-guided polypeptide, (iv) a CRISPR/Cas RNA-guided polypeptide,
(v) a CRISPR/Cas guide RNA complexed with a CRISPR/Cas RNA-guided
polypeptide, (vi) a nucleic acid molecule encoding a zinc finger
protein (ZFP), (vii) a ZFP, (viii) a nucleic acid molecule encoding
a transcription activator-like effector (TALE) protein, (ix) a TALE
protein, (x) a DNA donor template, (xi) a nucleic acid molecule
encoding a site-specific recombinase (e.g., Cre recombinase, Dre
recombinase, Flp recombinase, KD recombinase, B2 recombinase, B3
recombinase, R recombinase, Hin recombinase, Tre recombinase,
PhiC31 integrase, Bxb1 integrase, R4 integrase, lambda integrase,
HK022 integrase, HP1 integrase), (xii) a site-specific recombinase,
(xiii) a nucleic acid molecule encoding a resolvase and/or
invertase (e.g., Gin, Hin, .gamma..delta.3, Tn3, Sin, Beta), (xiv)
a resolvase and/or invertase (e.g., Gin, Hin, .gamma..delta.3, Tn3,
Sin, Beta), (xv) a transposon and/or a DNA derived from a
transposon (e.g., a bacterial transposon such as Tn3, Tn5, Tn7,
Tn9, Tn10, Tn903, Tn1681; a eukaryotic transposon such as a
Tc1/mariner super family transposon, a PiggyBac superfamily
transposon, an hAT superfamily transposon, PiggyBac, Sleeping
Beauty, Frog Prince, Minos, Himar1, mariner), and (xvi) a
transposase. 3. The method of 1 or 2, wherein (b) comprises a
nucleic acid or protein agent that induces proliferation of the
target cell. 4. The method of any one of 1-3, wherein (b) comprises
a nucleic acid or protein agent that biases differentiation of the
target cell. 5. The method of any one of 1-4, wherein (b) comprises
one or more of: SCF, a nucleic acid encoding SCF, HoxB4, a nucleic
acid encoding HoxB4, BCL-XL, a nucleic acid encoding BCL-XL, SIRT6,
a nucleic acid encoding SIRT6, a nucleic acid molecule (e.g., an
siRNA, an LNA) that suppresses miR-155, a nucleic acid molecule
(e.g., an siRNA, an shRNA, a microRNA) that reduces ku70
expression, and a nucleic acid molecule (e.g., an siRNA, an shRNA,
a microRNA) that reduces ku80 expression. 6. The method of any one
of 1-5, wherein (b) comprises a microRNA for blocking or driving
differentiation of the target cell. 7. The method of any one of
1-6, wherein (b) comprises a signaling protein for differentiation
of the target cell. 8. The method of any one of 1-7, wherein the
delivery vehicle is non-viral. 9. The method of any one of 1-7,
wherein the delivery vehicle is viral. 10. The method of any one of
1-9, wherein the delivery vehicle is not a nanoparticle. 11. The
method of any one of 1-9, wherein the delivery vehicle is a
nanoparticle, e.g., a nanoparticle as described herein. It will be
apparent to one of ordinary skill in the art that various changes
and modifications can be made without departing from the spirit or
scope of the invention.
EXPERIMENTAL
[0398] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of the invention nor are they
intended to represent that the experiments below are all or the
only experiments performed. Efforts have been made to ensure
accuracy with respect to numbers used (e.g., amounts, temperature,
etc.) but some experimental errors and deviations should be
accounted for. Unless indicated otherwise, parts are parts by
weight, molecular weight is weight average molecular weight,
temperature is in degrees Centigrade, and pressure is at or near
atmospheric.
[0399] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0400] The present invention has been described in terms of
particular embodiments found or proposed to comprise preferred
modes for the practice of the invention. It will be appreciated by
those of skill in the art that, in light of the present disclosure,
numerous modifications and changes can be made in the particular
embodiments exemplified without departing from the intended scope
of the invention. For example, due to codon redundancy, changes can
be made in the underlying DNA sequence without affecting the
protein sequence. Moreover, due to biological functional
equivalency considerations, changes can be made in protein
structure without affecting the biological action in kind or
amount. All such modifications are intended to be included within
the scope of the appended claims.
Example 1
[0401] To determine core formulation parameters, fluorescence
spectroscopy was used to monitor nucleic acid condensation (for
double stranded DNA payloads). The emission spectra of
intercalating Ethidium bromide (EtBr) was measured after the
addition of condensing agents at increasing charge ratios.
De-intercalation of ethidium bromide caused by polymer induced
nucleic acid condensation results in a drop in fluorescent signal.
This is because ethidium bromide exhibits a much higher quantum
fluorescent yield in the DNA-bound state, than unbound state. The
results are depicted in FIG. 1.
[0402] FIG. 1 depicts results from a fluorimetric assay testing
various parameters (e.g., cation: anion charge ratio) for
condensation of nucleic acid payloads. The result showed, e.g.,
that a charge ratio of 2 works well for the condensation of
plasmids encoding Cas9 and guide RNA molecules. 100 .mu.l of
Anionic solution was added to each well: 100 ng/.mu.l DNA, 80
ng/.mu.l poly(D-Glutamic Acid) (PDE), 0.5 ng/.mu.l Ethidium
Bromide. Condensing species were titrated at 12.5 .mu.l for each
data point. The total concentration (m/v) of condensing agent in
the solution was 0.4 ug/.mu.l, with different compositions of
histone tail peptide (HTP)[H3K4(me3)] and poly(L-arginine) (PLR)
denoted by their mass fraction in parenthesis. After each
titration, the plate was read at 605 nm with a 250 nm excitation.
For each tested charge ratio, the controls included: (i) "DNA
only", which was DNA with no EtBr; (ii) "DNA" which was DNA plus
EtBr with no PDE, PLR, or HTP present; and (iii) "DNA+PDE" which
was DNA plus EtBr plus PDE with no PLR or HTP present.
Example 2: An Example Synthesis Method
[0403] Procedures were performed within a sterile, dust free
environment (BSL-II hood). Gastight syringes were sterilized with
70% ethanol before rinsing 3 times with filtered nuclease free
water, and were stored at 4.degree. C. before use. Surfaces were
treated with RNAse inhibitor prior to use.
Nanoparticle Core
[0404] A first solution (an anionic solution) was prepared by
combining the appropriate amount of payload (in this case plasmid
DNA (EGFP-N1 plasmid) with an aqueous mixture (an `anionic polymer
composition`) of poly(D-glutamic Acid) and poly(L-glutamic acid).
This solution was diluted to the proper volume with 10 mM Tris-HCl
at pH 8.5. A second solution (a cationic solution), which was a
combination of a `cationic polymer composition` and a `cationic
polypeptide composition`, was prepared by diluting a concentrated
solution containing the appropriate amount of condensing agents to
the proper volume with 60 mM HEPES at pH 5.5. In this case, the
`cationic polymer composition` was poly(L-arginine) and the
`cationic polypeptide composition` was 16 .mu.g of H3K4(me3) (tail
of histone H3, tri methylated on K4).
[0405] Precipitation of nanoparticle cores in batches less than 200
.mu.l can be carried out by dropwise addition of the condensing
solution to the payload solution in glass vials or low protein
binding centrifuge tubes followed by incubation for 30 minutes at
4.degree. C. For batches greater than 200 .mu.l, the two solutions
can be combined in a microfluidic format using a standard mixing
chip (e.g. Dolomite Micromixer) or a hydrodynamic flow focusing
chip. In each case, optimal input flowrates can be determined such
that the resulting suspension of nanoparticle cores is
monodispersed, exhibiting a mean particle size below 100 nm.
[0406] In this case, the two equal volume solutions from above (one
of cationic condensing agents and one of anionic condensing agents)
were prepared for mixing. For the solution of cationic condensing
agents, polymer/peptide solutions were added to one protein low
bind tube (eppendorf) and were then diluted with 60 mM HEPES (pH
5.5) to a total volume of 100 .mu.l (as noted above). This solution
was kept at room temperature while preparing the anionic solution.
For the solution of anionic condensing agents, the anionic
solutions were chilled on ice with minimal light exposure. 10 .mu.g
of nucleic acid in aqueous solution (roughly 1 .mu.g/.mu.l) and 7
.mu.g of aqueous poly (D-Glutamic Acid) [0.1%] were diluted with 10
mM Tris-HCl (pH 8.5) to a total volume of 100 .mu.l (as noted
above).
[0407] Each of the two solutions was filtered using a 0.2 micron
syringe filter and transferred to its own Hamilton 1 ml Gastight
Syringe (Glass, (insert product number). Each syringe was placed on
a Harvard Pump 11 Elite Dual Syringe Pump. The syringes were
connected to appropriate inlets of a Dolomite Micro Mixer chip
using tubing, and the syringe pump was run at 120 .mu.l/min for a
100 .mu.l total volume. The resulting solution included the core
composition (which now included nucleic acid payload, anionic
components, and cationic components). The nanoparticle size (peak)
was 128.8 nm, and the zeta potential (peak) was +10.5 mV (100%)
(e.g., see FIG. 2).
Core Stabilization (Adding a Sheddable Layer)
[0408] To coat the core with a sheddable layer, the resulting
suspension of nanoparticle cores was then combined with a dilute
solution of sodium silicate in 10 mM Tris HCl (pH8.5, 10-500 mM) or
calcium chloride in 10 mM PBS (pH 8.5, 10-500 mM), and allowed to
incubate for 1-2 hours at room temperature. In this case, the core
composition was added to a diluted sodium silicate solution to coat
the core with an acid labile coating of polymeric silica (an
example of a sheddable layer). To do so, 10 .mu.l of stock Sodium
Silicate (Sigma) was first dissolved in 1.99 ml of Tris buffer (10
mM Tris pH=8.5, 1:200 dilution) and was mixed thoroughly. The
Silicate solution was filtered using a sterile 0.1 micron syringe
filter, and was transferred to a sterile Hamilton Gastight syringe,
which was mounted on a syringe pump. The core composition from
above was also transferred to a sterile Hamilton Gastight syringe,
which was also mounted on the syringe pump. The syringes were
connected to the appropriate inlets of a Dolomite Micro Mixer chip
using PTFE tubing, and the syringe pump was run at 120
.mu.l/min.
[0409] Stabilized (coated) cores can be purified using standard
centrifugal filtration devices (100 kDa Amicon Ultra, Millipore) or
dialysis in 30 mM HEPES (pH 7.4) using a high molecular weight
cutoff membrane. In this case, the stabilized (coated) cores were
purified using a centrifugal filtration device. The collected
coated nanoparticles (nanoparticle solution) were washed with
dilute PBS (1:800) or HEPES and filtered again (the solution can be
resuspended in 500 .mu.l sterile dispersion buffer or nuclease free
water for storage). Effective silica coating was demonstrated. The
stabilized cores had a size of 110.6 nm and zeta potential of -42.1
mV (95%) (FIG. 3).
Surface Coat (Outer Shell)
[0410] Addition of a surface coat (also referred to as an outer
shell), sometimes referred to as "surface functionalization," was
accomplished by electrostatically grafting ligand species (in this
case Rabies Virus Glycoprotein fused to a 9-Arg peptide sequence as
a cationic anchoring domain--`RVG9R`) to the negatively charged
surface of the stabilized (in this case silica coated)
nanoparticles. Beginning with silica coated nanoparticles that were
filtered and resuspended in dispersion buffer or water, the final
volume of each nanoparticle dispersion was determined, as was the
desired amount of polymer or peptide to add such that the final
concentration of protonated amine group was at least 75 uM. The
desired surface constituents were added and the solution was
sonicated for 20-30 seconds prior to incubate for 1 hour.
Centrifugal filtration was performed at 300 kDa (the final product
can be purified using standard centrifugal filtration devices,
e.g., 300-500 kDa from Amicon Ultra Millipore, or dialysis, e.g.,
in 30 mM HEPES (pH 7.4) using a high molecular weight cutoff
membrane), and the final resuspension was in either cell culture
media or dispersion buffer. In some cases, optimal outer shell
addition yields a monodispersed suspension of particles with a mean
particle size between 50 and 150 nm and a zeta potential between 0
and -10 mV. In this case, the nanoparticles with an outer shell had
a size of 115.8 nm and a Zeta potential of -3.1 mV (100%) (FIG.
4).
Example 3: Nanoparticle Uptake
[0411] In these studies (e.g., see FIG. 5), nanoparticles with
various surface chemistries and charge ratios were tested.
Formulations were (charge ratio refers to the nanoparticle
core):
[0412] HTT018B: charge ratio (cations/anions) was 2; surface coat
was poly(L-Arginine)
[0413] HTT019B: charge ratio was 5; surface coat was
poly(L-Arginine)
[0414] HTT020B: charge ratio was 2, surface coat was N-acetyl
Semax
[0415] HTT021B: charge ratio was 5, surface coat was N-acetyl
Semax
[0416] HTT022B: charge ratio was 2, surface coat was N-acetyl
Selank
[0417] HTT023B: charge ratio was 5, surface coat was N-acetyl
Selank
[0418] L3000GFP: lipofectamine (non-nanoparticle) delivery of a
nucleic acid encoding GFP (plasmid encoding GFP)
[0419] L3000CRISPR: lipofectamine (non-nanoparticle) delivery of
CRISPR/Cas components with no GFP or fluorescent tag.
[0420] Nanoparticles were generated. For HTT018B-023B, the core
components included a nucleic acid payload (CRISPR/Cas encoding
nucleic acids: one plasmid encoding a Cas9 guide RNA and a second
plasmid encoding a Cas9 protein) and poly(L-arginine) (a cationic
polymer composition) that was tagged with a fluorophore (FITC) so
that uptake could be assessed by fluorescent microscopy. For
L3000GFP (positive control), no nanoparticle was used and the
delivered nucleic acid was a plasmid encoding GFP. For L3000CRISPR
(negative control), no nanoparticle was used and the delivered
nucleic acid was a plasmid encoding CRISPR components, but no
plasmid used encoded GFP and nothing was tagged with FITC.
[0421] Neural stem cells were seeded at a density of 10.sup.5 cells
per well (96-well plate) and grown in Neurobasal medium
supplemented with fibroblast growth factor (FGF)(1:1000). The
nanoparticles and Lipofectamine 3000 (0.75 .mu.L reagent/.mu.g DNA)
were introduced to cells 24 hr after seeding with 400 ng of DNA
payload transfected per well. The nanoparticle samples were applied
to neural stem cells in culture and allowed to incubate for 4-24
hours before washing with PBS up to 3 times to remove any
non-internalized particles. Uptake was determined by imaging with
the appropriate laser excitation and filter selection. Cells were
imaged with a Zeiss LSM780 using a 20.times. objective. As an
alternative or in addition, quantitative uptake data can be
obtained using high content imaging and flow cytometry. The three
rows depict three different replicates.
[0422] As noted above, samples HTT18B, HTT20B, and HTT22B were
prepared with a charge ratio of 2, whereas, samples HTT19B, HTT21B,
and HTT23B where prepared with a charge ratio of 5. The data show
that a charge ratio of 2 (for condensation of the core) resulted in
higher internalization than a charge ratio of 5. Further, surface
coatings (outer shells) of the heptapeptide adaptogens Selank and
Semax promoted a higher degree of internalization than a surface
coat including the cell penetrating peptide poly(L-Arginine) [9.7
kDa].
Example 4: Characterizing Nanoparticle Internalization Behavior and
Subcellular Trafficking
[0423] Neural stem cells were contacted with nanoparticles that
include CRISPR/Cas9 plasmids as the nucleic acid payload. The
nanoparticle core included poly(L-arginine) (a cationic polymer
composition) that was tagged with a fluorophore (FITC) so that
uptake could be assessed by fluorescent microscopy. The endosome
and nucleus were stained using Lysotracker (Red) and Hoescht 3342
(blue) respectively. Nanoparticles and Lipfectamine 3000 (0.75
.mu.l reagent/.mu.g DNA) were introduced to cells 16 hours after
seeding with 400 ng of DNA payload transfected per well. Cells were
incubated with Hoescht 3342 and Lysotracker Red before imaging.
Cells were imaged 2.5 and 5 hours post-transfection with a
Cellomics CX5 using a 10.times. objective (FIG. 6, panels A-B).
Co-localization of the nanoparticle's fluorescent signal with that
of the stained endosome and nucleus were quantitatively measured
and extent of co-localization was denoted by the resulting Pearson
product-moment coefficient (FIG. 6, panels C-D).
[0424] For all nanoparticles listed in Table 2 (see FIG. 6), the
core included (i) the cationic polypeptide composition indicated in
the table, (ii) a nucleic acid payload, (iii) poly(L-arginine) [a
cationic amino acid polymer], and (iv) poly(L-glutamic acid) [an
anionic amino acid polymer]. The sheddable layer was a silica coat
and the surface coat was as indicated in Table 2.
TABLE-US-00007 TABLE 2 Sample Cationic polypeptide name composition
of the core Surface coat CAS9005 Histone tail peptide
poly(L-arginine) (PLR) (HTP) = H3K4(me3) [tail of histone H3, tri
methylated on K4] CAS9007 Same as above `RVG`: Rabies Virus
Glycoprotein (RVG) fused to a 9-Arg peptide sequence (as a cationic
anchoring domain) CAS9009 Same as above TAT (cell penetrating
peptide) HTT024 Same as above N-acetyl Selank CAS9006 SV40 NLS RVG
fused to a 9-Arg peptide sequence CAS9008 SV40 NLS TAT (cell
penetrating peptide)
[0425] FIG. 6, panel C depicts Pearson product-moment correlation
coefficients between nanoparticle polymers (the FITC tagged core
polymer) and Hoescht DNA stain or between nanoparticle polymers
(the FITC tagged core polymer) and Lyotracker. The correlation
coefficients were calculated while generating the images in panels
A and B. All values were normalized to the value of the negative
control (Cas9 Lipofectamine 3000). The decreased Pearson
correlation between Hoescht and FITC between the 2.5 hour and 5
hour time-points indicated nanoparticle polymer degradation,
release, or diffusion to compartments other than the nucleus over
time. FIG. 6, panel D depicts Pearson product-moment correlation
coefficients between nanoparticle polymers and endosomes
(Lysotracker Red). The correlation coefficients were calculated
while generating the images in panels A and B. All values were
normalized to the value of the negative control (Cas9 Lipofectamine
3000). Comparison of the 2.5 hour and 5 hour time points indicated
endosomal escape over time.
Example 5: Timed-Release
[0426] FIG. 7 depicts microscopy images of peripheral blood
mononuclear cells (PBMCs) that were been transfected with
nanoparticles, where the nucleic acid payload was mRNA encoding
GFP. The images demonstrate that mRNA expression was extended to 16
days with nanoparticles that include a core with, at a defined
ratio, a polymer of D-isomers of an anionic amino acid and a
polymer of L-isomers of an anionic amino acid. In this case, use of
a nanoparticle core with a 2:1 ratio of poly(D-Glutamic acid) to
poly(L-Glutamic Acid) resulted in maximum expression at 16 days
(panel A=4 days; panel B=16 days). The nanoparticle core included
(i) an anionic polymer composition: 7 .mu.g total of poly(glutamic
acid) (i.e., D- and L- isomers combined totaled 7 .mu.g); (ii) a
cationic polymer composition: poly(L-arginine); (iii) a cationic
polypeptide composition: H3K4(me3) [i.e., tail of histone H3, tri
methylated on K4]; and (iv) a nucleic acid payload: mRNA encoding
GFP. The nanoparticle core was encapsulated by a silica coat (a
sheddable layer) and the surface coat was poly(L-arginine)
(PLR).
Example 6: Targeting Ligand that Provides for Targeted Binding to a
Family B GPCR
[0427] FIG. 11 provides a schematic diagram of a family B GPCR,
highlighting separate domains to consider when evaluating a
targeting ligand, e.g., for binding to allosteric/affinity
N-terminal domains and orthosteric endosomal-sorting/signaling
domains. (Figure is adapted from Siu, Fai Yiu, et al., Nature
499.7459 (2013): 444-449). Such domains were considered when
selecting a site within the targeting ligand exendin-4 for cysteine
substitution.
[0428] In FIG. 12, a cysteine 11 substitution (5110) was identified
as one possible amino acid modification for conjugating exendin-4
to an anchoring domain (e.g., cationic anchoring domain) in such a
way that maintains affinity and also engages long endosomal
recycling pathways that promote nucleic acid release and limit
nucleic acid degradation. Following alignment of simulated
Exendin-4 (SEQ ID NO: 1) to known crystal structures of
glucagon-GCGR (4ERS) and GLP1-GLP1R-ECD complex (PDB: 3IOL), the
PDB renderings were rotated in 3-dimensional space in order to
anticipate the direction that a cross-linked complex must face in
order not to disrupt the two binding clefts. When the cross-linking
site of a secretin-family ligand was sufficiently orthogonal to the
two binding clefts of the corresponding secretin-family receptor,
then it was determined that high-affinity binding may occur as well
as concomitant long endosomal recycling pathway sequestration for
optimal payload release. Using this technique, Amino acid positions
10, 11, and 12 of Exendin-4 were identified as positions for
insertion of or substitution with a cysteine residue.
Example 7: Targeting Ligand that Provides for Targeted Binding to
an RTK
[0429] FIG. 13 shows a tbFGF fragment as part of a ternary
FGF2-FGFR1-HEPARIN complex (1fq9 on PDB). CKNGGFFLRIHPDGRVDGVREKS
(highlighted) (SEQ ID NO: 14) was determined to be important for
affinity to FGFR1. FIG. 14 shows that HFKDPK (SEQ ID NO: 5) was
determined as a peptide to use for ligand-receptor orthosteric
activity and affinity. FIG. 15 shows that LESNNYNT (SEQ ID NO: 6)
was also determined as a peptide to use for ligand-receptor
orthosteric activity and affinity.
Example 8
[0430] Table 3-Table 5 provide a guide for the components used in
the experiments that follow (e.g., condensation data;
physiochemical data; and flow cytometry and imaging data).
TABLE-US-00008 TABLE 3 Features of delivery molecules used in the
experiments below. Targeting Ligand Name/nomenclature Format:
A_B_C_D_E_F where A = Receptor Name: Name of receptor ligand is
targeting; B = Targeting Ligand Source: Name of ligand targeting
the receptor (Prefix "m" or "rm" for modified if Ligand is NOT wild
type); C = Linker Name; D = Charged Polypeptide Name; E = Linker
Terminus based on B; and F = Version Number (To distinguish between
two modified targeting ligands that come from the same WT but
differ in AA sequence); Anchor Linker Ligand Targeting Ligand (TL)/
SEQ Anchor Mers/ Mers/ Mers/ Peptide Catalogue Name Sequence ID NO:
Charge Total Mers Total Mers Total Mers PLR10 RRRRRRRRRR 10 100.00%
0.00% 0.00% CD45_mSiglec_(4GS)2_9R_C SNRWLDVKGGGGGSG 9 32.14%
35.71% 32.14% GGGSRRRRRRRRR CD28_mCD80_(4GS)2_9R_N RRRRRRRRRGGGGGS
9 26.47% 29.41% 44.12% GGGGSVVLKYEKDAF KR CD28_mCD80_(4GS)2_9R_C
VVLKYEKDAFKRGGG 9 26.47% 29.41% 44.12% GGSGGGGSRRRRRRR RR
CD28_mCD86_(4GS)2_9R_N_1 RRRRRRRRRGGGGSG 9 34.62% 38.46% 26.92%
GGGSENLVLNE CD28_mCD86_(4GS)2_9R_C ENLVLNEGGGGSGGG 9 34.62% 38.46%
26.92% GSRRRRRRRRR CD28_mCD86_(4GS)2_9R_N_2 RRRRRRRRRGGGGSG 9
31.03% 34.48% 34.48% GGGSPTGMIRIHQM CD137_m41BB_(4GS)2_9R_N
RRRRRRRRRGGGGGS 9 36.00% 40.00% 24.00% GGGGSAAQEE
CD3_mCD3Ab_(4GS)2_9R_N RRRRRRRRRGGGGSG 9 27.27% 30.30% 42.42%
GGGSTSVGKYPNTGY YGD CD3_mCD3Ab_(4GS)2_9R_C TSVGKYPNTGYYGDG 9 27.27%
30.30% 42.42% GGGSGGGGSRRRRRR RRR IL2R_m IL2_(4GS)2_9R_N
RRRRRRRRRGGGGSG 9 28.13% 31.25% 40.63% GGGSNPKLTRMLTFK FY
IL2R_mIL2_(4GS)2_9R_C NPKLTRMLTFKFYGG 9 28.13% 31.25% 40.63%
GGSGGGGSRRRRRRR RR PLK10_PEG22 KKKKKKKKKK- 10 31.25% 68.75% 0.00%
PEG22 ALL_LIGANDS_EQUIMOLAR N/A 9 30.25% 33.61% 36.13%
ESELLg_mESEL(4GS)2_9R_N RRRRRRRRRGGGGSG 9 22.50% 25.00% 52.50%
GGGSMIASQFLSALT LVLLIKESGA ESELLg_mESEL(4GS)2_9R_C MIASQFLSALTLVLL
9 22.50% 25.00% 52.50% IKESGSGGGGSGGGG SRRRRRRRRR
cKit_mSCF_(4GS)2_9R_N RRRRRRRRRGGGGSG 10 31.25% 68.75% 0.00%
GGGSEKFILKVRPAF KAV EPOR_mEPO_6R_N RRRRRRTYSCHFGPL 6 25.00% 0.00%
TWVCKPQGG EPOR_mEPO_6R_C TYSCHFGPLTWVCKP 6 25.00% 0.00% QGGRRRRRR
TfR_TfTP_6R_N RRRRRRTHRPPMWSP 6 33.33% 0.00% VWP TfR_TfTP_6R_C
THRPPMWSPVWPRRR 6 33.33% 0.00% RRR mH3_K4Me3_1 ART-K(Me3)- 6
100.00% 0.00% 0.00% QTARKSTGGKAPRKQ LA mH4_K16Ac_1 SGRGKGGKGLGKGG 8
100.00% 0.00% 0.00% A-K(Ac)-RHRK mH2A_1 SGRGKQGGKARAKAK 8 100.00%
0.00% 0.00% TRSSR SCF_rmAc-cKit_(4GS)2_9R_C Ac-SNYSAibADKAi 9
19.15% 21.28% 59.57% bANAibADDAibAEA ibAKENSGGGGSGGG GSRRRRRRRRR
cKit_rmSCF_(4GS)2_9R_N RRRRRRRRRGGGGSG 10 GGGSEKFILKVRPAF KAV
TABLE-US-00009 TABLE 4 Payloads used in the experiments below.
Single or Double Protein Payloads Nucleotide Stranded? Mol. Wt.
BLOCK-iT Alexa 20 2 N/A Fluor 555 siRNA NLS-Cas9-EGFP + 98 1
186229.4531 gRNA Cy5 EGFP mRNA 998 1 N/A VWF-GFP pDNA + 13000 2 N/A
Cy5 PNA
TABLE-US-00010 TABLE 5 Guide Key for the components used in the
experiments below. KEY: N = "nanoparticle"; cat. = "cationic"; an.
= "anionic"; spec. = "species"; c:p = "Carboxyl:Phospate"; Project
Targeting Ligand Code N Payload (PI) (TL) TCell.001 1
NLS_Cas9_gRNA_EGFP_RNP N/A TCell.001 2 NLS_Cas9_gRNA_EGFP_RNP N/A
TCell.001 3 NLS_Cas9_gRNA_EGFP_RNP CD45_mSiglec_(4GS)2_9R_C
TCell.001 4 NLS_Cas9_gRNA_EGFP_RNP CD28_mCD80_(4GS)2_9R_N TCell.001
5 NLS_Cas9_gRNA_EGFP_RNP CD28_mCD80_(4GS)2_9R_C TCell.001 6
NLS_Cas9_gRNA_EGFP_RNP CD28_mCD86_(4GS)2_9R_N_1 TCell.001 7
NLS_Cas9_gRNA_EGFP_RNP CD28_mCD86_(4GS)2_9R_C TCell.001 8
NLS_Cas9_gRNA_EGFP_RNP CD28_mCD86_(4GS)2_9R_N_2 TCell.001 9
NLS_Cas9_gRNA_EGFP_RNP CD137_m41BB_(4GS)2_9R_N TCell.001 10
NLS_Cas9_gRNA_EGFP_RNP CD137_m41BB_(4GS)2_9R_C TCell.001 11
NLS_Cas9_gRNA_EGFP_RNP CD3_mCD3Ab_(4GS)2_9R_N TCell.001 12
NLS_Cas9_gRNA_EGFP_RNP CD3_mCD3Ab_(4GS)2_9R_C TCell.001 13
NLS_Cas9_gRNA_EGFP_RNP IL2R_mIL2_(4GS)2_9R_N TCell.001 14
NLS_Cas9_gRNA_EGFP_RNP IL2R_mIL2_(4GS)2_9R_C TCell.001 15
NLS_Cas9_gRNA_EGFP_RNP ALL_LIGANDS_EQUIMOLAR (C7-C18) TCell.001 16
Cy5_EGFP_mRNA N/A TCell.001 17 Cy5_EGFP_mRNA N/A TCell.001 18
Cy5_EGFP_mRNA CD45_mSiglec_(4GS)2_9R_C TCell.001 19 Cy5_EGFP_mRNA
CD28_mCD80_(4GS)2_9R_N TCell.001 20 Cy5_EGFP_mRNA
CD28_mCD80_(4GS)2_9R_C TCell.001 21 Cy5_EGFP_mRNA
CD28_mCD86_(4GS)2_9R_N_1 TCell.001 22 Cy5_EGFP_mRNA
CD28_mCD86_(4GS)2_9R_C TCell.001 23 Cy5_EGFP_mRNA
CD28_mCD86_(4GS)2_9R_N_2 TCell.001 24 Cy5_EGFP_mRNA
CD137_m41BB_(4GS)2_9R_N TCell.001 25 Cy5_EGFP_mRNA
CD137_m41BB_(4GS)2_9R_C TCell.001 26 Cy5_EGFP_mRNA
CD3_mCD3Ab_(4GS)2_9R_N TCell.001 27 Cy5_EGFP_mRNA
CD3_mCD3Ab_(4GS)2_9R_C TCell.001 28 Cy5_EGFP_mRNA
IL2R_mIL2_(4GS)2_9R_N TCell.001 29 Cy5_EGFP_mRNA
IL2R_mIL2_(4GS)2_9R_C TCell.001 30 Cy5_EGFP_mRNA
ALL_LIGANDS_EQUIMOLAR (C7-C18) TCell.001 31 VWF_GFP_Cy5_pDNA N/A
TCell.001 32 VWF_GFP_Cy5_pDNA N/A TCell.001 33 VWF_GFP_Cy5_pDNA
CD45_mSiglec_(4GS)2_9R_C TCell.001 34 VWF_GFP_Cy5_pDNA
CD28_mCD80_(4GS)2_9R_N TCell.001 35 VWF_GFP_Cy5_pDNA
CD28_mCD80_(4GS)2_9R_C TCell.001 36 VWF_GFP_Cy5_pDNA
CD28_mCD86_(4GS)2_9R_N_1 TCell.001 37 VWF_GFP_Cy5_pDNA
CD28_mCD86_(4GS)2_9R_C TCell.001 38 VWF_GFP_Cy5_pDNA
CD28_mCD86_(4GS)2_9R_N_2 TCell.001 39 VWF_GFP_Cy5_pDNA
CD137_m41BB_(4GS)2_9R_N TCell.001 40 VWF_GFP_Cy5_pDNA
CD137_m41BB_(4GS)2_9R_C TCell.001 41 VWF_GFP_Cy5_pDNA
CD3_mCD3Ab_(4GS)2_9R_N TCell.001 42 VWF_GFP_Cy5_pDNA
CD3_mCD3Ab_(4GS)2_9R_C TCell.001 43 VWF_GFP_Cy5_pDNA
IL2R_mIL2_(4GS)2_9R_N TCell.001 44 VWF_GFP_Cy5_pDNA
IL2R_mIL2_(4GS)2_9R_C TCell.001 45 VWF_GFP_Cy5_pDNA
ALL_LIGANDS_EQUIMOLAR (C7-C18) TCell.001 46
BLOCK_iT_Alexa_Fluor_555_siRNA N/A TCell.001 47
BLOCK_iT_Alexa_Fluor_555_siRNA N/A TCell.001 48
BLOCK_iT_Alexa_Fluor_555_siRNA CD45_mSiglec_(4GS)2_9R_C TCell.001
49 BLOCK_iT_Alexa_Fluor_555_siRNA CD28_mCD80_(4GS)2_9R_N TCell.001
50 BLOCK_iT_Alexa_Fluor_555_siRNA CD28_mCD80_(4GS)2_9R_C TCell.001
51 BLOCK_iT_Alexa_Fluor_555_siRNA CD28_mCD86_(4GS)2_9R_N_1
TCell.001 52 BLOCK_iT_Alexa_Fluor_555_siRNA CD28_mCD86_(4GS)2_9R_C
TCell.001 53 BLOCK_iT_Alexa_Fluor_555_siRNA
CD28_mCD86_(4GS)2_9R_N_2 TCell.001 54
BLOCK_iT_Alexa_Fluor_555_siRNA CD137_m41BB_(4GS)2_9R_N TCell.001 55
BLOCK_iT_Alexa_Fluor_555_siRNA CD137_m41BB_(4GS)2_9R_C TCell.001 56
BLOCK_iT_Alexa_Fluor_555_siRNA CD3_mCD3Ab_(4GS)2_9R_N TCell.001 57
BLOCK_iT_Alexa_Fluor_555_siRNA CD3_mCD3Ab_(4GS)2_9R_C TCell.001 58
BLOCK_iT_Alexa_Fluor_555_siRNA IL2R_mIL2_(4GS)2_9R_N TCell.001 59
BLOCK_iT_Alexa_Fluor_555_siRNA IL2R_mIL2_(4GS)2_9R_C TCell.001 60
BLOCK_iT_Alexa_Fluor_555_siRNA ALL_LIGANDS_EQUIMOLAR (C7-C18)
TCell.002 61 NLS_Cas9_gRNA_RNP N/A TCell.002 62 NLS_Cas9_gRNA_RNP
IL2R_mIL2_(4GS)2_9R_N TCell.002 63 NLS_Cas9_gRNA_RNP
CD3_mCD3Ab_(4GS)2_9R_N TCell.002 64 NLS_Cas9_gRNA_RNP
CD45_mSiglec_(4GS)2_9R_C TCell.002 65 NLS_Cas9_gRNA_RNP
CD28_mCD86_(4GS)2_9R_N_2 TCell.002 66 NLS_Cas9_gRNA_RNP
CD3_mCD3Ab_(4GS)2_9R_N + CD28_mCD86_(4GS)2_9R_N_2 TCell.002 67
NLS_Cas9_gRNA_RNP CD3_mCD3Ab_(4GS)2_9R_N + CD28_mCD86_(4GS)2_9R_N_3
+ CD45_mSiglec_(4GS)2_9R_C TCell.002 68 NLS_Cas9_gRNA_RNP
CD3_mCD3Ab_(4GS)2_9R_N + CD28_mCD86_(4GS)2_9R_N_3 +
CD45_mSiglec_(4GS)2_9R_C + IL2R_mIL2_(4GS)2_9R_N HSC.004 69
Cy5_EGFP_mRNA N/A HSC.004 70 Cy5_EGFP_mRNA N/A HSC.004 71
Cy5_EGFP_mRNA N/A HSC.004 72 Cy5_EGFP_mRNA ESELLg_mESEL(4GS)2_9R_N
HSC.004 73 Cy5_EGFP_mRNA ESELLg_mESEL(4GS)2_9R_N +
cKit_rmSCF_(4GS)2_9R_N HSC.004 74 Cy5_EGFP_mRNA
cKit_rmSCF_(4GS)2_9R_N CynoBM.002 75 NLS_Cas9_gRNA_EGFP_RNP N/A
CynoBM.002 76 NLS_Cas9_gRNA_EGFP_RNP N/A CynoBM.002 77
NLS_Cas9_gRNA_EGFP_RNP IL2R_mIL2_(4GS)2_9R_N CynoBM.002 78
NLS_Cas9_gRNA_EGFP_RNP ESELLg_mESEL(4GS)2_9R_N CynoBM.002 79
NLS_Cas9_gRNA_EGFP_RNP SCF_mcKit_(4GS)2_9R_N CynoBM.002 80
NLS_Cas9_gRNA_EGFP_RNP d CynoBM.002 81 NLS_Cas9_gRNA_EGFP_RNP
IL2R_mIL2_(4GS)2_9RN + ESELLg_mESEL(4GS)2_9R_N +
cKit_mSCF_(4GS)2_9R_N CynoBM.002 82 NLS_Cas9_gRNA_EGFP_RNP + N/A
Cy5_EGFP_mRNA CynoBM.002 83 NLS_Cas9_gRNA_EGFP_RNP +
IL2R_mIL2_(4GS)2_9R_N Cy5_EGFP_mRNA CynoBM.002 84
NLS_Cas9_gRNA_EGFP_RNP + ESELLg_mESEL(4GS)2_9R_N Cy5_EGFP_mRNA
CynoBM.002 85 NLS_Cas9_gRNA_EGFP_RNP + cKit_mSCF_(4GS)2_9R_N
Cy5_EGFP_mRNA CynoBM.002 86 NLS_Cas9_gRNA_EGFP_RNP +
IL2R_mIL2_(4GS)2_9RN + Cy5_EGFP_mRNA ESELLg_mESEL(4GS)2_9R_N +
cKit_mSCF_(4GS)2_9R_N Blood.001 87 Cy5_EGFP_mRNA
CD45_mSiglec_(4GS)2_9R_C Blood.002 88 Cy5_EGFP_mRNA
CD45_mSiglec_(4GS)2_9R_C Blood.002 89 Cy5_EGFP_mRNA
CD45_mSiglec_(4GS)2_9R_C Blood.002 90 Cy5_EGFP_mRNA N/A Blood.002
91 Cy5_EGFP_mRNA N/A Blood.002 92 Vehicle CD45_mSiglec_(4GS)2_9R_C
Project Cat. An. C:P +/- Code Spec. Spec. Ratio Ratio TCell.001 1
PLR10 pLE100: 2:1 2:1 pDE100 TCell.001 2 PLK10_PEG22 pLE100: 2:1
2:1 pDE100 TCell.001 3 TL pLE100: 2:1 2:1 PDE100 TCell.001 4 TL
pLE100: 2:1 2:1 PDE100 TCell.001 5 TL pLE100: 2:1 2:1 pDE100
TCell.001 6 TL pLE100: 2:1 2:1 pDE100 TCell.001 7 TL pLE100: 2:1
2:1 pDE100 TCell.001 8 TL pLE100: 2:1 2:1 pDE100 TCell.001 9 TL
pLE100: 2:1 2:1 pDE100 TCell.001 10 TL pLE100: 2:1 2:1 pDE100
TCell.001 11 TL pLE100: 2:1 2:1 PDE100 TCell.001 12 TL pLE100: 2:1
2:1 pDE100 TCell.001 13 TL pLE100: 2:1 2:1 pDE100 TCell.001 14 TL
pLE100: 2:1 2:1 PDE100 TCell.001 15 TL pLE100: 2:1 2:1 pDE100
TCell.001 16 PLR10 pLE100: 1.35:1 0.82:1 pDE100 TCell.001 17
PLK10_PEG22 pLE100: 1.35:1 0.82:1 pDE100 TCell.001 18 TL pLE100:
1.35:1 0.82:1 pDE100 TCell.001 19 TL pLE100: 1.35:1 0.82:1 pDE100
TCell.001 20 TL pLE100: 1.35:1 0.82:1 pDE100 TCell.001 21 TL
pLE100: 1.35:1 0.82:1 pDE100 TCell.001 22 TL pLE100: 1.35:1 0.82:1
PDE100 TCell.001 23 TL pLE100: 1.35:1 0.82:1 pDE100 TCell.001 24 TL
pLE100: 1.35:1 0.82:1 pDE100 TCell.001 25 TL pLE100: 1.35:1 0.82:1
PDE100 TCell.001 26 TL pLE100: 1.35:1 0.82:1 pDE100 TCell.001 27 TL
pLE100: 1.35:1 0.82:1 pDE100 TCell.001 28 TL pLE100: 1.35:1 0.82:1
pDE100 TCell.001 29 TL pLE100: 1.35:1 0.82:1 pDE100 TCell.001 30 TL
pLE100: 1.35:1 0.82:1 PDE100 TCell.001 31 PLR10 pLE100: 2:1 2:1
pDE100 TCell.001 32 PLK10_PEG22 pLE100: 2:1 2:1 pDE100 TCell.001 33
TL pLE100: 2:1 2:1 PDE100 TCell.001 34 TL pLE100: 2:1 2:1 pDE100
TCell.001 35 TL pLE100: 2:1 2:1 PDE100 TCell.001 36 TL pLE100: 2:1
2:1 pDE100 TCell.001 37 TL pLE100: 2:1 2:1 pDE100 TCell.001 38 TL
pLE100: 2:1 2:1 pDE100 TCell.001 39 TL pLE100: 2:1 2:1 pDE100
TCell.001 40 TL pLE100: 2:1 2:1 pDE100 TCell.001 41 TL pLE100: 2:1
2:1 PDE100 TCell.001 42 TL pLE100: 2:1 2:1 pDE100 TCell.001 43 TL
pLE100: 2:1 2:1 pDE100 TCell.001 44 TL pLE100: 2:1 2:1 PDE100
TCell.001 45 TL pLE100: 2:1 2:1 PDE100 TCell.001 46 PLR10 pLE100:
2:1 2:1 pDE100 TCell.001 47 PLK10_PEG22 pLE100: 2:1 2:1 pDE100
TCell.001 48 TL pLE100: 2:1 2:1 pDE100 TCell.001 49 TL pLE100: 2:1
2:1 PDE100 TCell.001 50 TL pLE100: 2:1 2:1 pDE100 TCell.001 51 TL
pLE100: 2:1 2:1 pDE100 TCell.001 52 TL pLE100: 2:1 2:1 pDE100
TCell.001 53 TL pLE100: 2:1 2:1 pDE100 TCell.001 54 TL pLE100: 2:1
2:1 pDE100 TCell.001 55 TL pLE100: 2:1 2:1 PDE100 TCell.001 56 TL
pLE100: 2:1 2:1 pDE100 TCell.001 57 TL pLE100: 2:1 2:1 pDE100
TCell.001 58 TL pLE100: 2:1 2:1 PDE100 TCell.001 59 TL pLE100: 2:1
2:1 pDE100 TCell.001 60 TL pLE100: 2:1 2:1 pDE100 TCell.002 61
PLR10 pLE100: 2:1 2:1 pDE100 TCell.002 62 TL pLE100: 2:1 2:1
pDE100
TCell.002 63 TL pLE100: 2:1 2:1 pDE100 TCell.002 64 TL pLE100: 2:1
2:1 pDE100 TCell.002 65 TL pLE100: 2:1 2:1 pDE100 TCell.002 66 TL
pLE100: 2:1 2:1 PDE100 TCell.002 67 TL pLE100: 2:1 2:1 pDE100
TCell.002 68 TL pLE100: 2:1 2:1 PDE100 HSC.004 69 PLR10 pLE20 2:1
2:1 HSC.004 70 PLR50 pLE20 2:1 2:1 HSC.004 71 PLK10_PEG22 pLE20 2:1
2:1 HSC.004 72 TL pLE20 2:1 2:1 HSC.004 73 TL pLE20 2:1 2:1 HSC.004
74 TL pLE20 2:1 2:1 CynoBM.002 75 PLR10 pLE100: 2:1 2:1 PDE100
CynoBM.002 76 mH4_K 16Ac_1:mH2A_1 pLE100: 2:1 2:1 PDE100 CynoBM.002
77 TL pLE100: 2:1 2:1 pDE100 CynoBM.002 78 TL pLE100: 2:1 2:1
pDE100 CynoBM.002 79 TL pLE100: 2:1 2:1 pDE100 CynoBM.002 80 TL
pLE100: 2:1 2:1 pDE100 CynoBM.002 81 TL pLE100: 2:1 2:1 pDE100
CynoBM.002 82 PLR50 pLE100: 2:1 2:1 pDE100 CynoBM.002 83 TL pLE100:
2:1 2:1 pDE100 CynoBM.002 84 TL pLE100: 2:1 2:1 pDE100 CynoBM.002
85 TL pLE100: 2:1 2:1 PDE100 CynoBM.002 86 TL pLE100: 2:1 2:1
pDE100 Blood.001 87 TL pLE100 1.35:1 0.82:1 Blood.002 88 TL pLE100
1.35:1 0.82:1 Blood.002 89 TL pLE100 1.35:1 0.82:1 Blood.002 90
PLK30_PEG113 pLE100 Blood.002 91 PLR50 pLE100 Blood.002 92 TL
pLE100 N/A 1.93:1 *Subcellular trafficking peptides used in the
nanoparticle formulations were nuclear localization signal peptides
conjugated to certain payloads (e.g., "NLS_Cas9 . . . ") *Cationic
species used in the nanoparticle formulations were conjugated to
the targeting ligands (TL) as a poly(arginine) chain with amino
acid length 9 (9R). Nanoparticles without targeting ligands
contained the non-conjugated cationic species poly(arginine) AA
chain with length 10 (PLR10) or PEGylated poly(lysine) with AA
chain length of 10. All cationic species in the table have L:D
isomer ratios of 1:0. *HSC = hematopoietic stem cells; BM = bone
marrow cells; Tcell = T cells; blood = whole blood; cynoBM =
cynomolgus bone marrow
Materials and Methods
Ligand Synthesis
[0431] Most targeting ligand sequences were designed in-house and
custom manufactured by 3rd party commercial providers. Peptide
ligands were derived from native polypeptide sequences and in some
cases, mutated to improve binding affinity. Computational analysis
of binding kinetics and the determination of optimal mutations was
achieved through the use of Rosetta software. In the case where
targeting ligands were manufactured in-house, the method and
materials were as follows: [0432] Peptides were synthesized using
standard Fmoc-based solid-phase peptide synthesis (SPPS). Peptides
were synthesized on Rink-amide AM resin. Amino acid couplings were
performed with
O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HCTU) coupling reagent and N-methylmorpholine
(NMM) in dimethyl formamide (DMF). Deprotection and cleavage of
peptides were performed with trifluoroacetic acid (TFA),
triisopropyl silane (TIPS), and water. Crude peptide mixtures were
purified by reverse-phase HPLC (RP-HPLC). Pure peptide fractions
were frozen and lyophilized to yield purified peptides.
Nanoparticle Synthesis
[0433] Nanoparticles were synthesized at room temperature, 37C or a
differential of 37C and room temperature between cationic and
anionic components. Solutions were prepared in aqueous buffers
utilizing natural electrostatic interactions during mixing of
cationic and anionic components. At the start, anionic components
were dissolved in Tris buffer (30 mM-60 mM; pH=7.4-9) or HEPES
buffer (30 mM, pH=5.5) while cationic components were dissolved in
HEPES buffer (30 mM-60 mM, pH=5-6.5).
[0434] Specifically, payloads (e.g., genetic material (RNA or DNA),
genetic material-protein-nuclear localization signal polypeptide
complex (ribonucleoprotein), or polypeptide) were reconstituted in
a basic, neutral or acidic buffer. For analytical purposes, the
payload was manufactured to be covalently tagged with or
genetically encode a fluorophore. With pDNA payloads, a Cy5-tagged
peptide nucleic acid (PNA) specific to TATATA tandem repeats was
used to fluorescently tag fluorescent reporter vectors and
fluorescent reporter-therapeutic gene vectors. A timed-release
component that may also serve as a negatively charged condensing
species (e.g. poly(glutamic acid)) was also reconstituted in a
basic, neutral or acidic buffer. Targeting ligands with a wild-type
derived or wild-type mutated targeting peptide conjugated to a
linker-anchor sequence were reconstituted in acidic buffer. In the
case where additional condensing species or nuclear localization
signal peptides were included in the nanoparticle, these were also
reconstituted in buffer as 0.03% w/v working solutions for cationic
species, and 0.015% w/v for anionic species. Experiments were also
conducted with 0.1% w/v working solutions for cationic species and
0.1% w/v for anionic species. All polypeptides, except those
complexing with genetic material, were sonicated for ten minutes to
improve solubilization.
[0435] Each separately reconstituted component of the nanoparticle
was then mixed in the order of addition that was being
investigated. Different orders of additions investigated include:
[0436] 1) payload<cationic species [0437] 2) payload<cationic
species (anchor)<cationic species (anchor-linker-ligand) [0438]
3) payload<anionic species<cationic species [0439] 4)
payload<cationic species<anionic species [0440] 5)
payload<cationic species (anchor)<cationic species
(anchor-linker-ligand)<anionic species [0441] 6)
payload<anionic species<cationic species (anchor)+cationic
species (anchor-linker-ligand) [0442] 7) payload+anionic
species<cationic species (anchor)+cationic species
(anchor-linker-ligand) [0443] 8) payload 1 (ribonucleoprotein or
other genetic/protein material)<cationic species (histone
fragment, NLS or charged polypeptide anchor without
linker-ligand)<anionic species [0444] 9) payload 1
(ribonucleoprotein or other genetic/protein material)<cationic
species (histone fragment, NLS or charged polypeptide anchor
without linker-ligand)<anionic species<cationic species
(histone fragment, NLS, or charged polypeptide anchor with or
without linker-ligand) [0445] 10) payload 1 (ribonucleoprotein or
other genetic/protein material)<cationic species (histone
fragment, NLS or charged polypeptide anchor without
linker-ligand)<payload 2/3/4 (one or more payloads)<cationic
species (histone fragment, NLS, or charged polypeptide anchor with
or without linker-ligand) [0446] 11) payload 1 (ribonucleoprotein
or other genetic/protein material)<cationic species (histone
fragment, NLS or charged polypeptide anchor without
linker-ligand)<payload 2/3/4 (one or more payloads)+anionic
species<cationic species (histone fragment, NLS, or charged
polypeptide anchor with or without linker-ligand) [0447] 12)
payload 1/2/3/4 (one or more ribonucleoprotein, protein or nucleic
acid payloads)+anionic species<cationic species (histone
fragment, NLS, or charged polypeptide anchor with or without
linker-ligand) [0448] 13) payload 1/2/3/4 (one or more
ribonucleoprotein, protein or nucleic acid payloads)<cationic
species (histone fragment, NLS, or charged polypeptide anchor with
or without linker-ligand)
Cell Culture
[0449] T cells
[0450] 24 hours prior to transfection, a cryovial containing 20M
human primary Pan-T cells (Stemcell #70024) was thawed and seeded
in 4.times.66 wells of 4 96-well plates at 200 .mu.l and 75,000
cells/well (1.5E6 cells/ml). Cells were cultured in antibiotic free
RPMI 1640 media (Thermofisher #11875119) supplemented with 10% FBS
and L-glutamine, and maintained by exchanging the media every 2
days.
Hematopoietic Stem Cells (HSC)
[0451] 24 hours prior to transfection a cryovial containing 500 k
human primary CD34+ cells (Stemcell #70002) was thawed and seeded
in 48 wells of a 96-well plate, at 200 .mu.l and 10-12 k cells per
well. The culture media consisted of Stemspan SFEM 11 (Stemcell
#09605) supplemented with 10% FBS, 25 ng/ml TPO, 50 ng/ml Flt-3
ligand, and 50 ng/ml SCF and the cells were maintained by
exchanging the media every 2 days.
Cynomolgus Bone Marrow (HSC)
[0452] 48 hours prior to transfection, a cryovial containing 1.25M
Cynomolgus monkey bone marrow cells (IQ Biosciences # IQB-MnBM1)
was thawed and 48 wells of a round bottom 96-well plate, were
seeded at 200 .mu.l and -30 k cells/well. The cells are cultured in
antibiotic free RPMI 1640 media supplemented with 12% FBS, and
maintained by exchanging the media every 2 days.
Human Whole Blood
[0453] 5 mL of whole blood was drawn through venous puncture. 1 mL
was mixed with 14 mL of PBS. Nanoparticles were either directly
transfected into 15 mL tubes, or 100 .mu.l of blood was titrated
into each well of a 96-well plate prior to nanoparticle
transfection.
Transfection
[0454] After forming stock solutions of nanoparticles, 10 .mu.l of
nanoparticles were added per well of 96-well plates and incubated
without changes to cell culture conditions or supplementation of
media (See Table 6). 96-well plates were maintained during live
cell imaging via a BioTek Cytation 5 under a CO2 and temperature
controlled environment.
TABLE-US-00011 TABLE 6 Dosage per well Volume of Nanoparticle
Payload (96 well plate) Suspension mRNA 100 ng mRNA 10 ul CRISPR
RNP 100 ng sgRNA, 10 ul pDNA 200 ng pDNA 10 ul siRNA 50 ng 10
ul
Analysis
Condensation and Inclusion Curves
[0455] Condensation curves were generated by mixing 50 .mu.l
solutions containing 0.0044 ug/.mu.l of hemoglobin subunit beta
(HBB) gRNA or von Willebrand factor (VWF)-EGFP-pDNA with pDNA
binding site or mRNA or siRNA with 1 .mu.l of SYBR 0.4.times.
suspended in 30 mM Tris buffer (pH=7.4-8.5). HBB gRNA was present
as complexed in RNP. The fluorescence emission from intercalated
SYBR Gold was monitored before and after a single addition of
PLE20, PLE35, PLE100, or PLE100:PDE100 (1:1 D:L ratio) where the
carboxylate-to-phosphate (C:P) ratio ranged between 1 and 150.
Afterwards, cationic species were added in order to reach the
desired amine to phosphate (N:P) or amine to phosphate+carboxylate
[N:(P+C)] ratios. Representative cationic species included PLR10,
PLR50, PLR150, anchor-linker peptides, various mutated targeting
ligands conjugated to GGGGSGGGGS (SEQ ID NO: xx) linker conjugated
to a charged poly(arginine) chain (i.e. internal name:
SCF_mcKit_(4GS)2_9R_C), Histone_H3K4(Me3) peptide [1-22]
(mH3_K4Me3_1), Histone_H4K16(Ac) peptide [1-20] (mH4_K16Ac_1),
Histone_H2A peptide [1-20] (mH2A_1), corresponding to different
positive to negative charge ratios (CR). In some experiments,
cationic species were added prior to anionic species according to
the above instructions.
[0456] Inclusion curves were obtained after performing multiple
additions of SYBR GOLD 0.2.times. diluted in Tris buffer 30 mM
(pH=7.4) to nanoparticles suspended in 60 mM HEPES (pH=5.5)
solutions containing known amounts (100 to 600 ng) of
VWF-EGFP-pDNA, gRNA HBB, Alexa555 Block-IT-siRNA encapsulated in
different nanoparticle formulations.
[0457] Fluorescence emissions from intercalated SYBR Gold in the
GFP channel were recorded in a flat bottom, half area, 96
well-plate using a Synergy Neo2 Hybrid Multi-mode reader (Biotek,
USA) or a CLARIOstar Microplate reader (BMG, Germany).
Nanoparticle Tracking Analysis (Zeta)
[0458] The hydrodynamic diameter and zeta potential of the
nanoparticle formulations were investigated by nanoparticle
tracking analysis using a ZetaView instrument (Particle Metrix,
Germany). Samples are diluted 1:100 in PBS (1:12) before injection
into the instrument. To obtain the measurement, the camera settings
are adjusted to the optimal sensitivity and particles/frame
(.about.100-150) before analysis.
Fluorescence Microscopy--BioTek Cytation 5
[0459] A Cytation 5 high-content screening live-cell imaging
microscope (BioTek, USA) was utilized to image transfection
efficiency prior to evaluation by flow cytometry. Briefly, cells
were imaged prior to transfection, in 15 m increments
post-transfection for 4 h, and then in 2 h increments for the
following 12 hours utilizing the GFP and/or Cy5 channels as well as
bright field under a 10.times. objective. Images were subsequently
gathered as representative of continuous kinetics or discrete 1-18,
24, 36, or 48-hour time-points.
Flow-Cytometry
[0460] Cell labeling experiments were conducted performing a
washing step to remove cell media followed by incubation of the
cells with Zombie NIR viability kit stain and/or CellEvent.TM.
Caspase-3/7 Green (Invitrogen, U.S.A.) dissolved in PBS at room
temperature for 30 minutes. The total volume of the viability
labeling mixture was 25 .mu.l per well. A panel of fluorescent
primary antibodies was then added to the mixture (0.25 .mu.l of
each antibody per well) and left incubating for 15 minutes.
Positive controls and negative single-channel controls were
generated utilizing UltraComp eBeads Compensation Beads and
Negative Beads or Cy5 nuclear stains of live cells. All incubation
steps were performed on a rotary shaker and in the dark. Attune
multiparametric flow cytometry measurements were conducted on live
cells using an Attune NxT Flow Cytometer (ThermoFisher, USA) after
appropriate compensations among different channels have been
applied. Representative populations of cells were chosen by
selection of appropriate gates of forward and side scattering
intensities. The detection of cell fluorescence was continued until
at least 10000 events had been collected.
Results/Data
FIG. 19-FIG. 44: Condensation Data
[0461] FIG. 19. (a) SYBR Gold exclusion assay showing fluorescence
intensity variations as a function of positive to negative charge
ratio (CR) in nanoparticles containing VWF-EGFP pDNA with PNA
payload initially intercalated with SYBR Gold. The carboxylate to
phosphate (C:P) ratios shown in the legend are based on the
nanoparticle's ratio of carboxylate groups on anionic polypeptides
species (PLE100) to phosphate groups on the genetic material of the
payload. CR was increased via stepwise addition of cationic PLR150.
The fluorescence decrease observed show that increasing the CR
through addition of PLR150 causes SYBR to be displaced from the
payload as the particle condenses. Additionally, condensation
remains consistent across various c:p ratios. Blank solutions
contain SYBR Gold in absence of the payload. (b) Fluorescence
intensity variations as a function of the positive to negative
charge ratio (CR) in nanoparticles without PLE100.
[0462] FIG. 20. SYBR Gold exclusion assay showing fluorescence
intensity variations as a function of positive to negative charge
ratio (CR*) in nanoparticles containing NLS-CAS9-NLS RNP complexed
w/ HBB gRNA payload initially intercalated with SYBR Gold.
Additionally, determination of CR* does not include the negatively
charged portion of the gRNA shielded by complexation with cas9. The
carboxylate to phosphate (C:P) ratios shown in the legend are based
on the nanoparticle's ratio of carboxylate groups on anionic
polypeptides species (PLE100) to phosphate groups on the genetic
material of the payload.
[0463] CR was increased via stepwise addition of cationic PLR150.
Blank solutions contain SYBR Gold in absence of the payload. The
fluorescence decrease observed show that increasing the CR through
addition of PLR150 causes SYBR to be displaced from the payload as
the particle condenses. Additionally, condensation remains
consistent across various c:p ratios.
[0464] FIG. 21. SYBR Gold exclusion assay showing fluorescence
intensity variations as a function of positive to negative charge
ratio (CR) in nanoparticles containing gRNA HBB payloads initially
intercalated with SYBR Gold. The carboxylate to phosphate (C:P)
ratios shown in the legend are based on the nanoparticle's ratio of
carboxylate groups on anionic polypeptides species (PLE100) to
phosphate groups on the genetic material of the payload.
[0465] CR was increased via stepwise addition of PLR150. Blank
solutions contain SYBR Gold in absence of the payload. The
fluorescence decrease observed show that increasing the CR through
addition of PLR150 causes SYBR to be displaced from the payload as
the particle condenses. Additionally, condensation with respect to
CR remains consistent across various C:P ratios.
TABLE-US-00012 TABLE 7 Hydrodynamic diameter and zeta potential for
some formulations were measured at the condensation end- points and
are reported in the following table. Hydrodynamic diameter Zeta
Potential Payload C:P [nm] [mV] pDNA 0 120 .+-. 49 -- HBB gRNA 0 99
.+-. 32 6.7 .+-. 0.6 RNP (NLS-Cas9-NLS 0 90 .+-. 36 -0.6 .+-. 0.9
and HBB gRNA) RNP (NLS-Cas9-NLS 15 110 .+-. 49 26.7 .+-. 1.sup. and
HBB gRNA pDNA 15 88 .+-. 49 11.7 .+-. 0.6
FIG. 22-FIG. 24: Condensation Curves with Peptide SCF_rmAc-cKit
(4GS)2_9R_C as Cationic Material
[0466] FIG. 22. (a)(b) SYBR Gold exclusion assay showing
fluorescence intensity variations as a function of positive to
negative charge ratio (CR) in nanoparticles containing HBB gRNA
payload initially intercalated with SYBR Gold. The carboxylate to
phosphate (C:P) ratios shown in the legend are based on the
nanoparticle's ratio of carboxylate groups on anionic polypeptides
species (PLE100) to phosphate groups on the genetic material of the
payload. CR was increased via stepwise addition of cationic mutated
cKit targeting ligand conjugated to a (GGGS)2 linker conjugated to
positively charged poly(arginine) (internal ligand name:
SCF_rmAc-cKit(4GS)2_9R_C). The fluorescence decrease observed show
that increasing the CR through addition of
SCF_rmAc-cKit_(4GS)2_9R_C causes SYBR to be displaced from the
payload as the particle condenses. Additionally, condensation
remains consistent across various c:p ratios. Blank solutions
contain SYBR Gold in absence of the payload.
[0467] FIG. 23. (a)(b) SYBR Gold exclusion assay showing
fluorescence intensity variations as a function of positive to
negative charge ratio (CR) in nanoparticles containing NLS-CAS9-NLS
RNP complexed w/ HBB gRNA payload initially intercalated with SYBR
Gold. The carboxylate to phosphate (C:P) ratios shown in the legend
are based on the nanoparticle's ratio of carboxylate groups on
anionic polypeptides species (PLE100) to phosphate groups on the
genetic material of the payload.
[0468] CR was increased via stepwise addition of cationic mutated
cKit targeting ligand conjugated to a (GGGS)2 linker conjugated to
positively charged poly(arginine) (internal ligand name:
SCF_rmAc-cKit_(4GS)2_9R_C). The fluorescence decrease observed show
that increasing the CR through addition of
SCF_rmAc-cKit_(4GS)2_9R_C causes SYBR to be displaced from the
payload as the particle condenses. Additionally, condensation
remains consistent across various c:p ratios. Blank solutions
contain SYBR Gold in absence of the payload.
[0469] FIG. 24. (a)(b) SYBR Gold exclusion assay showing
fluorescence intensity variations as a function of positive to
negative charge ratio (CR) in nanoparticles containing VWF-EGFP
pDNA with PNA payload initially intercalated with SYBR Gold. The
carboxylate to phosphate (C:P) ratios shown in the legend are based
on the nanoparticle's ratio of carboxylate groups on anionic
polypeptides species (PLE100) to phosphate groups on the genetic
material of the payload.
[0470] CR was increased via stepwise addition of cationic mutated
cKit targeting ligand conjugated to a (GGGS)2 linker conjugated to
positively charged poly(arginine) (internal ligand name:
SCF_rmAc-cKit_(4GS)2_9R_C). The fluorescence decrease observed show
that increasing the CR through addition of
SCF_rmAc-cKit_(4GS)2_9R_C causes SYBR to be displaced from the
payload as the particle condenses. Additionally, condensation
remains consistent across various c:p ratios. Blank solutions
contain SYBR Gold in absence of the payload.
FIG. 25-FIG. 26: Condensation Curves with Histone H3K4Me as
Cationic Material
[0471] FIG. 25. SYBR Gold exclusion assay showing fluorescence
intensity variations as a function of positive to negative charge
ratio (CR) in nanoparticles containing VWF-EGFP pDNA with PNA
payload initially intercalated with SYBR Gold. The carboxylate to
phosphate (C:P) ratios shown in the legend are based on the
nanoparticle's ratio of carboxylate groups on anionic polypeptides
species (PLE100) to phosphate groups on the genetic material of the
payload. CR was increased via stepwise addition of cationic mutated
Histone_H3K4(Me3) peptide [1-22] (internal peptide name
mH3_K4Me3_1). The fluorescence changes observed show that
increasing the CR through addition of mH3_K4Me3_1, in the presence
of PLE100, fail to sufficiently cause SYBR to be displaced from the
payload. Blank solutions contain SYBR Gold in absence of the
payload.
[0472] FIG. 26. (a) SYBR Gold exclusion assay showing fluorescence
intensity variations as a function of positive to negative charge
ratio (CR) in nanoparticles containing NLS-CAS9-NLS RNP complexed
w/ HBB gRNA payload initially intercalated with SYBR Gold. The
carboxylate to phosphate (C:P) ratios shown in the legend are based
on the nanoparticle's ratio of carboxylate groups on anionic
polypeptides species (PLE100) to phosphate groups on the genetic
material of the payload. CR was increased via stepwise addition of
cationic mutated Histone_H3K4(Me3) peptide [1-22] (internal peptide
name mH3_K4Me3_1). The fluorescence changes observed show that
increasing the CR through addition of mH3_K4Me3_1, in the presence
of PLE100, fails to consistently cause SYBR to be displaced from
the payload. However, Histone_H3K4(Me3) is shown to be an effective
condensing agent at CR.ltoreq.8:1 in the absence of anionic
polypeptide.
FIG. 27-FIG. 30: Condensation Curves with Peptide CD45
aSiglec_(4GS)2_9R_C as Cationic Material
[0473] FIG. 27. SYBR Gold exclusion assay showing fluorescence
intensity variations as a function of positive to negative charge
ratio (CR) in nanoparticles containing VWF-EGFP pDNA with PNA
payload initially intercalated with SYBR Gold. The carboxylate to
phosphate (C:P) ratios shown in the legend are based on the
nanoparticle's ratio of carboxylate groups on anionic polypeptides
species (PLE100) to phosphate groups on the genetic material of the
payload. CR was increased via stepwise addition of cationic mutated
CD45 receptor targeting ligand conjugated to a (GGGS)2 linker
conjugated to positively charged poly(arginine) (internal ligand
name: CD45_mSiglec_(4GS)2_9R_C). Empty symbols represent blank
solutions containing SYBR Gold in absence of the payload.
[0474] The fluorescence decrease observed show that increasing the
CR through addition of CD45_mSiglec_(4GS)2_9R_C causes SYBR to be
displaced from the payload as the particle condenses. Additionally,
condensation remains consistent across various C:P ratios.
[0475] FIG. 28. SYBR Gold exclusion assay showing fluorescence
intensity variations as a function of positive to negative charge
ratio (CR) in nanoparticles containing Cy5-EGFP mRNA payload
initially intercalated with SYBR Gold. The carboxylate to phosphate
(C:P) ratios shown in the legend are based on the nanoparticle's
ratio of carboxylate groups on anionic polypeptides species
(PLE100) to phosphate groups on the genetic material of the
payload.
[0476] CR was increased via stepwise addition of cationic mutated
CD45 receptor targeting ligand conjugated to a (GGGS)2 linker
conjugated to positively charged poly(arginine) (internal ligand
name: CD45_mSiglec_(4GS)2_9R_C). The fluorescence decrease observed
show that increasing the CR through addition of
CD45_mSiglec_(4GS)2_9R_C causes SYBR to be displaced from the
payload as the particle condenses. Additionally, condensation
remains consistent across various c:p ratios.
[0477] FIG. 29. SYBR Gold exclusion assay showing fluorescence
intensity variations as a function of positive to negative charge
ratio (CR) in nanoparticles containing BLOCK-iT Alexa Fluor 555
siRNA payload initially intercalated with SYBR Gold. The
carboxylate to phosphate (C:P) ratios shown in the legend are based
on the nanoparticle's ratio of carboxylate groups on anionic
polypeptides species (PLE100) to phosphate groups on the genetic
material of the payload. CR was increased via stepwise addition of
cationic mutated CD45 receptor targeting ligand conjugated to a
(GGGS)2 linker conjugated to positively charged poly(arginine)
(internal ligand name: CD45_mSiglec_(4GS)2_9R_C). The fluorescence
decrease observed show that increasing the CR through addition of
CD45_mSiglec_(4GS)2_9R_C causes SYBR to be displaced from the
payload as the particle condenses. Additionally, condensation
remains consistent across various C:P ratios.
[0478] FIG. 30. (a) SYBR Gold exclusion assay showing fluorescence
intensity variations as a function of positive to negative charge
ratio (CR) in nanoparticles containing NLS-Cas9-EGFP RNP complexed
to HBB gRNA payload initially intercalated with SYBR Gold. The
carboxylate to phosphate (C:P) ratios shown in the legend are based
on the nanoparticle's ratio of carboxylate groups on anionic
polypeptides species (PLE100) to phosphate groups on the genetic
material of the payload.
[0479] CR was increased via stepwise addition of cationic mutated
CD45 receptor targeting ligand conjugated to a (GGGS)2 linker
conjugated to positively charged poly(arginine) (internal ligand
name: CD45_mSiglec_(4GS)2_9R_C). Filled symbols represent blank
solutions containing SYBR Gold in absence of the payload.
[0480] The fluorescence decrease observed show that increasing the
CR through addition of CD45_mSiglec_(4GS)2_9R_C causes SYBR to be
displaced from the payload as the particle condenses. Additionally,
condensation remains consistent across various c:p ratios.
(b) Representative image of hydrodynamic diameter distribution for
nanoparticles without PLE and having a charge ratio=22. The mean
diameter is <d>=134 nm.+-.65.
TABLE-US-00013 TABLE 8 Hydrodynamic diameter and zeta potential for
some formulations were measured at the condensation end-points and
are reported in the following table. Hydrodynamic diameter Zeta
Potential Payload C:P Cationic Peptide [nm] [mV] RNP (NLS- 0
CD45_mSiglec_(4GS)2_9R_C 134 .+-. 65 13 .+-. 1 Cas9-EGFP and gRNA)
RNP (NLS- 10 CD45_mSiglec_(4GS)2_9R_C 166 .+-. 75 19.2 .+-. 1.sup.
Cas9-EGFP and gRNA RNP (NLS- 20 CD45_mSiglec_(4GS)2_9R_C 179 .+-.
92 21 .+-. 1 Cas9-EGFP and gRNA
FIG. 31-FIG. 34: Inclusion Curves
[0481] FIG. 31. SYBR Gold inclusion assay showing fluorescence
intensity variations as a function of stepwise SYBR addition to
different nanoparticles formulations all containing 150 ng of
BLOCK-iT Alexa Fluor 555 siRNA payload. The delta change in
fluorescence from 0 .mu.l to 50 .mu.l of SYBR indicates the
stability of the nanoparticle formulations. The less stably
condensed a formulation, the more likely SYBR Gold is to
intercalate with the genetic payload. Lipofectamine RNAiMAX is used
here as a positive control. Tables 2-4.
[0482] FIG. 32. SYBR Gold inclusion assay showing fluorescence
intensity variations as a function of stepwise SYBR addition to
different nanoparticles formulations all containing 300 ng the HBB
gRNA payload. The delta change in fluorescence from 0 .mu.l to 50
.mu.l of SYBR indicates the stability of the nanoparticle
formulations. The less stably condensed a formulation, the more
likely SYBR Gold is to intercalate with the genetic payload.
Lipofectamine CRISPRMAX is used here as a positive control. Tables
2-4.
[0483] FIG. 33. SYBR Gold inclusion assay showing fluorescence
intensity variations as a function of stepwise SYBR addition to
different nanoparticles formulations all containing the Cy5 EGFP
mRNA payload. The delta change in fluorescence from 0 .mu.l to 50
.mu.l of SYBR indicates the stability of the nanoparticle
formulations. The less stably condensed a formulation, the more
likely SYBR Gold is to intercalate with the genetic payload.
Lipofectamine Messenger MAX is used here as a positive control.
Tables 2-4.
[0484] FIG. 34. SYBR Gold inclusion assay showing fluorescence
intensity variations as a function of stepwise SYBR addition to
different nanoparticles formulations all containing 600 ng of
VWF-EGFP pDNA with Cy5 tagged peptide nucleic acid (PNA) Binding
Site payload. The delta change in fluorescence from 0 .mu.l to 50
.mu.l of SYBR indicates the stability of the nanoparticle
formulations. The less stably condensed a formulation, the more
likely SYBR Gold is to intercalate with the genetic payload.
Lipofectamine 3000 is used here as a positive control. Tables
2-4.
FIG. 35-FIG. 44: SYBR Exclusion/Condensation Assays on TC.001 (See
Tables 2-4)
[0485] These data show that formulations used in experiment TC.001
are stable, moreover they show that H2A and H4 histone tail
peptides, unlike H3, are effective condensing agents on their own
for all listed payloads. It also shows that H2A and H4 can be
further combined with anchor-linker-ligands. Finally, evidence is
presented that the subsequent addition of anionic polymers (in this
embodiment, PLE100) does not affect particle stability, or enhances
stability as demonstrated through size and zeta potential
measurements on various anchor-linker-ligand peptides conjugated to
nucleic acid or ribonucleoprotein payloads prior to addition to
anionic polymers.
[0486] FIG. 35. SYBR Gold exclusion assay showing fluorescence
intensity decrease by addition of cationic polypeptide
CD45_mSiglec_(4GS)2_9R_C followed by PLE100 and by further addition
of the cationic polypeptide to RNP. The fluorescence background
signal id due to GFP fluorescence from the RNP.
[0487] FIG. 36. SYBR Gold exclusion assay showing fluorescence
intensity variations by addition of cationic polypeptide
CD45_mSiglec_(4GS)2_9R_C followed by PLE100 and by further addition
of the cationic polypeptide to siRNA and SYBR Gold.
[0488] FIG. 37. SYBR Gold exclusion assay showing fluorescence
intensity variations by addition of cationic polypeptide histone
peptide H2A followed by CD45_mSiglec_(4GS)2_9R_C and by further
addition of PLE100 to RNP of NLS-Cas9-EGFP with HBB gRNA and SYBR
Gold.
[0489] FIG. 38. SYBR Gold exclusion assay showing fluorescence
intensity variations by addition of cationic polypeptide histone
peptide H4 together with CD45_mSiglec_(4GS)2_9R_C and by further
addition of PLE100 to RNP of NLS-Cas9-EGFP with HBB gRNA and SYBR
Gold.
[0490] FIG. 39. SYBR Gold exclusion assay showing fluorescence
intensity variations by addition of cationic polypeptide
CD45_mSiglec_(4GS)2_9R_C fand by further addition of PLE100 to
mRNA.
[0491] FIG. 40. SYBR Gold exclusion assay showing fluorescence
intensity variations by addition histone H4 and by further addition
of CD45-mSiglec-(4GS)2_9R_c and PLE100 to mRNA.
[0492] FIG. 41. SYBR Gold exclusion assay showing fluorescence
intensity variations by addition histone H2A and by further
addition of CD45-mSiglec-(4GS)2_9R_c and PLE100 to mRNA.
[0493] FIG. 42. SYBR Gold exclusion assay from intercalation with
VWF_EGFP pDNA showing fluorescence intensity variations by addition
of cationic polypeptide CD45_mSiglec_(4GS)2_9R_C followed by
PLE100.
[0494] FIG. 43. SYBR Gold exclusion assay from intercalation with
VWF_EGFP pDNA showing fluorescence intensity variations by addition
of histone H4, followed by cationic polypeptide
CD45_mSiglec_(4GS)2_9R_C followed by PLE100.
[0495] FIG. 44. SYBR Gold exclusion assay from intercalation with
VWF_EGFP pDNA showing fluorescence intensity variations by addition
of histone H4, followed by cationic polypeptide
CD45_mSiglec_(4GS)2_9R_C followed by PLE100.
FIG. 45-FIG. 83: Physicochemical Data
[0496] Particle size and zeta potential are routine measurements
used in the characterization of colloidal nanomaterials. These
measurements are primarily acquired through light scattering
techniques such as DLS (dynamic light scattering). Nanoparticle
tracking analysis (NTA) utilizes laser scattering microscopy and
image analysis to obtain measurements of particle size and zeta
potential with high resolution.
[0497] Analysis
[0498] Dispersity is a measure of sample heterogeneity and is
determined by the distribution, where a low standard of deviation
and single peak indicates particle uniformity.
[0499] Targeting ligands consisting of polypeptides with a ligand,
(GGGGS)2 linker, and electrostatic anchor domain were synthesized
by solid phase peptide synthesis and used to functionalize the
silica surface (sheddable layer) of particles carrying pEGFP-N1
plasmid DNA payload. The resulting particle size and zeta potential
distributions were obtained by nanoparticle tracking analysis using
a ZetaVIEW instrument (Particle Metrix, Germany).
[0500] FIG. 45. (A) Core Polyplex Size distribution, consisting of
pEGFP-N1 plasmid complexed with H3K4(Me3) and poly(L-Arginine) (29
kD, n=150). (B) Polyplex of FIG. 45A with silica sheddable layer
exhibiting characteristic negative zeta potential and mean particle
size of 124 nm. (C) E Selectin ligand with N terminal anchor and
glycine-serine linker ((GGGGS)2) coated upon the particles shown in
FIG. 45B.
[0501] FIG. 46. Branched Histone Peptide Conjugate Pilot Particle.
Histone H3 peptide with a C-terminal Cysteine was conjugated to 48
kD poly(L-Lysine) with 10% side-chain thiol substitutions. The
final product, purified by centrifugal filtration and molecular
weight exclusion, was used to complex plasmid DNA (pEGFP-N1). The
resulting measurements, portrayed in FIG. 46, show a narrow size
distribution. Size Distribution of H3-Poly(L-Lysine) conjugate in
complex with plasmid DNA (pEGFP-N1)
For FIG. 47-FIG. 83, the Data are Indexed by Experiment Number
(Project Code). In Many Cases, this can be Cross-Referenced to the
Project Code of Table 5 (HSC=Hematopoietic Stem Cells; BM=Bone
Marrow Cells; Tcell=T Cells; Blood=Whole Blood; cynoBM=Cynomolgus
Bone Marrow).
[0502] FIG. 47 provides data related to project HSC.001.001.
[0503] FIG. 48 provides data related to project HSC.001.002, which
used H3-poly(L-Lysine) conjugate complexed to PNA-tagged pDNA and
an E-Selectin targeting peptide (ESELLg_mESEL_(4GS)2_9R_N).
[0504] FIG. 49-FIG. 52 provide data for experiments in which
various targeting ligands or stealth molecules were coated upon
silica-coated particles and silica-coated nanodiamonds (for
diagnostic enhanced fluorescent applications). Size and Zeta
Potential distributions are presented with associated statistics.
Targeting ligands were ESELLg_mESEL_(4GS)2_9R_N,
ESELLg_mESEL_(4GS)2_9R_C, CD45_mSiglec_(4GS)2_9R_C, and
Cy5mRNA-SiO2-PEG, respectively.
[0505] Performance of nanoformulations and targeting ligands was
significantly improved in all data that follows--elimination of
silica layer and replacement with a charged anionic sheddable
polypeptide matrix significantly enhanced transfection efficiencies
of nanoparticles across all formulations, with a variety of
payloads and ligand-targeting approaches. However, the
multilayering techniques used in the data above, as well as
enhanced condensation with branched histone complexes and
subsequent peptide matrix engineering (working examples are
presented in Tcell.001, HSC.004, CYNOBM.002, and Blood.002)
demonstrate the flexibility of the techniques (e.g., multilayering)
and core biomaterials (e.g., see entirety of disclosure and
subsequent experiments). All techniques described herein may be
applied to any particle core, whether diagnostic or therapeutic, as
well as to self-assembled materials. For example, branched histones
may be conjugated to linker-ligand domains or co-condensed with a
plurality of embodiments and uses thereof.
[0506] FIG. 53-FIG. 57 depict particles carrying Cy5-EGFP mRNA
payload, complexed with a sheddable poly(glutamic acid) surface
matrix and CD45 ligand. Nanoparticles produced using this
formulation were highly uniform in particle size and zeta
potential. Particles with poly(glutamic acid) added after
SIGLEC-derived peptide association with mRNA (BLOOD.002.88) were
more stable and monodisperse than particles with poly(glutamic
acid) added before SIGLEC-derived association with mRNA and
poly(glutamic acid), indicating that a particular order of addition
can be helpful in forming more stable particles. Additionally,
particles formed from poly(glutamic acid) complexed with
SIGLEC-derived peptides without a phosphate-containing nucleic acid
were highly anionic monodispersed (BLOOD.002.92). Particles formed
from PLR50 with PLE100 added after PLR association with mRNA were
highly stable, monodispersed and cationic (BLOOD.002.91). In
contrast, PLK-PEG association with mRNA prior to PLE100 addition
resulted in very small particles with heterogenous charge
distributions. The efficacy of these order of addition and
SIGLEC-derivative peptaides was demonstrated by flow cytometry data
wherein ligand-targeted SIGLEC-derivative particles resulted in
nearly two orders of magnitude more Cy5 intensity in whole blood
cells despite similar transfection efficiencies to PEGylated
controls.
[0507] FIG. 53 provides data from BLOOD.002.88. Nanoparticles had
zeta potential of -3.32+/-0.29 mV with 90% having diameters less
than 180 nm. These nanoparticles resulted in 58.6% efficient
Cy5_EGFP_mRNA uptake in whole blood according to flow cytometry
data. The narrow and uniform peak is exemplary of excellent charge
distributions and was reproducible in forming net anionic particles
in TCELL.001.18. This demonstrates broad applicability of
SIGLEC-derived targeting peptides for systemic delivery (e.g., see
flow cytometry and imaging data below).
[0508] FIG. 54 provides data from BLOOD.002.89. Nanoparticles
hadzeta potential of -0.25+/-0.12 mV with 90% having diameters less
than 176 nm. These nanoparticles resulted in 58.6% efficient
Cy5_EGFP_mRNA uptake in whole blood respectively according to flow
cytometry data. This demonstrates broad applicability of Siglec
derived targeting peptide for systemic delivery (e.g., see flow
cytometry and imaging data below).
[0509] FIG. 55 provides data from BLOOD.002.90. Nanoparticles had
zeta potential of 2.54+/-0.03 mV with 90% having diameters less
than 99 nm. These nanoparticles resulted in 79.9% efficient
Cy5_EGFP_mRNA uptake in whole blood respectively according to flow
cytometry data (e.g., see flow cytometry and imaging data
below).
[0510] FIG. 56 provides data from BLOOD.002.91. Nanoparticles had
zeta potential of 27.10 FWHM 18.40 mV with 90% having diameters
less than 130 nm. These nanoparticles resulted in 96.7% efficient
Cy5_EGFP_mRNA uptake in whole blood respectively according to flow
cytometry data (e.g., see flow cytometry and imaging data below).
Strongly positively charged zeta potentials led to high
efficiencies and intensities of Cy5+ signal on whole blood cells.
Briefly, in this embodiment, a larger dose of PLR50 (15 .mu.l of
PLR50 0.1% w/v solution) was added to 100 .mu.l pH 5.5 30 mM HEPES
with 2.5 ug Cy5 mRNA (TriLink). After 5 minutes at 37.degree. C.,
1.5 .mu.l of PLE100 0.1% was added to the solution. In contrast,
other experiments involved adding larger relative volumes (5-20% of
total solution volume) of PLE100 to a preformed cationic
polymer+anionic material core.
[0511] FIG. 57 provides data from BLOOD.002.92. Nanoparticles had
zeta potential of -22.16 FWHM 18.40 mV with 90% having diameters
less than 130 nm. These nanoparticles did not result in detectable
Cy5_EGFP_mRNA uptake in whole blood according to flow cytometry
data, as they were not labeled with a fluorophore (e.g., see flow
cytometry and imaging data below). The effective condensation of
these nanoparticles without a payload (vehicle) also has
implications in non-genetic material payload delivery, such as
conjugation of the charged polymer to a small molecule or
chemotherapeutic agent.
[0512] FIG. 58-FIG. 73 depict results from experiments performed to
characterize representative particles containing CRISPR
ribonucleoprotein (RNP) (TCELL.001.01-TCELL.001.15), mRNA
(TCELL.001.16-TCELL.001.30), plasmid DNA
(TCELL.001.31-TCELL.001.45) and siRNA (TCELL.001.46-TCELL.001.60)
and patterned with identical ligands in corresponding groups.
[0513] FIG. 58 provides data from TCELL.001.1. Nanoparticles had
zeta potential of -3.24+/-0.32 mV with 90% having diameters less
than 77 nm. These nanoparticles resulted in 99.16% and 98.47%
efficient CRISPR-GFP-RNP uptake in viable CD4+ and CD8a+ pan T
cells respectively according to flow cytometry data (e.g., see flow
cytometry and imaging data below). These formulations were also
reflective of physicochemical properties of all CYNOBM.002.75, as
well as the cores serving as substrates for subsequent layering in
CYNOBM.002.82-CYNOBM.002.85, wherein the PLR10-coated particle was
complexed with a sheddable anionic coat of one or more anionic
polypeptides, nucleic acids and/or charged macromolecules of a
range of D:L ratios, molecular weights, and compositions.
TCELL.001.1 was subsequently coated in PLE100+mRNA prior to
addition of charged polymers or charged anchor-linker-ligands in
CYNOBM.002.82-CYNOBM.002.85.
[0514] FIG. 59 provides data from TCELL.001.3. Nanoparticles had
zeta potential of -0.98+/-0.08 mV with 90% having diameters less
than 65 nm. Despite ideal size ranges, these nanoparticles resulted
in 11.6% and 13.2% efficient CRISPR-GFP-RNP uptake in viable CD4+
and CD8a+ pan T cells, respectively, according to flow cytometry
data in contrast to the strongly anionic similarly-sized particles
in TCELL.001.1 that achieved .about.99% efficiency in the same cell
populations. The relationship of particle size and stable negative
zeta potential and methods and uses thereof are shown to be
predicable constraints through the experiments described herein. An
ideal nanoparticle has a majority of particles <70 nm with zeta
potentials of <-5 mV, and the sheddable anionic coating methods
described herein as well as multistage-layering sheddable matrices
for codelivery described in CYNOBM.002 achieve stable and extremely
efficient transfection of sensitive primary cells from human and
cynomolgus blood, bone marrow, and specific cells within the
aforementioned. The reduced efficiency of TCELL.001.3 is a marked
contrast to the results of TCELL.01.27, where the same ligands
achieved stable condensation of mRNA at an altered
amine-to-phosphate-to-carboxylate ratio than the one used for this
particular CRISPR formulation (e.g., see flow cytometry and imaging
data below).
[0515] FIG. 60 provides data from TCELL.001.13. Nanoparticles have
zeta potential of 2.19+/-0.08 mV with 90% having diameters less
than 101 nm. See flow cytometry/imaging data below for the
efficiency of CRISPR-GFP-RNP uptake in viable CD4+ and CD8a+ pan T
cells.
[0516] FIG. 61 provides data from TCELL.001.14. Nanoparticles have
zeta potential of -9.37+/-0.16 mV with 90% having diameters less
than 111 nm. These nanoparticles resulted in 25.7% and 28.6%
efficient CRISPR-GFP-RNP uptake in viable CD4+ and CD8a+ pan T
cells respectively according to flow cytometry data. (e.g., see
flow cytometry and imaging data below).
[0517] FIG. 62 provides data from TCELL.001.16.
[0518] FIG. 63 provides data from TCELL.001.18. The size and zeta
potential of these particles demonstrate average particle sizes of
80.9 nm with zeta potentials of -20.26+/-0.15 mV and 90% of
particles with 39.2-129.8 nm diameters, indicating strong particle
stability at a 1.35 carboxylate-to-phosphate (C:P) and 0.85
amine-to-phosphate ratio wherein poly(glutamic acid) was added
following inclusion of the cationic anchor-linker-ligand. Please
reference all zeta potential, size, flow cytometry and microscopy
data of TCELL.001.2, TCELL.001.18, and CYNOBM.002 for additional
general patterns, engineering constraints, observations and
empirical measurements as relate to attaining high-efficiency
primary cell transfections (e.g., see Table 5 and flow cytometry
and imaging data below).
[0519] FIG. 64 provides data from TCELL.001.28. FIG. 65 provides
data from TCELL.001.29. FIG. 66 provides data from TCELL.001.31.
FIG. 67 provides data from TCELL.001.33. FIG. 68 provides data from
TCELL.001.43. FIG. 69 provides data from TCELL.001.44. FIG. 70
provides data from TCELL.001.46. FIG. 71 provides data from
TCELL.001.48. FIG. 72 provides data from TCELL.001.58. FIG. 73
provides data from TCELL.001.59.
[0520] FIG. 74-FIG. 83 depict results characterizing the
formulations used in cynomolgus bone marrow cells.
[0521] FIG. 74 provides data from CYNOBM.002.82. Particles
successfully deleted the BCL11a erythroid enhancer in whole bone
marrow erythroid progenitor cells as evidenced by fetal hemoglobin
protein expression in 3% of live cells. CYNOBM.002.82 nanoparticles
had zeta potential of 2.96+/-0.14 mV with 90% having diameters less
than 132 nm and 50% of particles with diameters less than 30 nm.
These nanoparticles resulted in .about.48%, .about.53%, and
.about.97% efficient CRISPR-GFP-RNP+Cy5_EGFP_mRNA colocalized
uptake of CRISPR RNP and Cy5 mRNA in viable CD3+, CD45+, and CD34+
bone marrow subpopulations, respectively, despite only 11.4%
overall bone marrow viable subpopulation targeting according to
flow cytometry data.
[0522] In contrast, CYNOBM.002.75, with an identical core template
consisting of PLR10, PLE100, PDE100 and Cas9 RNP but without an
mRNA co-delivery component or additional layer of PLR50, exhibited
.about.20%, .about.14%, and .about.100% efficient CRISPR-GFP-RNP
uptake in viable CD3+, CD45+, and CD34+ bone marrow subpopulations,
respectively, and 18.0% overall bone marrow viable subpopulation
targeting according to flow cytometry data.
[0523] With these data, it can be inferred that larger particles
may be less amenable to selective targeting even when minor
enhancements were seen in overall transfection efficiency within a
mixed bone marrow primary population. The effects of bimodal
distributions of particles on primary cell culture transfections
remains to be determined. In prior work, osteoblasts were found to
endocytose 150-200 nm particles with high efficiency. Strikingly,
the majority of population of particles with CYNOBM.002.82 was
below the 85 nm peak, similarly to TCELL.001.1 but with a
positively charged positive matrix of PLR50 surrounding the
underlying polypeptide-ribonucleoprotein-mRNA-protein matrix of
PLE, PDE, mRNA and Cas9 RNP.
[0524] Additionally, 3.0% of overall viable cells were positive for
fetal hemoglobin, with none of these cells being CD34+, suggesting
successful clonal expansion of BCL11a erythroid progenitor knockout
populations within CD34- erythroid progenitor cells. (e.g., see
flow cytometry and imaging data below). The results may also
implicate successful targeting in endothelial cells, osteoblasts,
osteoclasts, and other cells of the bone marrow.
[0525] FIG. 75 provides data from CYNOBM.002.83. Particles
successfully deleted the BCL11a erythroid enhancer in whole bone
marrow erythroid progenitor cells as evidenced by fetal hemoglobin
protein expression in 1.9% of live cells, with none of these cells
being CD34+. The nanoparticles had a zeta potential of -2.47+/-0.33
mV with 90% having diameters less than 206 nm, leading to improved
transfection efficiency vs. CYNOBM.002.03 with the same IL2-mimetic
peptide coating. The large charge distribution with tails at
approximately -50 mV and +25 mV were indicative of a polydisperse
particle population with a variance of particle stabilities,
similarly to CYNOBM.002.83, and in contrast to CYNOBM.002.84 which
has a stable anionic single-peak zeta potential of -18 mV and
corresponding increase in cellular viability compared to other
CRISPR+mRNA co-delivery particle groups
(CYNOBM.002.82-CYNOBM.002.85). The next-best nanoparticle group in
terms of overall cynomolgus bone marrow co-delivery was
CYNOBM.002.86, which demonstrated similar highly net-negatively
charged zeta potential of -20 mV and a corresponding high
efficiency of transfection, CD34 clonal expansion, and fetal
hemoglobin production from BCL11a erythroid enhancer knockout.
These nanoparticles resulted in .about.100% efficient
CRISPR-GFP-RNP+Cy5_EGFP_mRNA uptake in viable CD34+ bone marrow
cells, within mixed cell populations, as well as 8.1% of whole bone
marrow viable subpopulations according to flow cytometry data. The
flow cytometry data indicates induction of selective CD34+
proliferation in cynomolgus bone marrow cells suggesting successful
clonal expansion of BCL11a erythroid progenitor knockout
populations within CD34- erythroid progenitor cells. (e.g., see
flow cytometry and imaging data below). The results also implicate
successful targeting in endothelial cells, osteoblasts,
osteoclasts, and/or other cells of the bone marrow.
[0526] FIG. 76 provides data from CYNOBM.002.84. Particles
successfully deleted the BCL11a erythroid enhancer in whole bone
marrow erythroid progenitor cells as evidenced by fetal hemoglobin
protein expression in 9.5% of live whole bone marrow cells and no
positive fetal hemoglobin measurements in CD34+, CD45 or CD3+
subpopulations despite moderate transfection efficiencies, as
measured by Cy5-mRNA+ and CRISPR-GFP-RNP+ gates in each selective
subpopulation. CYNOBM.002.84 nanoparticles had zeta potential of
-18.07+/-0.71 mV with 90% having diameters less than 205 nm. The
high net-negative charge indicates stable particle formation. These
nanoparticles resulted in 76.5%, 71%, and .about.100% efficient
CRISPR-GFP-RNP+Cy5_EGFP_mRNA uptake in viable CD3+, CD45+, and
CD34+ bone marrow cells, respectively, as well as 25.5% of whole
bone marrow viable subpopulations according to flow cytometry data.
Additionally, 9.5% of overall viable cells were positive for fetal
hemoglobin, with none of these cells being CD34+, suggesting
successful clonal expansion of BCL11a erythroid progenitor knockout
populations within CD34- erythroid progenitor cells. (e.g., see
flow cytometry and imaging data below). The results also implicate
successful targeting in endothelial cells, osteoblasts,
osteoclasts, and/or other cells of the bone marrow.
[0527] FIG. 77 provides data from CYNOBM.002.85. Nanoparticles had
zeta potential of -12.54+/-0.25 mV with 90% having diameters less
than 186 nm. These nanoparticles resulted in .about.33%,
.about.23%, and .about.100% efficient CRISPR-GFP-RNP+Cy5_EGFP_mRNA
uptake in viable CD3+, CD45+, and CD34+ bone marrow cells,
respectively, according to flow cytometry data. (e.g., see flow
cytometry and imaging data below). The results may implicate
successful targeting in endothelial, osteoblasts, osteoclasts, and
other cells of the bone marrow. Particle sizes and charge
distributions were consistent with subsequent CYNOBM.002 groups and
their expected biological performance in cynomolgus bone marrow
CRISPR and/or mRNA delivery.
[0528] FIG. 78 provides data from CYNOBM.002.86. Nanoparticles had
zeta potential of -20.02+/-0.10 mV with 90% having diameters less
than 120 nm. These nanoparticles resulted in 20.1% efficient
codelivery of CRISPR-GFP-RNP+Cy5_EGFP_mRNA in viable cynomolgus
bone marrow, with .about.68%, 70%, and .about.97% efficient CD3+,
CD45+, and CD34+ respective targeting according to flow cytometry
data. (e.g., see flow cytometry and imaging data below). The
results may implicate successful targeting in endothelial,
osteoblasts, osteoclasts, and other cells of the bone marrow. A
highly negatively charged zeta potential and of 90% of particles
counts <200 nm predicts high efficiency.
[0529] FIG. 79 provides data from CYNOBM.002.76. Nanoparticles had
zeta potential of -12.02+/-0.59 mV with 90% having diameters less
than 135 nm. These nanoparticles resulted in 18.4%, 10.3%, and
.about.100% efficient CRISPR-GFP-RNP uptake in viable CD3+, CD45+,
and CD34+ bone marrow cells, respectively, according to flow
cytometry data (e.g., see flow cytometry and imaging data below).
Additionally, particles exhibit limited toxicity as expected from a
histone-mimetic particle with highly negative zeta potential
10th-50th percentile particle sizes of 25.8-80.6 nm with no large
aggregates as seen in CYNOBM.002.78, which exhibits similar zeta
potential distributions and sizes with the addition of a large
volume peak at .about.500 nm.
[0530] FIG. 80 provides data from CYNOBM.002.77. Nanoparticles had
90% of their diameters below 254 nm with a large portion in the
171-254 nm range. (e.g., see flow cytometry and imaging data
below). Additionally, the 10th-50th percentile particles by number
were 70-172 nm, indicating a reasonable size distribution within
this population. Consistent with other studies where a large number
of particles >200 nm existed in solution and/or had a large,
distributed zeta potential and/or a non-anionic zeta potential,
these particles lead to significant cell death. These nanoparticles
resulted in high uptake percentages overall, but a large number of
cells (>90%) being dead. Ultimately, the particles resulted in
negligible uptake at the limits of detection of CRISPR-GFP-RNP in
viable CD3+, CD45+, and CD34+ bone marrow cells, and 3.8% CRISPR
uptake within whole bone marrow viable subpopulations according to
flow cytometry data. In contrast, 90% of CYNOBM.002.83 (a CRISPR
& mRNA codelivery variant) particles with the same surface
coating were below 200 nm with the number average being 121 nm.
Other particles in CYNOBM.002.75-CYNOBM.002.81, which were produced
via a different method than particles in TCELL.001.01-TCELL.001.15
with similar formulations, had more favorable size and zeta
potential distributions and resulted in high transfection
efficiencies (up to 99%) in viable human CD4+ and CD8a+
T-cells.
[0531] FIG. 81 provides data from CYNOBM.002.78. Nanoparticles had
zeta potential of -11.72+/-0.79 mV with 90% having diameters less
than 223 nm. (e.g., see flow cytometry and imaging data below).
Similarly, 90% of CYNOBM.002.84 (a CRISPR & mRNA codelivery
variant) particles with the same surface coating were below 200 nm
with the number average being 125 nm, though the zeta potential of
CYNOBM.002.84 is significantly more negative (-18.07 mV vs.-11.72
mV), indicating enhanced stability with an anionic sheddable
interlayer step intermediate to initial Cas9 RNP charge
homogenization with PLR10 and subsequent coating with ligands or
additional, optionally molecular weight staggered polymers or
polypeptides. The differential physicochemical properties of these
monodelivery vs. co-delivery (or interlayer vs. direct conjugation
of ligands to RNP) nanoparticles and their respective size ranges
is strongly correlated to transfection efficiency and toxicity.
[0532] FIG. 82 provides data from CYNOBM.002.79. Nanoparticles had
diameters less than 200 nm. These nanoparticles resulted in very
low (3.7%) GFP-RNP uptake in bone marrow overall, but the cells
retained exceptional viability (70.0% vs. 71.6% for negative
controls) in the culture. Despite very low overall uptake, the
particles demonstrated selective uptake for .about.9.0% of viable
CD3+ cells, 4.4% of viable CD45+ cells, and .about.100% of viable
CD34+ cells according to flow cytometry data, which is at the
limits of detection for cell counts in the CD34+ subpopulation.
(e.g., see flow cytometry and imaging data below). The results
implicate specific targeting of CD34+ hematopoietic stem cells
within mixed cell populations.
[0533] FIG. 83 provides data from CYNOBM.002.80. Nanoparticles had
zeta potential of 1.36+1-1.69 mV. These nanoparticles resulted in
8% transfection efficiency and .about.100% efficient CRISPR-GFP-RNP
uptake in viable CD34+ bone marrow cells according to flow
cytometry data, which is at the limits of detection for cell
counts. (e.g., see flow cytometry and imaging data below). The
results may implicate successful targeting in endothelial,
osteoblasts, osteoclasts, and other cells of the bone marrow. The
even peak at .about.0 mV with wide surfaces is indicative of a
zwitterionic particle surface. A high degree of cellular viability
indicates that particles were well tolerated with this size and
that a c-Kit-receptor-derived particle surface is likely to mimic
presentation of native stem cell population surface markers within
the bone marrow during cell-cell interactions.
[0534] FIG. 84-FIG. 120: Flow Cytometry and Imaging Data
[0535] FIG. 84. Untransfected controls for CynoBM.002 samples in
cynomolgus bone marrow. Microscope images--Top: digital phase
contrast; middle: GFP; bottom: merge. Flow cytometry data--with
viability, CD34, CD3, and CD45 stains.
[0536] FIG. 85. Lipofectamine CRISPRMAX delivery of NLS-Cas9-EGFP
BCL11a gRNA RNPs attains 2.5% transfection efficiency in viable
cells and causes significant toxicity, with percentage of CD45 and
CD3 relative subpopulations significantly decreased compared to
negative controls in cynomolgus bone marrow. Lipofectamine
CRISPRMAX does not exhibit cell-selectivity as exemplified by 7.4%
efficient targeting of remaining CD3+ cells and negligible
remaining populations of CD45+ and CD34+ cells. Microscope
images--Top: digital phase contrast; middle: GFP; bottom:
merge.
[0537] FIG. 86. CynoBM.002 RNP-Only controls show NLS-Cas9-EGFP
BCL11a gRNA RNPs attaining negligible transfection efficiencies in
cynomolgus bone marrow without a delivery vector, but with both
payloads pre-combined prior to transfection. A high degree of
colocalization despite no delivery vector and minimal events is
indicative of association of the ribonucleoprotein complex with
mRNA, and exemplary of anionic functionalization of CRISPR RNPs.
(In this instance, the mRNA acts as a loosely-associated sheddable
coat for the RNP and could be further layered upon with cationic
materials). Calculating colocalization coefficient. X: % CRISPR
uptake in live cells:
[0538] Y: % mRNA uptake in live cells
[0539] C: % of cells with CRISPR AND mRNA
[0540] Z: value of X or Y, whichever is greater
Colocalization Coefficient=C/Z
Cas9-mRNA Colocalization Coefficient: 92.2%
[0541] FIG. 87. CynoBM.002.82 demonstrated that
non-specifically-targeted NLS-Cas9-EGFP achieves 11.3% efficient
mRNA delivery and 11.4% efficient CRISPR delivery to cynomolgus
bone marrow with a 98.9% colocalization coefficient. Subcellular
localization demonstrated that noon-specifically targeted
NLS-Cas9-EGFP BCL11a gRNA RNPs co-localize with Cy5 mRNA and attain
high transfection efficiencies. A high degree of colocalization
determines that discrete particles were loaded with both payloads.
Additionally, Cas9 can be seen neatly localized in a separate
compartment from the mRNA, wherein the mRNA forms a ringed
structure around the nuclear-associated Cas9. This indicates
cytosolic (mRNA) vs. nuclear (CRISPR) localization of the two
payloads. Microscope images--Top: digital phase contrast; middle:
Cy5 mRNA; bottom: merge. and top: Cas9-GFP RNP; bottom: Cy5 mRNA
colocalized with Cas9-GFP RNP.
[0542] See above data for physicochemical parameters and additional
observations. CYNOBM.002.82 had zeta potential of 2.96+/-0.14 mV
with 90% having diameters less than 132 nm and 50% of particles
with diameters less than 30 nm. These nanoparticles resulted in
45.5%, 56.0%, and 97.3% efficient CRISPR-GFP-RNP+Cy5_EGFP_mRNA
uptake in viable CD3+, CD45+, and CD34+ bone marrow subpopulations,
respectively, despite only 11.4% overall bone marrow viable
subpopulation targeting. Cas9-mRNA Colocalization Coefficient:
94.8%. Viable CD34+ and CRISPR+: 97.2% of Viable CD34+. Fetal
Hemoglobin Positive: 3.022% of viable cells
[0543] FIG. 88. CynoBM.002.83 achieves 8.1% efficient mRNA delivery
and 8.1% efficient CRISPR delivery to cynomolgus bone marrow with a
93.0% colocalization coefficient. Subcellular localization
demonstrated that homovalently-targeted IL2-derived peptides
associated with NLS-Cas9-EGFP BCL11a gRNA RNPs co-localize with Cy5
mRNA and attain high transfection efficiencies. A high degree of
colocalization determines that discrete particles were loaded with
both payloads. Additionally, Cas9 can be seen neatly localized in a
separate compartment from the mRNA, wherein the mRNA forms a ringed
structure around the nuclear-associated Cas9. This indicates
cytosolic (mRNA) vs. nuclear (CRISPR) localization of the two
payloads. Microscope images--Top: digital phase contrast; middle:
Cy5 mRNA; bottom: merge. and top: Cas9-GFP RNP; bottom: Cy5 mRNA
colocalized with Cas9-GFP RNP.
[0544] See above data for physicochemical parameters and additional
observations. These nanoparticles resulted in .about.27%, 41%, and
.about.100% efficient CRISPR-GFP-RNP+Cy5_EGFP_mRNA uptake in viable
CD3+, CD45+, and CD34+ bone marrow cells, respectively. Cas9-mRNA
Colocalization Coefficient: 93.0%. Fetal Hemoglobin Positive: 1.9%
of viable cells
[0545] FIG. 89. CYNOBM.002.84 particles successfully delete the
BCL11a erythroid enhancer in whole bone marrow erythroid progenitor
cells as evidenced by fetal hemoglobin protein expression in 9.5%
of live whole bone marrow cells and no positive fetal hemoglobin
measurements in CD34+, CD45 or CD3+ subpopulations despite moderate
transfection efficiencies, as measured by Cy5-mRNA+ and
CRISPR-GFP-RNP+ gates in each selective subpopulation. Subcellular
localization demonstrated that homovalently-targeted
E-selectin-derived peptides associated with NLS-Cas9-EGFP BCL11a
gRNA RNPs co-localize with Cy5 mRNA and attain high transfection
efficiencies. A high degree of colocalization determines that
discrete particles were loaded with both payloads. Additionally,
Cas9 can be seen neatly localized in a separate compartment from
the mRNA, wherein the mRNA forms a ringed structure around the
nuclear-associated Cas9. This indicates cytosolic (mRNA) vs.
nuclear (CRISPR) localization of the two payloads. Microscope
images--Top: digital phase contrast; middle: Cy5 mRNA; bottom:
merge. and top: Cas9-GFP RNP; bottom: Cy5 mRNA colocalized with
Cas9-GFP RNP.
[0546] See above data for physicochemical parameters and additional
observations. These nanoparticles resulted in 76.5%, 71%, and
.about.100% efficient CRISPR-GFP-RNP+Cy5_EGFP_mRNA colocalized
uptake in viable CD3+, CD45+, and CD34+ bone marrow cells,
respectively, as well as .about.25.5% of whole bone marrow viable
subpopulations according to flow cytometry data. Additionally, 9.5%
of overall viable cells were positive for fetal hemoglobin, with
none of these cells being CD34+, CD3+, or CD45+, suggesting
successful clonal expansion of BCL11a erythroid progenitor knockout
populations within CD34- erythroid progenitor cells. Cas9-mRNA
Colocalization Coefficient: 97.1%. Fetal Hemoglobin (HbF) Positive:
9.5% of viable cells 14% CD34+ cells; 0% colocalization of CD34+
and HbF+
[0547] FIG. 90. CynoBM.002.85 achieved 5.2% efficient mRNA delivery
and 5.3% efficient CRISPR delivery to cynomolgus bone marrow with a
87.2% colocalization coefficient. Despite 5.3% efficient CRISPR
delivery to viable cells, CynoBM.002.85 did not lead to a
concomitant increase in fetal hemoglobin positive cells as seen in
other codelivery embodiments. Subcellular localization demonstrated
that homovalently-targeted SCF-derived peptides associated with
NLS-Cas9-EGFP BCL11a gRNA RNPs co-localize with Cy5 mRNA and attain
high transfection efficiencies. A high degree of colocalization
determined that discrete particles were loaded with both payloads.
Additionally, Cas9 could be seen neatly localized in a separate
compartment from the mRNA, wherein the mRNA forms a ringed
structure around the nuclear-associated Cas9. This indicates
cytosolic (mRNA) vs. nuclear (CRISPR) localization of the two
payloads. Microscope images--Top: digital phase contrast; middle:
Cy5 mRNA; bottom: merge. and top: Cas9-GFP RNP; bottom: Cy5 mRNA
colocalized with Cas9-GFP RNP.
[0548] See above data for additional physicochemical
characteristics. These nanoparticles resulted in .about.33%,
.about.23%, and .about.100% efficient CRISPR-GFP-RNP+Cy5_EGFP_mRNA
uptake in viable CD3+, CD45+, and CD34+ bone marrow cells,
respectively. Cas9-mRNA Colocalization Coefficient: 87.2%. Fetal
Hemoglobin Positive: 0.9% of viable cells
[0549] FIG. 91. CynoBM.002.86 achieved 20.1% efficient mRNA
delivery and 21.8% efficient CRISPR delivery to cynomolgus bone
marrow with a 98.6% colocalization coefficient. Subcellular
localization demonstrated that heterotrivalently-targeted IL2-,
E-selectin- and SCF-derived NLS-Cas9-EGFP BCL11a gRNA RNPs
co-localized with Cy5 mRNA and attain high transfection
efficiencies. A high degree of colocalization determined that
discrete particles were loaded with both payloads. Additionally,
Cas9 could be seen neatly localized in a separate compartment from
the mRNA, wherein the mRNA forms a ringed structure around the
nuclear-associated Cas9. This indicates cytosolic (mRNA) vs.
nuclear (CRISPR) localization of the two payloads. Microscope
images--Top: digital phase contrast; middle: Cy5 mRNA; bottom:
merge. and top: Cas9-GFP RNP; bottom: Cy5 mRNA colocalized with
Cas9-GFP RNP.
[0550] See above data for additional physicochemical
characteristics. Cas9-mRNA Colocalization Coefficient: 91.3%. Fetal
Hemoglobin Positive: 7.6% of viable cells
[0551] FIG. 92. CynoBM.002.75 demonstrated that
non-specifically-targeted NLS-Cas9-EGFP BCL11a gRNA RNPs with
sheddable anionic polypeptide coats attain 18.0% transfection
efficiency in viable cynomolgus bone marrow. Overall, 20% of viable
CD3+ T-cells were CRISPR+ in the mixed population cynomolgus bone
marrow culture model herein, in contrast to 97-99% of viable CD4
and CD8a T-cells in human primary Pan T-cells being CRISPR+ in
TCELL.001. Particle sizes of an identical formulation were smaller
and more uniform in TCELL1, which was synthesized via
fluid-handling robotics as opposed to by hand. See above data for
additional qualitative and quantitative commentary and data
comparisons. Top: digital phase contrast; middle: GFP; bottom:
merge.
[0552] FIG. 93. CynoBM.002.76 demonstrated that
dual-histone-fragment-associated and non-specifically-targeted
NLS-Cas9-EGFP BCL11a gRNA RNPs attain 13.1% transfection efficiency
and limited toxicity versus negative controls in cynomolgus bone
marrow. 18%, 10%, and 0% of CD3+, CD45+ and CD34+ viable
subpopulations were CRISPR+. See above data for additional
physicochemical characteristics and observations. Top: digital
phase contrast; middle: GFP; bottom: merge.
[0553] FIG. 94. CynoBM.002.77 demonstrated that
homovalently-targeted IL2-derived peptides associated with
NLS-Cas9-EGFP BCL11a gRNA RNPs attain 3.8% transfection efficiency
and enhanced viability over negative controls in cynomolgus bone
marrow.-90% of transfected cells were dead. Ultimately, the
particles resulted in negligible uptake at the limits of detection
of CRISPR-GFP-RNP in viable CD3+, CD45+, and CD34+ bone marrow
cells, indicating that the remaining 3.8% of live CRISPR+ cells
were not from those subpopulations. Size data supports a causative
role for toxicity in large particle polydispersity and .about.999
nm 90th volume percentile particle sizes. See above data for
additional physicochemical properties. Top: digital phase contrast;
middle: GFP; bottom: merge.
[0554] FIG. 95. CynoBM.002.78 demonstrated that
homovalently-targeted E-selectin-derived peptides associated with
NLS-Cas9-EGFP BCL11a gRNA RNPs attain .about.71% transfection
efficiency overall (including dead cells), with only 4.5% of live
cells remaining transfected in cynomolgus bone marrow. This is
indicative of particle toxicity and may be correlated to a large
size distribution, despite 50% of the particles by number being
33.1-113.1 nm. The >250 nm particles, comprising the majority of
particle mass and volume in solution, likely led to the reduced
viability of this experiment. CD45+ and CD3+ subpopulation
densities were manifold reduced in this embodiment as well. See
above data for more detailed physicochemical characteristics and
qualitative observations comparing nanoparticle groups from the
same transfection. Top: digital phase contrast; middle: GFP;
bottom: merge.
[0555] FIG. 96. CynoBM.002.79 demonstrated that
homovalently-targeted SCF-derived peptides associated with
NLS-Cas9-EGFP BCL11a gRNA RNPs attain 3.7% transfection
efficiencies and excellent viability over negative controls in
cynomolgus bone marrow. These nanoparticles resulted in very low
(3.7%) GFP-RNP uptake in bone marrow overall, but the cells
retained exceptional viability (69.0% vs. 71.6% for negative
controls) in the culture. Despite very low overall uptake, the
particles demonstrated selective uptake for .about.5% of viable
CD3+ cells, .about.4% of viable CD45+ cells, and .about.100% of
viable CD34+ cells (the latter which were at the limits of
detection in number). The high degree of cellular viability coupled
with a strongly negative zeta potential and significantly more
CD45+ cells than other groups is implicative of a SCF-mimetic
particle surface's multifactorial role in establishing stem cell
niche targeting and proliferation and/or survival techniques. See
above data for additional physicochemical parameters. Top: digital
phase contrast; middle: GFP; bottom: merge.
[0556] FIG. 97. CynoBM.002.80 demonstrated that
homovalently-targeted c-Kit-(CD117)-derived peptides associated
with NLS-Cas9-EGFP BCL11a gRNA RNPs attain 8.097% transfection
efficiencies. Transfection efficiencies were 3.3%, 2.4%, and at the
limits of detection for CD3+, CD45+ and CD34+ viable
subpopulations, respectively, indicating low selectivity for CD3+
and CD45+ cells. See above data for more quantitative and
qualitative data. Top: digital phase contrast; middle: GFP; bottom:
merge. (cont.): flow cytometry data.
[0557] FIG. 98. CynoBM.002.81 demonstrated that
heterotrivalently-targeted IL2-, E-selectin- and SCF-derived
NLS-Cas9-EGFP BCL11a gRNA RNPs attain 5% transfection efficiency in
cynomolgus bone marrow with .about.10% of transfected cells being
live CD34+ cells despite only 0.48% of cells being CD34+. This
indicates nearly 100% efficient selective transfection of CD34+
cells. Top: digital phase contrast; middle: GFP; bottom: merge.
[0558] FIG. 99. Qualitative images of CynoBM.002 RNP-Only control
show NLS-Cas9-EGFP BCL11a gRNA RNPs attaining mild positive signal
in cynomolgus bone marrow without a delivery vector. Top: digital
phase contrast; middle: GFP; bottom: merge.
[0559] FIG. 100. HSC.004 (nanoparticles 69-74, see Table 5)
High-Content Screening. Fluorescence microscopy images (Cy5 mRNA)
of HSC.004 Cy5 mRNA delivery 12-15 h post-transfection in Primary
Human CD34+ Hematopoietic Stem Cells. With this particular
embodiment of mRNA formulation, heterobivalent targeting with SCF
peptides and E-selectin, as well as homovalent targeting with
E-selectin but not SCF peptides, achieves higher transfection
efficiencies than Lipofectamine MessengerMAX. HSC.001.69: A1-A6;
HSC.001.70: B1-B6; HSC.001.71: C1-C6; HSC.001.72: D1-D6;
HSC.001.73: E1-E6; HSC.001.74: F1-F6; HSC.004 Lipofectamine
MessengerMAX Dose 1: G1-G2 & G4-G5; TC.001 Lipofectamine
MessengerMAX Dose 2: H1-H2 & H4-H5; TC.001 Negative: G3, G6,
H3, H6
[0560] FIG. 101. TCELL.001 (nanoparticles 1-15, see Table 5)
High-Content Screening. Robotic formulations were performed for
TC.001.1-TC.001.60, representing 15 ligands across 4 payloads
(CRISPR RNP, mRNA, siRNA and pDNA). Shown are embodiments of T-cell
CRISPR delivery and qualitative transfection
efficiencies--thumbnail images of 12-15 h post-transfection
composite microscopy of TCELL.001 CRISPR-EGFP RNP delivery to
Primary Human Pan T-cells. Plate layout: TC.001.1: A1-C1; TC.001.3:
D1-F1; TC.001.4: A2-C2; TC.001.5: D2-F2; TC.001.6: A3-C3; TC.001.7:
D3-F3; TC.001.8: A4-C4; TC.001.9: D4-F4; TC.001.10: A5-C5;
TC.001.11: D5-F5; TC.001.12: A6-C6; TC.001.13: D6-F6; TC.001.14:
A7-A9; TC.001.15: B7-B9; TC.001.2: A10-A12; TC.001 Lipofectamine
CRISPRMAX Dose 1: B10-B12; TC.001 Lipofectamine CRISPRMAX Dose 2:
C7-C9; TC.001 RNP Only: C10-C12; TC.001 Negative: D7-E12.
[0561] FIG. 102. TCELL.001 Lipofectamine CRISPRMAX. Lipofectamine
CRISPRMAX attained 4.7% and 4.8% efficient delivery of
NLS-Cas9-EGFP RNP in viable CD4+ and CD8a+ subpopulations,
respectively, of human primary Pan T-cells at 24 h
post-transfection. Overall, 12.5% of CRISPR+ cells and 65.9% of
overall cells were viable.
[0562] FIG. 103: TCell.001.1 demonstrated 99.163% efficient and
98.447% efficient non-specifically-targeted CRISPR-GFP
Ribonucleoprotein uptake in viable CD4+ and CD8a+ subpopulations,
respectively, of human primary Pan T-cells at 24 h
post-transfection. Overall, 60.2% of CRISPR+ cells and 57.2% of
overall cells were viable.
[0563] FIG. 104. TCell.001.2, a non-specifically-targeted PEGylated
control, demonstrated 5.5% efficient and 6.9% efficient CRISPR-GFP
Ribonucleoprotein uptake in viable CD4+ and CD8a+ subpopulations,
respectively, of human primary Pan T-cells at 24 h
post-transfection. Overall, 5.6% of CRISPR+ cells and 40.5% of
overall cells were viable.
[0564] FIG. 105. TCell.001.3 demonstrated that
homovalently-targeted sialoadhesin-derived peptides associated with
CRISPR-GFP Ribonucleoprotein generate 11.6% and 13.2% efficient
uptake in viable CD4+ and CD8a+ subpopulations, respectively, of
human primary Pan T-cells at 24 h post-transfection. Overall, 40.0%
of CRISPR+ cells and 79.2% of overall cells were viable.
[0565] FIG. 106. TCell.001.4 demonstrated that
homovalently-targeted CD80-derived peptides associated with
CRISPR-GFP Ribonucleoprotein generate 6.8% and 8.8% efficient
uptake in viable CD4+ and CD8a+ subpopulations, respectively, of
human primary Pan T-cells at 24 h post-transfection. Overall, 12.9%
of CRISPR+ cells and 60.2% of overall cells were viable.
[0566] FIG. 107. TCell.001.5 demonstrated that
homovalently-targeted CD80-derived peptides associated with
CRISPR-GFP Ribonucleoprotein generate 10.3% and 10.9% efficient
CRISPR-GFP Ribonucleoprotein uptake in viable CD4+ and CD8a+
subpopulations, respectively, of human primary Pan T-cells at 24 h
post-transfection. Overall, 48.3% of CRISPR+ cells and 85.1% of
overall cells were viable. Note that across 9 wells of negative
controls (n=3 negatives for TCELL.001 flow cytometry), viabilities
were 81.4%, 84.7%, and 82.5%, which demonstrated that a
C-terminally anchored, CD80-derived CD28-targeting peptide may have
mild survival-promoting effects on non-transfected cells in culture
solution. In contrast, TCell.001.4, an identical N-terminally
anchored peptide, displayed marked toxicity, as did TC.001.6 and
TC.001.7, which are also CD80-derived fragments with different
allosterism for the CD28 transmembrane receptor.
[0567] FIG. 108. TCell.001.6 demonstrated that
homovalently-targeted CD86-derived peptides associated with
CRISPR-GFP Ribonucleoprotein generate 1.7% and 2.9% efficient
uptake in viable CD4+ and CD8a+ subpopulations, respectively, of
human primary Pan T-cells at 24 h post-transfection. Overall, 6.8%
of CRISPR+ cells and 69.1% of overall cells were viable.
[0568] FIG. 109. TCell.001.7 demonstrated that
homovalently-targeted CD86-derived peptides associated with
CRISPR-GFP Ribonucleoprotein generate 1.6% and 2.1% efficient
uptake in viable CD4+ and CD8a+ subpopulations, respectively, of
human primary Pan T-cells at 24 h post-transfection. Overall, 10.3%
of CRISPR+ cells and 76.4% of overall cells were viable.
[0569] FIG. 110. TCell.001.8 demonstrated that
homovalently-targeted CD86-derived peptides associated with
CRISPR-GFP Ribonucleoprotein generate 14.5% and 16.0% efficient
uptake in viable CD4+ and CD8a+ subpopulations, respectively, of
human primary Pan T-cells at 24 h post-transfection. Overall, 39.1%
of CRISPR+ cells and 76.3% of overall cells were viable.
[0570] FIG. 111. TCell.001.9 demonstrated that
homovalently-targeted 4-1BB-derived peptides associated with
CRISPR-GFP Ribonucleoprotein generate 3.6% and 3.2% efficient
uptake in viable CD4+ and CD8a+ subpopulations, respectively, of
human primary Pan T-cells at 24 h post-transfection. Overall, 27.5%
of CRISPR+ cells and 87.8% of overall cells were viable. Note that
across 9 wells of negative controls (n=3 negatives for TCELL.001
flow cytometry), viabilities were 81.4%, 84.7%, and 82.5%, which
demonstrated that a C-terminally anchored, 4-1BB-derived
CD137-targeting peptide, which has innate survival signaling with
T-cells, has mild survival-promoting effects on non-transfected
cells in culture solution.
[0571] FIG. 112. TCell.001.10 demonstrated that
homovalently-targeted 4-1BB-derived peptides associated with
CRISPR-GFP Ribonucleoprotein generate 5.8% and 5.4% efficient
uptake in viable CD4+ and CD8a+ subpopulations, respectively, of
human primary Pan T-cells at 24 h post-transfection. Overall, 30.8%
of CRISPR+ cells and 84.2% of overall cells were viable. Note that
across 9 wells of negative controls (n=3 negatives for TCELL.001
flow cytometry), viabilities were 81.4%, 84.7%, and 82.5%, which
demonstrated that a C-terminally anchored, 4-1BB-derived
CD137-targeting peptide, which has innate survival signaling with
T-cells, demonstrates no overall toxicity in culture solution.
[0572] FIG. 113. TCell.001.11 demonstrated that
homovalently-targeted CD3-Ab-derived peptides associated with
CRISPR-GFP Ribonucleoprotein generate 12.9% and 12.4% efficient
uptake in viable CD4+ and CD8a+ subpopulations, respectively, of
human primary Pan T-cells at 24 h post-transfection. Overall, 50.0%
of CRISPR+ cells and 77.6% of overall cells were viable.
[0573] FIG. 114. TCell.001.12 demonstrated that
homovalently-targeted CD3-Ab-derived peptides associated with
CRISPR-GFP Ribonucleoprotein generate 9.0% and 9.5% efficient
uptake in viable CD4+ and CD8a+ subpopulations, respectively, of
human primary Pan T-cells at 24 h post-transfection. Overall, 38.9%
of CRISPR+ cells and 80.7% of overall cells were viable.
[0574] FIG. 115. TCell.001.13 demonstrated that
homovalently-targeted IL2-derived peptides associated with
CRISPR-GFP Ribonucleoprotein generate 25.7% and 28.6% efficient
uptake in viable CD4+ and CD8a+ subpopulations, respectively, of
human primary Pan T-cells at 24 h post-transfection. Overall, 40.3%
of CRISPR+ cells and 68.1% of overall cells were viable.
[0575] FIG. 116. TCell.001.14 demonstrated that
homovalently-targeted IL2-derived peptides associated with
CRISPR-GFP Ribonucleoprotein generate 24.9% and 25.8% efficient
uptake in viable CD4+ and CD8a+ subpopulations, respectively, of
human primary Pan T-cells at 24 h post-transfection. Overall, 45.9%
of CRISPR+ cells and 70.1% of overall cells were viable.
[0576] FIG. 117. TCell.001.15, a dodecavalently-targeted 12-ligand
variant, does not lead to endocytic uptake or CRISPR delivery.
Overall, 59.8% of overall cells were viable.
[0577] FIG. 118. TCELL.001 Negative Controls. Representative
results from one of 9 wells of negative (non-transfected) control.
Overall, 81.4%, 84.7%, and 82.5% of total cells were viable 52 h
after cell seeding (24 h post-transfection).
[0578] FIG. 119. Blood.002 attains 60%-97% mRNA delivery efficiency
in the lymphocyte gate of whole human blood through utilizing a
SIGLEC derivative for glycosylated cell surface marker targeting;
shown is Cy5-tagged EGFP mRNA assayed via an Attune NxT flow
cytometer. Ligand targeting is a significant enhancer of cellular
signal versus a PEGylated control. See above data for additional
physicochemical properties predictive of nanoparticle behavior.
Blood.002 Control: Untransfected. Blood.002.88: CD45- and
Neu5Ac-targeting SIGLEC derivative (cationic anchor-linker-ligand
peptide added before anionic polymer). Blood.002.89: CD45- and
Neu5Ac-targeting SIGLEC derivative (cationic anchor-linker-ligand
peptide added after anionic polymer). Blood.002.90: PEGylated
control (cationic anchor-PEG added before anionic polymer).
Blood.002.91: Non-specifically-targeted variant. Blood.002.92:
CD45- and Neu5Ac-targeting SIGLEC derivative without payload
(anchor-linker-ligand is directly conjugated to anionic polymer,
negative fluorescent control).
[0579] FIG. 120. TCell.001.27 demonstrated that
homovalently-targeted SIGLEC-derived peptides direct 45% efficient
Cy5 mRNA uptake in viable CD8a+ and CD4+ subpopulations of human
primary Pan T-cells at 5 h post-transfection, as measured via flow
cytometry. The size and zeta potential of these particles
demonstrated average particle sizes of 171 nm with zeta potentials
of -25.5+/-0.15 mV, indicating strong particle stability at a 1.35
carboxylate-to-phosphate (C:P) and 0.85 amine-to-phosphate ratio
wherein poly(glutamic acid) is added following inclusion of the
cationic anchor-linker-ligand. See above data for zeta potential
and size data, TCell.001.2 and TCell.001.18. See above data for
additional quantitative details. Top-right: bright field;
middle-right: Cy5 mRNA; bottom-right: merge Top: bright field of
negative control; bottom: Cy5 channel of negative control.
Example 9
[0580] FIG. 121. Rationale for Ribonucleoprotein and Protein
Delivery. Charge density plots of CRISPR RNP allow for determining
whether an anionic or cationic peptide/material should be added to
form a stable charged layer on the protein surface. In one
embodiment, exposed nucleic acid (anionic) and anionic charge
pockets serve as strong electrostatic anchoring sites for charged
cations prior to addition of charged anions, or as their own
ligand-linker anionic anchors. Scale bar: charge.
[0581] FIG. 122. Rationale for Ribonucleoprotein and Protein
Delivery. Charge density plots of Sleeping Beauty Transposons allow
for determining whether an anionic or cationic peptide/material
should be added to form a stable charged layer on the protein
surface. In another embodiment, cationic charge pockets serve as
strong electrostatic anchoring sites for charged anions, either as
their own ligand-linker-anionic anchor domains, or prior to
addition of charged cations. Scale bar: charge.
[0582] FIG. 123. (1) Exemplary anionic peptides (9-10 amino acids
long, approximately to scale to 10 nm diameter CRISPR RNP)
anchoring to cationic sites on the CRISPR RNP surface prior to (2)
addition of cationic anchors as (2a) anchor-linker-ligands or
standalone cationic anchors, with or without addition of (2b)
subsequent multilayering chemistries, co-delivery of multiple
nucleic acid or charged therapeutic agents, or layer stabilization
through cross-linking.
[0583] Handwriting in drawing from left to right converted to text:
`cationic anchor`. `spacer`. ligand'. `And/Or`. `2d`. `cationic
polymer and/or polypeptide`. `2b. followed by interlayer
chemistry`.
[0584] FIG. 124. Rationale for Payload Co-delivery with Charged
Protein Core Templates. Examples of orders of addition and
electrostatic matrix compositions based on core templates, which
may include Cas9 RNP or any homogenously or zwitterionically
charged surface. A method for homogenizing the charge of a
zwitterionic surface utilizing a variety of polymers is shown. A
.about.10 nm core particle consisting of CRISPR-Cas9 RNP bound to
gRNA is shown with zwitterionic domains. Briefly, a cationic
polymer or anionic polymer may be added to homogenize the surface
charge prior to addition of oppositely charged polymers. Stagged
molecular weight of anionic constituents is demonstrated to
increase the transfection efficiency and gene editing efficiency of
particles with RNP cores and mRNA-PLE interlayers with a variety of
surface coatings in CYNOBM.002.82-CYNOBM.002-86 vs. single payload
delivery variants in CYNOBM.002.75-CYNOBM.002.81. Charged core
template embodiments encompass any charged surface including a
charged dendrimer or oligosaccharide-dendrimer, recombinant or
synthetic histone dimer/trimer/tetramer/octamer, nanodiamond, gold
nanoparticle, quantum dot, MRI contrast agent, or combination
thereof with the above.
[0585] Handwriting in drawing from left to right converted to text:
The negatively charged coating may be layered upon by with cationic
polymer or anchor-linker-ligand, wherein the anchor is cationic.`
`amino sugar`. `charged glycosaminoglycan`. `pDNA`. `CODELIVERY`.
`exposed gRNA`. `net negative sheddable polymer coat`. `glycan`.
`cationic protein domain on cas9`. `.about.10 nm cas9 RNP`.
`cationic protein domain on cas9`. `PLR`. `PDE (5-100)`.
`PLE(5-100)`. `anionic protein domain on cas9`. `mRNA`. `branched
cationic polymer on glycopeptide`. `histone`. `siRNA`. The
negatively charged coating may also be domain of an anionic
anchor-linker-ligand or a standalone anionic matrix composition.
Staggered mw of consistent polymers increases colloidal stability
and gene editing efficiency.
Example 10
[0586] FIG. 125. Peptide Engineering--Novel IL2-Mimetic Fragment
for IL2R Targeting.
[0587] Interleukin-2 (left) bound to the Interleukin-2 Receptor
(right) (PDB: 1Z92) The sequence
ASN(33)-PRO(34)-LYS(35)-LEU(36)-THR(37)-ARG(38)-MET(39)-LEU(40)-THR(41)-P-
HE(42)-LYS(43)-PHE(44)-TYR(45) is selected from IL2 (PDB 1Z92),
correlating to the areas of active binding to the IL2 receptor
alpha chain. Engineering complementary binding through selecting
the interacting motifs of IL2R with IL2: here, the sequence
CYS(3)-ASP(4)-ASP(5)-ASP(6)-MET(25)-LEU(26)-ASN(27)-CYS(28)-GLU(29)
is selected for two binding motifs from IL2 receptor.
[0588] FIG. 126: PEPTIDE ENGINEERING--A Novel Antibody-Derived
"Active Binding Pocket" Engineering Proof of Concept with CD3. The
sequence
THR(30)-GLY(31)-ASN(52)-PRO(53)-TYR(54)-LYS(55)-GLY(56)-VAL(57)-SER(58)-T-
HR(59)-TYR(101)-TYR(102)-GLY(103)-ASP(104) is selected from a CD3
antibody (PDB 1XIW), correlating to the areas of active binding to
CD3 epsilon and delta chains. The order of the amino acids is
rearranged in order to reflect binding kinetics of a 2-dimensional
plane of peptides in the binding pocket which no longer have
tertiary structure maintained by the larger protein. This
dimensional reduction results in:
THR(59)-SER(58)-VAL(57)-GLY(56)-LYS(55)-TYR(54)-PRO(53)-ASN(52)-THR(30)-G-
LY(31)-TYR(101)-TYR(102)-GLY(103)-ASP(104).
[0589] FIG. 127: PEPTIDE ENGINEERING--A Novel SIGLEC Derivative for
CD45 Glycosylation Targeting. PDB rendering of sialoadhesin
N-terminal in complex with N-Acetylneuraminic acid (Neu5Ac) (RCS
PDB 1ODA). A sialoadhesin fragment proximal to sialoadhesin in the
rendering was utilized for targeting glycosylated CD45 and other
complex cell-surface glycoproteins. It generates successful
targeting of T-cells with CRISPR RNP in TCELL.001.3, as well as
mRNA in whole blood lymphocyte gates in BLOOD.002.1-BLOOD.002.2.
The sequence for the ligand is SNRWLDVK (SEQ ID NO: xx).
[0590] FIG. 128: PEPTIDE ENGINEERING--A Novel SCF Fragment for
c-Kit Targeting. Dashed circles--signal peptide domains of Stem
Cell Factor (RCS PDB 1SCF) represent dimeric domains necessary for
c-Kit activity. Effect of ligand presentation on cellular uptake
due to particular nanoparticle surface size+ SCF coating densities
can be compared and contrasted between CynoBM.002.79 (.about.5%
efficiency) and CynoBM.002.85 (.about.56% efficiency).
Additionally, a contrast is displayed with qualitative imagery of
human CD34+ hematopoietic stem cell transfections, where
E-selectin+ SCF Fragment (HSC.004.73) achieves high efficiencies,
but the SCF Fragment on its own does not (HSC.004.74). The marked
difference in behavior is suggestive of a particular role of the
dimeric peptide in generating endocytic cues and subsequent nuclear
targeting of nucleic acid and/or ribonucleoprotein materials. The
sequence for the ligand is EKFILKVRPAFKAV (SEQ ID NO: xx) (mSCF);
and EKFILKVRPAFKAV (SEQ ID NO: xx) (rmSCF).
[0591] FIG. 129: PEPTIDE ENGINEERING--A Novel cKit Receptor
Fragment for Membrane-Bound SCF Targeting. Rational design of a
stem cell factor targeting peptide derived from c-Kit to mimic
behavior of hematopoietic stem cell rolling behavior on endothelial
and bone marrow cells and increase systemic transfection efficiency
(see CynoBM.002.80). Sequence evaluated for folding: Name SCFN,
Sequence: RRRRRRRRRGGGGSGGGGSEGICRNRVTNNVKDVTKLVANLPK (SEQ ID NO:
xx). Sequences were evaluated with Rosetta and NAMD simulation
packages--Rosetta Results: A shortened sequence was placed into
Rosetta for ab initio folding (GGSEGICRNRVTNNVKDVTKLVANLPK)(SEQ ID
NO: xx).
[0592] FIG. 130: PEPTIDE ENGINEERING--cKit Receptor Fragment
(Continued). Molecular dynamics simulations with anchor segment of
anchor-linker-ligands held in place to allow for simulating
entropically favorable conformation as would be presented on the
nanoparticle surface. Each result contains the same scoring factor
which means it's difficult to determine if any of these structures
would be preferred. Also Rosetta does not do folding dynamics so it
is highly possible that these sequences will not fold into a
helix-like structure.
[0593] NAMD results: Because Rosetta doesn't do folding dynamics,
it was checked if the full sequences would quickly fold into a
secondary structure. Simulations were performed in NAMD using
replica exchange molecular dynamics (REMD) on 16 or 32 replicas
between 300-500 K and simulated to 10 ns on each replica. The
anchor section (poly-R) was fixed as linear to simulate bound
protein to particle. Lowest energy snapshots are shown.
[0594] Further analysis of the sequence derived from KIT showed
that it likely doesn't have a lot of inherent order. Orange cartoon
section belongs to the sequence initially selected from KIT.
[0595] FIG. 131: PEPTIDE ENGINEERING--cKit Receptor Fragment
(Continued). Stabilization of a random coiled peptide with strong
ligand-linker self-folding into a stable helical peptide for
effective ligand presentation through modification of key
hydrophobic domains with amino isobutyric acid.
[0596] Blue chains represented a more ordered helix present in KIT,
ranging from residues 71 to 94:SNYSIIDKLVNIVDDLVECVKENS. NAMD
simulations of KIT residues 71 to 94 with anchor and linker:
RRRRRRRRRGGGGSGGGGSSNYSIIDKLVNIVDDLVECVKENS
[0597] Converged to a structure in which the strand heavily
interacts with the linker residues. For residues 71 to 94 there are
hydrophobic residues that stabilize the helix by interacting with
two other helices in KIT. Hydrophobic residues are shown in red
(underline): SNYSIIDKLVNIVDDLVECVKENS. The sequence was changed to
remove the hydrophobic residues and replaced with amino isobutyric
acid (Aib), which helps induce helical folds, to arrive at the
following sequence: KIT7194_AIB1: SNYS AibADK AibANAibA DD
AibAEAibAKENS. Sequence containing Aib was synthesized on Rink
resin and isolated at the free amine and an acylated amine (Ac).
Secondary structure was examined by circular dichroism.
[0598] FIG. 132: PEPTIDE ENGINEERING--cKit Receptor Fragment
(Continued)
[0599] Circular dichroism of SCF_mcKit_(4GS)2_9R_N and
SCF_mcKit(Ac)_(4GS)2_9R_N. Acetylation of ligand ends can be
utilized to neutralize the charge of a charged polypeptide end.
Top: CD of KIT7194_AIB1 shows a slight dip around 222 and large dip
around 208, consistent with the secondary structure of an
alpha-helix and helices that contain Aib units. Bottom:
KIT7194_AIB1_Ac shows a similar CD to that of KIT7194_AIB1.
Sometime acylation can assist in folding but it does not seem
necessary. Acetylation can also help with ligand interaction is the
terminal amine need to be neutral rather than charged. Full
anchor-linker-KIT7194_AIB1 construct: RRRRRRRRR-GGGGSGGGGS-SNYS
AibADK AibANAibA DD AibAEAibAKENS.
[0600] FIG. 133: PEPTIDE ENGINEERING--cKit Receptor Fragment.
Stable conformation of SCF_mcKit(Ac)_(4GS)2_9R_N following
modification of key hydrophobic residues with amino isobutyric
acid.
Sequence CWU 1
1
277139PRTArtificial sequenceSynthetic sequence 1His Gly Glu Gly Thr
Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val
Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser
Gly Ala Pro Pro Pro Ser 35 239PRTArtificial sequenceSynthetic
sequence 2His Gly Glu Gly Thr Phe Thr Ser Asp Leu Cys Lys Gln Met
Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35
386PRTArtificial sequenceSynthetic sequence 3Lys Arg Leu Tyr Cys
Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro 1 5 10 15 Asp Gly Arg
Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys 20 25 30 Leu
Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val 35 40
45 Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala
50 55 60 Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu
Glu Ser 65 70 75 80 Asn Asn Tyr Asn Thr Tyr 85 422PRTArtificial
sequenceSynthetic sequence 4Lys Asn Gly Gly Phe Phe Leu Arg Ile His
Pro Asp Gly Arg Val Asp 1 5 10 15 Gly Val Arg Glu Lys Ser 20
56PRTArtificial sequenceSynthetic sequence 5His Phe Lys Asp Pro Lys
1 5 68PRTArtificial sequenceSynthetic sequence 6Leu Glu Ser Asn Asn
Tyr Asn Thr 1 5 721PRTArtificial sequenceSynthetic sequence 7Met
Ile Ala Ser Gln Phe Leu Ser Ala Leu Thr Leu Val Leu Leu Ile 1 5 10
15 Lys Glu Ser Gly Ala 20 838PRTArtificial sequenceSynthetic
sequence 8Met Val Phe Pro Trp Arg Cys Glu Gly Thr Tyr Trp Gly Ser
Arg Asn 1 5 10 15 Ile Leu Lys Leu Trp Val Trp Thr Leu Leu Cys Cys
Asp Phe Leu Ile 20 25 30 His His Gly Thr His Cys 35
938PRTArtificial sequenceSynthetic sequence 9Met Ile Phe Pro Trp
Lys Cys Gln Ser Thr Gln Arg Asp Leu Trp Asn 1 5 10 15 Ile Phe Lys
Leu Trp Gly Trp Thr Met Leu Cys Cys Asp Phe Leu Ala 20 25 30 His
His Gly Thr Asp Cys 35 1028PRTArtificial sequenceSynthetic sequence
10Met Ile Phe Pro Trp Lys Cys Gln Ser Thr Gln Arg Asp Leu Trp Asn 1
5 10 15 Ile Phe Lys Leu Trp Gly Trp Thr Met Leu Cys Cys 20 25
1112PRTArtificial sequenceSynthetic sequence 11Thr His Arg Pro Pro
Met Trp Ser Pro Val Trp Pro 1 5 10 127PRTArtificial
sequenceSynthetic sequence 12Arg Arg Glu Thr Ala Trp Ala 1 5
1386PRTArtificial sequenceSynthetic sequence 13Lys Arg Leu Tyr Cys
Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro 1 5 10 15 Asp Gly Arg
Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys 20 25 30 Leu
Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val 35 40
45 Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala
50 55 60 Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu
Glu Ser 65 70 75 80 Asn Asn Tyr Asn Thr Tyr 85 1423PRTArtificial
sequenceSynthetic sequence 14Cys Lys Asn Gly Gly Phe Phe Leu Arg
Ile His Pro Asp Gly Arg Val 1 5 10 15 Asp Gly Val Arg Glu Lys Ser
20 159PRTArtificial sequenceSynthetic sequence 15Arg Arg Arg Arg
Arg Arg Arg Arg Arg 1 5 166PRTArtificial sequenceSynthetic sequence
16His His His His His His 1 5 179PRTArtificial sequenceSynthetic
sequence 17Gly Ala Pro Gly Ala Pro Cys Ala Pro 1 5
1810PRTArtificial sequenceSynthetic sequence 18Gly Ala Pro Gly Ala
Pro Cys Ala Pro Cys 1 5 10 1910PRTArtificial sequenceSynthetic
sequence 19Cys Gly Ala Pro Gly Ala Pro Gly Ala Pro 1 5 10
205PRTArtificial sequenceSynthetic sequence 20Gly Ser Gly Gly Ser 1
5 216PRTArtificial sequenceSynthetic sequence 21Gly Gly Ser Gly Gly
Ser 1 5 224PRTArtificial sequenceSynthetic sequence 22Gly Gly Gly
Ser 1 234PRTArtificial sequenceSynthetic sequence 23Gly Gly Ser Gly
1 245PRTArtificial sequenceSynthetic sequence 24Gly Gly Ser Gly Gly
1 5 255PRTArtificial sequenceSynthetic sequence 25Gly Ser Gly Ser
Gly 1 5 265PRTArtificial sequenceSynthetic sequence 26Gly Ser Gly
Gly Gly 1 5 275PRTArtificial sequenceSynthetic sequence 27Gly Gly
Gly Ser Gly 1 5 285PRTArtificial sequenceSynthetic sequence 28Gly
Ser Ser Ser Gly 1 5 2911PRTArtificial sequenceSynthetic sequence
29Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly 1 5 10
3011PRTArtificial sequenceSynthetic sequence 30Gly Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 1 5 10 3112PRTArtificial sequenceSynthetic
sequence 31Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Cys 1 5 10
3212PRTArtificial sequenceSynthetic sequence 32Cys Gly Gly Gly Gly
Gly Ser Gly Gly Gly Gly Gly 1 5 10 3312PRTArtificial
sequenceSynthetic sequence 33Gly Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Cys 1 5 10 3412PRTArtificial sequenceSynthetic sequence
34Cys Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10
354PRTArtificial sequenceSynthetic sequence 35Lys Ala Leu Ala 1
364PRTArtificial sequenceSynthetic sequence 36Gly Ala Leu Ala 1
375PRTArtificial sequenceSynthetic sequence 37Cys Lys Ala Leu Ala 1
5 385PRTArtificial sequenceSynthetic sequence 38Lys Ala Leu Ala Cys
1 5 395PRTArtificial sequenceSynthetic sequence 39Cys Gly Ala Leu
Ala 1 5 405PRTArtificial sequenceSynthetic sequence 40Gly Ala Leu
Ala Cys 1 5 4119PRTArtificial sequenceSynthetic sequence 41Arg Arg
Arg Arg Arg Arg Arg Arg Arg Gly Ala Pro Gly Ala Pro Gly 1 5 10 15
Ala Pro Cys 4219PRTArtificial sequenceSynthetic sequence 42Cys Gly
Ala Pro Gly Ala Pro Gly Ala Pro Arg Arg Arg Arg Arg Arg 1 5 10 15
Arg Arg Arg 4323PRTArtificial sequenceSynthetic sequence 43Cys Lys
Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg Val 1 5 10 15
Asp Gly Val Arg Glu Lys Ser 20 4423PRTArtificial sequenceSynthetic
sequence 44Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg
Val Asp 1 5 10 15 Gly Val Arg Glu Lys Ser Cys 20 4525PRTArtificial
sequenceSynthetic sequence 45Arg Arg Arg Arg Arg Arg Arg Arg Arg
Gly Ala Pro Gly Ala Pro Gly 1 5 10 15 Ala Pro Arg Arg Glu Thr Ala
Trp Ala 20 25 4625PRTArtificial sequenceSynthetic sequence 46Arg
Arg Glu Thr Ala Trp Ala Gly Ala Pro Gly Ala Pro Gly Ala Pro 1 5 10
15 Arg Arg Arg Arg Arg Arg Arg Arg Arg 20 25 4721PRTArtificial
sequenceSynthetic sequence 47Arg Arg Arg Arg Arg Arg Arg Arg Arg
Gly Ala Pro Gly Ala Pro Gly 1 5 10 15 Ala Pro Arg Gly Asp 20
4821PRTArtificial sequenceSynthetic sequence 48Arg Gly Asp Gly Ala
Pro Gly Ala Pro Gly Ala Pro Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg
Arg Arg 20 494PRTArtificial sequenceSynthetic sequence 49Cys Arg
Gly Asp 1 504PRTArtificial sequenceSynthetic sequence 50Arg Gly Asp
Cys 1 5130PRTArtificial sequenceSynthetic sequence 51Arg Arg Arg
Arg Arg Arg Arg Arg Arg Gly Ala Pro Gly Ala Pro Gly 1 5 10 15 Ala
Pro Thr His Arg Pro Pro Met Trp Ser Pro Val Trp Pro 20 25 30
5230PRTArtificial sequenceSynthetic sequence 52Thr His Arg Pro Pro
Met Trp Ser Pro Val Trp Pro Gly Ala Pro Gly 1 5 10 15 Ala Pro Gly
Ala Pro Arg Arg Arg Arg Arg Arg Arg Arg Arg 20 25 30
5313PRTArtificial sequenceSynthetic sequence 53Cys Thr His Arg Pro
Pro Met Trp Ser Pro Val Trp Pro 1 5 10 5414PRTArtificial
sequenceSynthetic sequence 54Cys Pro Thr His Arg Pro Pro Met Trp
Ser Pro Val Trp Pro 1 5 10 5513PRTArtificial sequenceSynthetic
sequence 55Thr His Arg Pro Pro Met Trp Ser Pro Val Trp Pro Cys 1 5
10 5639PRTArtificial sequenceSynthetic sequence 56Arg Arg Arg Arg
Arg Arg Arg Arg Arg Gly Ala Pro Gly Ala Pro Gly 1 5 10 15 Ala Pro
Met Ile Ala Ser Gln Phe Leu Ser Ala Leu Thr Leu Val Leu 20 25 30
Leu Ile Lys Glu Ser Gly Ala 35 5739PRTArtificial sequenceSynthetic
sequence 57Met Ile Ala Ser Gln Phe Leu Ser Ala Leu Thr Leu Val Leu
Leu Ile 1 5 10 15 Lys Glu Ser Gly Ala Gly Ala Pro Gly Ala Pro Gly
Ala Pro Arg Arg 20 25 30 Arg Arg Arg Arg Arg Arg Arg 35
5822PRTArtificial sequenceSynthetic sequence 58Cys Met Ile Ala Ser
Gln Phe Leu Ser Ala Leu Thr Leu Val Leu Leu 1 5 10 15 Ile Lys Glu
Ser Gly Ala 20 5922PRTArtificial sequenceSynthetic sequence 59Met
Ile Ala Ser Gln Phe Leu Ser Ala Leu Thr Leu Val Leu Leu Ile 1 5 10
15 Lys Glu Ser Gly Ala Cys 20 6040PRTArtificial sequenceSynthetic
sequence 60Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly Ala Pro Gly Ala
Pro Gly 1 5 10 15 Ala Pro Lys Asn Gly Gly Phe Phe Leu Arg Ile His
Pro Asp Gly Arg 20 25 30 Val Asp Gly Val Arg Glu Lys Ser 35 40
6140PRTArtificial sequenceSynthetic sequence 61Lys Asn Gly Gly Phe
Phe Leu Arg Ile His Pro Asp Gly Arg Val Asp 1 5 10 15 Gly Val Arg
Glu Lys Ser Gly Ala Pro Gly Ala Pro Gly Ala Pro Arg 20 25 30 Arg
Arg Arg Arg Arg Arg Arg Arg 35 40 6220PRTArtificial
sequenceSynthetic sequence 62Ser Gly Arg Gly Lys Gln Gly Gly Lys
Ala Arg Ala Lys Ala Lys Thr 1 5 10 15 Arg Ser Ser Arg 20
6339PRTArtificial sequenceSynthetic sequence 63Ser Gly Arg Gly Lys
Gln Gly Gly Lys Ala Arg Ala Lys Ala Lys Thr 1 5 10 15 Arg Ser Ser
Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg 20 25 30 Leu
Leu Arg Lys Gly Gly Gly 35 64130PRTArtificial sequenceSynthetic
sequence 64Met Ser Gly Arg Gly Lys Gln Gly Gly Lys Ala Arg Ala Lys
Ala Lys 1 5 10 15 Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val
Gly Arg Val His 20 25 30 Arg Leu Leu Arg Lys Gly Asn Tyr Ala Glu
Arg Val Gly Ala Gly Ala 35 40 45 Pro Val Tyr Leu Ala Ala Val Leu
Glu Tyr Leu Thr Ala Glu Ile Leu 50 55 60 Glu Leu Ala Gly Asn Ala
Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile 65 70 75 80 Pro Arg His Leu
Gln Leu Ala Ile Arg Asn Asp Glu Glu Leu Asn Lys 85 90 95 Leu Leu
Gly Lys Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile 100 105 110
Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Ser His His Lys Ala Lys 115
120 125 Gly Lys 130 6510PRTArtificial sequenceSynthetic sequence
65Cys Lys Ala Thr Gln Ala Ser Gln Glu Tyr 1 5 10 6625PRTArtificial
sequenceSynthetic sequence 66Lys Lys Thr Ser Ala Thr Val Gly Pro
Lys Ala Pro Ser Gly Gly Lys 1 5 10 15 Lys Ala Thr Gln Ala Ser Gln
Glu Tyr 20 25 67143PRTArtificial sequenceSynthetic sequence 67Met
Ser Gly Arg Gly Lys Thr Gly Gly Lys Ala Arg Ala Lys Ala Lys 1 5 10
15 Ser Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
20 25 30 Arg Leu Leu Arg Lys Gly His Tyr Ala Glu Arg Val Gly Ala
Gly Ala 35 40 45 Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr
Ala Glu Ile Leu 50 55 60 Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn
Lys Lys Thr Arg Ile Ile 65 70 75 80 Pro Arg His Leu Gln Leu Ala Ile
Arg Asn Asp Glu Glu Leu Asn Lys 85 90 95 Leu Leu Gly Gly Val Thr
Ile Ala Gln Gly Gly Val Leu Pro Asn Ile 100 105 110 Gln Ala Val Leu
Leu Pro Lys Lys Thr Ser Ala Thr Val Gly Pro Lys 115 120 125 Ala Pro
Ser Gly Gly Lys Lys Ala Thr Gln Ala Ser Gln Glu Tyr 130 135 140
6811PRTArtificial sequenceSynthetic sequence 68Pro Glu Pro Ala Lys
Ser Ala Pro Ala Pro Lys 1 5 10 6911PRTArtificial sequenceSynthetic
sequence 69Pro Glu Pro Ala Lys Ser Ala Pro Ala Pro Lys 1 5 10
7015PRTArtificial sequenceSynthetic sequence 70Ala Gln Lys Lys Asp
Gly Lys Lys Arg Lys Arg Ser Arg Lys Glu 1 5 10 15
71126PRTArtificial sequenceSynthetic sequence 71Met Pro Glu Pro Ala
Lys Ser Ala Pro Ala Pro Lys Lys Gly Ser Lys 1 5 10 15 Lys Ala Val
Thr Lys Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys Arg 20 25 30 Ser
Arg Lys Glu Ser Tyr Ser Ile Tyr Val Tyr Lys Val Leu Lys Gln 35 40
45 Val His Pro Asp Thr Gly Ile Ser Ser Lys Ala Met Gly Ile Met Asn
50 55 60 Ser Phe Val Asn Asp Ile Phe Glu Arg Ile Ala Gly Glu Ala
Ser Arg 65 70 75 80 Leu Ala His Tyr Asn Lys Arg Ser Thr Ile Thr Ser
Arg Glu Ile Gln 85 90 95 Thr Ala Val Arg Leu Leu Leu Pro Gly Glu
Leu Ala Lys His Ala Val 100 105 110 Ser Glu Gly Thr Lys Ala Val Thr
Lys Tyr Thr Ser Ser Lys 115 120 125 728PRTArtificial
sequenceSynthetic sequence 72Ala Arg Thr Lys Gln Thr Ala Arg 1 5
7310PRTArtificial sequenceSynthetic sequence 73Ala Arg Thr Lys Gln
Thr Ala Arg Lys Ser 1 5 10 7410PRTArtificial sequenceSynthetic
sequence 74Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser 1 5 10
7510PRTArtificial sequenceSynthetic sequence 75Ala Arg Thr Lys Gln
Thr Ala Arg Lys Ser 1 5 10 7615PRTArtificial sequenceSynthetic
sequence 76Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys
Ala 1 5 10 15 7720PRTArtificial sequenceSynthetic sequence 77Ala
Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1 5 10
15 Arg Lys Trp Cys 20 7819PRTArtificial sequenceSynthetic sequence
78Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1
5 10 15 Arg Lys Gln 7920PRTArtificial sequenceSynthetic sequence
79Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1
5 10 15 Arg Lys Gln Leu 20 8020PRTArtificial sequenceSynthetic
sequence 80Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys
Ala Pro 1 5 10 15 Arg Lys Gln Leu 20 8121PRTArtificial
sequenceSynthetic sequence 81Ala Arg Thr Lys Gln Thr Ala Arg Lys
Ser Thr
Gly Gly Lys Ala Pro 1 5 10 15 Arg Lys Gln Leu Ala 20
8221PRTArtificial sequenceSynthetic sequence 82Ala Arg Thr Lys Gln
Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1 5 10 15 Arg Lys Gln
Leu Ala 20 8321PRTArtificial sequenceSynthetic sequence 83Ala Arg
Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1 5 10 15
Arg Lys Gln Leu Ala 20 8421PRTArtificial sequenceSynthetic sequence
84Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1
5 10 15 Arg Lys Gln Leu Ala 20 8521PRTArtificial sequenceSynthetic
sequence 85Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys
Ala Pro 1 5 10 15 Arg Lys Gln Leu Ala 20 8621PRTArtificial
sequenceSynthetic sequence 86Ala Arg Thr Lys Gln Thr Ala Arg Lys
Ser Thr Gly Gly Lys Ala Pro 1 5 10 15 Arg Lys Gln Leu Ala 20
8721PRTArtificial sequenceSynthetic sequence 87Ala Arg Thr Lys Gln
Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1 5 10 15 Arg Lys Gln
Leu Ala 20 8821PRTArtificial sequenceSynthetic sequence 88Ala Arg
Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1 5 10 15
Arg Lys Gln Leu Ala 20 8921PRTArtificial sequenceSynthetic sequence
89Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1
5 10 15 Arg Lys Gln Leu Ala 20 9021PRTArtificial sequenceSynthetic
sequence 90Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys
Ala Pro 1 5 10 15 Arg Lys Gln Leu Ala 20 9122PRTArtificial
sequenceSynthetic sequence 91Ala Arg Thr Lys Gln Thr Ala Arg Lys
Ser Thr Gly Gly Lys Ala Pro 1 5 10 15 Arg Lys Gln Leu Ala Cys 20
9224PRTArtificial sequenceSynthetic sequence 92Ala Arg Thr Lys Gln
Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1 5 10 15 Arg Lys Gln
Leu Ala Thr Lys Ala 20 9324PRTArtificial sequenceSynthetic sequence
93Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1
5 10 15 Arg Lys Gln Leu Ala Thr Lys Ala 20 9425PRTArtificial
sequenceSynthetic sequence 94Ala Arg Thr Lys Gln Thr Ala Arg Lys
Ser Thr Gly Gly Lys Ala Pro 1 5 10 15 Arg Lys Gln Leu Ala Thr Lys
Ala Ala 20 25 9525PRTArtificial sequenceSynthetic sequence 95Ala
Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1 5 10
15 Arg Lys Gln Leu Ala Thr Lys Ala Ala 20 25 9615PRTArtificial
sequenceSynthetic sequence 96Thr Lys Gln Thr Ala Arg Lys Ser Thr
Gly Gly Lys Ala Pro Arg 1 5 10 15 9715PRTArtificial
sequenceSynthetic sequence 97Thr Lys Gln Thr Ala Arg Lys Ser Thr
Gly Gly Lys Ala Pro Arg 1 5 10 15 9815PRTArtificial
sequenceSynthetic sequence 98Thr Lys Gln Thr Ala Arg Lys Ser Thr
Gly Gly Lys Ala Pro Arg 1 5 10 15 9911PRTArtificial
sequenceSynthetic sequence 99Lys Ser Thr Gly Gly Lys Ala Pro Arg
Lys Gln 1 5 10 10019PRTArtificial sequenceSynthetic sequence 100Gln
Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro Arg Lys Gln Leu 1 5 10
15 Ala Ser Lys 10125PRTArtificial sequenceSynthetic sequence 101Ala
Pro Arg Lys Gln Leu Ala Thr Lys Ala Ala Arg Lys Ser Ala Pro 1 5 10
15 Ala Thr Gly Gly Val Lys Lys Pro His 20 25 10224PRTArtificial
sequenceSynthetic sequence 102Ala Thr Lys Ala Ala Arg Lys Ser Ala
Pro Ala Thr Gly Gly Val Lys 1 5 10 15 Lys Pro His Arg Tyr Arg Pro
Gly 20 1039PRTArtificial sequenceSynthetic sequence 103Lys Ala Ala
Arg Lys Ser Ala Pro Ala 1 5 10412PRTArtificial sequenceSynthetic
sequence 104Lys Ala Ala Arg Lys Ser Ala Pro Ala Thr Gly Gly 1 5 10
10513PRTArtificial sequenceSynthetic sequence 105Lys Ala Ala Arg
Lys Ser Ala Pro Ala Thr Gly Gly Cys 1 5 10 10612PRTArtificial
sequenceSynthetic sequence 106Lys Ala Ala Arg Lys Ser Ala Pro Ala
Thr Gly Gly 1 5 10 10712PRTArtificial sequenceSynthetic sequence
107Lys Ala Ala Arg Lys Ser Ala Pro Ala Thr Gly Gly 1 5 10
10812PRTArtificial sequenceSynthetic sequence 108Lys Ala Ala Arg
Lys Ser Ala Pro Ala Thr Gly Gly 1 5 10 10912PRTArtificial
sequenceSynthetic sequence 109Lys Ala Ala Arg Lys Ser Ala Pro Ala
Thr Gly Gly 1 5 10 11024PRTArtificial sequenceSynthetic sequence
110Ala Thr Lys Ala Ala Arg Lys Ser Ala Pro Ser Thr Gly Gly Val Lys
1 5 10 15 Lys Pro His Arg Tyr Arg Pro Gly 20 11124PRTArtificial
sequenceSynthetic sequence 111Ala Thr Lys Ala Ala Arg Lys Ser Ala
Pro Ser Thr Gly Gly Val Lys 1 5 10 15 Lys Pro His Arg Tyr Arg Pro
Gly 20 11235PRTArtificial sequenceSynthetic sequence 112Ala Arg Thr
Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1 5 10 15 Arg
Lys Gln Leu Ala Thr Lys Ala Ala Arg Lys Ser Ala Pro Ala Thr 20 25
30 Gly Gly Val 35 11311PRTArtificial sequenceSynthetic sequence
113Ser Thr Gly Gly Val Lys Lys Pro His Arg Tyr 1 5 10
11411PRTArtificial sequenceSynthetic sequence 114Ser Thr Gly Gly
Val Lys Lys Pro His Arg Tyr 1 5 10 11511PRTArtificial
sequenceSynthetic sequence 115Ser Thr Gly Gly Val Lys Lys Pro His
Arg Tyr 1 5 10 11620PRTArtificial sequenceSynthetic sequence 116Gly
Thr Val Ala Leu Arg Glu Ile Arg Arg Tyr Gln Lys Ser Thr Glu 1 5 10
15 Leu Leu Ile Arg 20 11750PRTArtificial sequenceSynthetic sequence
117Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro
1 5 10 15 Arg Lys Gln Leu Ala Thr Lys Ala Ala Arg Lys Ser Ala Pro
Ala Thr 20 25 30 Gly Gly Val Lys Lys Pro His Arg Tyr Arg Pro Gly
Thr Val Ala Leu 35 40 45 Arg Glu 50 11832PRTArtificial
sequenceSynthetic sequence 118Thr Glu Leu Leu Ile Arg Lys Leu Pro
Phe Gln Arg Leu Val Arg Glu 1 5 10 15 Ile Ala Gln Asp Phe Lys Thr
Asp Leu Arg Phe Gln Ser Ala Ala Ile 20 25 30 11911PRTArtificial
sequenceSynthetic sequence 119Glu Ile Ala Gln Asp Phe Lys Thr Asp
Leu Arg 1 5 10 12011PRTArtificial sequenceSynthetic sequence 120Glu
Ile Ala Gln Asp Phe Lys Thr Asp Leu Arg 1 5 10 12111PRTArtificial
sequenceSynthetic sequence 121Glu Ile Ala Gln Asp Phe Lys Thr Asp
Leu Arg 1 5 10 12221PRTArtificial sequenceSynthetic sequence 122Arg
Leu Val Arg Glu Ile Ala Gln Asp Phe Lys Thr Asp Leu Arg Phe 1 5 10
15 Gln Ser Ser Ala Val 20 12321PRTArtificial sequenceSynthetic
sequence 123Arg Leu Val Arg Glu Ile Ala Gln Asp Phe Lys Thr Asp Leu
Arg Phe 1 5 10 15 Gln Ser Ser Ala Val 20 12421PRTArtificial
sequenceSynthetic sequence 124Arg Leu Val Arg Glu Ile Ala Gln Asp
Phe Lys Thr Asp Leu Arg Phe 1 5 10 15 Gln Ser Ser Ala Val 20
12521PRTArtificial sequenceSynthetic sequence 125Arg Leu Val Arg
Glu Ile Ala Gln Asp Phe Lys Thr Asp Leu Arg Phe 1 5 10 15 Gln Ser
Ser Ala Val 20 12621PRTArtificial sequenceSynthetic sequence 126Lys
Arg Val Thr Ile Met Pro Lys Asp Ile Gln Leu Ala Arg Arg Ile 1 5 10
15 Arg Gly Glu Arg Ala 20 127136PRTArtificial sequenceSynthetic
sequence 127Met Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly
Lys Ala 1 5 10 15 Pro Arg Lys Gln Leu Ala Thr Lys Val Ala Arg Lys
Ser Ala Pro Ala 20 25 30 Thr Gly Gly Val Lys Lys Pro His Arg Tyr
Arg Pro Gly Thr Val Ala 35 40 45 Leu Arg Glu Ile Arg Arg Tyr Gln
Lys Ser Thr Glu Leu Leu Ile Arg 50 55 60 Lys Leu Pro Phe Gln Arg
Leu Met Arg Glu Ile Ala Gln Asp Phe Lys 65 70 75 80 Thr Asp Leu Arg
Phe Gln Ser Ser Ala Val Met Ala Leu Gln Glu Ala 85 90 95 Cys Glu
Ser Tyr Leu Val Gly Leu Phe Glu Asp Thr Asn Leu Cys Val 100 105 110
Ile His Ala Lys Arg Val Thr Ile Met Pro Lys Asp Ile Gln Leu Ala 115
120 125 Arg Arg Ile Arg Gly Glu Arg Ala 130 135 1287PRTArtificial
sequenceSynthetic sequence 128Ser Gly Arg Gly Lys Gly Gly 1 5
12912PRTArtificial sequenceSynthetic sequence 129Arg Gly Lys Gly
Gly Lys Gly Leu Gly Lys Gly Ala 1 5 10 13021PRTArtificial
sequenceSynthetic sequence 130Ser Gly Arg Gly Lys Gly Gly Lys Gly
Leu Gly Lys Gly Gly Ala Lys 1 5 10 15 Arg His Arg Lys Val 20
13120PRTArtificial sequenceSynthetic sequence 131Lys Gly Leu Gly
Lys Gly Gly Ala Lys Arg His Arg Lys Val Leu Arg 1 5 10 15 Asp Asn
Trp Cys 20 13230PRTArtificial sequenceSynthetic sequence 132Ser Gly
Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly Ala Lys 1 5 10 15
Arg His Arg Lys Val Leu Arg Asp Asn Gly Ser Gly Ser Lys 20 25 30
13320PRTArtificial sequenceSynthetic sequence 133Ser Gly Arg Gly
Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly Ala Lys 1 5 10 15 Arg His
Arg Lys 20 13420PRTArtificial sequenceSynthetic sequence 134Ser Gly
Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly Ala Lys 1 5 10 15
Arg His Arg Lys 20 13520PRTArtificial sequenceSynthetic sequence
135Ser Gly Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly Ala Lys
1 5 10 15 Arg His Arg Lys 20 13620PRTArtificial sequenceSynthetic
sequence 136Ser Gly Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly
Ala Lys 1 5 10 15 Arg His Arg Lys 20 13720PRTArtificial
sequenceSynthetic sequence 137Ser Gly Arg Gly Lys Gly Gly Lys Gly
Leu Gly Lys Gly Gly Ala Lys 1 5 10 15 Arg His Arg Lys 20
13820PRTArtificial sequenceSynthetic sequence 138Lys Gly Leu Gly
Lys Gly Gly Ala Lys Arg His Arg Lys Val Leu Arg 1 5 10 15 Asp Asn
Trp Cys 20 139103PRTArtificial sequenceSynthetic sequence 139Met
Ser Gly Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly Ala 1 5 10
15 Lys Arg His Arg Lys Val Leu Arg Asp Asn Ile Gln Gly Ile Thr Lys
20 25 30 Pro Ala Ile Arg Arg Leu Ala Arg Arg Gly Gly Val Lys Arg
Ile Ser 35 40 45 Gly Leu Ile Tyr Glu Glu Thr Arg Gly Val Leu Lys
Val Phe Leu Glu 50 55 60 Asn Val Ile Arg Asp Ala Val Thr Tyr Thr
Glu His Ala Lys Arg Lys 65 70 75 80 Thr Val Thr Ala Met Asp Val Val
Tyr Ala Leu Lys Arg Gln Gly Arg 85 90 95 Thr Leu Tyr Gly Phe Gly
Gly 100 14010PRTArtificial sequenceSynthetic sequence 140Cys Lys
Ala Thr Gln Ala Ser Gln Glu Tyr 1 5 10 14122PRTArtificial
sequenceSynthetic sequence 141Ala Arg Thr Lys Gln Thr Ala Arg Lys
Ser Thr Gly Gly Lys Ala Pro 1 5 10 15 Arg Lys Gln Leu Ala Cys 20
14220PRTArtificial sequenceSynthetic sequence 142Ala Arg Thr Lys
Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro 1 5 10 15 Arg Lys
Trp Cys 20 14313PRTArtificial sequenceSynthetic sequence 143Lys Ala
Ala Arg Lys Ser Ala Pro Ala Thr Gly Gly Cys 1 5 10
14420PRTArtificial sequenceSynthetic sequence 144Lys Gly Leu Gly
Lys Gly Gly Ala Lys Arg His Arg Lys Val Leu Arg 1 5 10 15 Asp Asn
Trp Cys 20 145136PRTArtificial sequenceSynthetic sequence 145Met
Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala 1 5 10
15 Pro Arg Lys Gln Leu Ala Thr Lys Val Ala Arg Lys Ser Ala Pro Ala
20 25 30 Thr Gly Gly Val Lys Lys Pro His Arg Tyr Arg Pro Gly Thr
Val Ala 35 40 45 Leu Arg Glu Ile Arg Arg Tyr Gln Lys Ser Thr Glu
Leu Leu Ile Arg 50 55 60 Lys Leu Pro Phe Gln Arg Leu Met Arg Glu
Ile Ala Gln Asp Phe Lys 65 70 75 80 Thr Asp Leu Arg Phe Gln Ser Ser
Ala Val Met Ala Leu Gln Glu Ala 85 90 95 Cys Glu Ser Tyr Leu Val
Gly Leu Phe Glu Asp Thr Asn Leu Cys Val 100 105 110 Ile His Ala Lys
Arg Val Thr Ile Met Pro Lys Asp Ile Gln Leu Ala 115 120 125 Arg Arg
Ile Arg Gly Glu Arg Ala 130 135 1464PRTArtificial sequenceSynthetic
sequence 146Ala Ala Ala Ala 1 1477PRTArtificial sequenceSynthetic
sequence 147Thr Lys Pro Arg Pro Gly Pro 1 5 1487PRTArtificial
sequenceSynthetic sequence 148Met Glu His Phe Pro Gly Pro 1 5
14928PRTArtificial sequenceSynthetic sequence 149Pro Glu Asp Glu
Ile Trp Leu Pro Glu Pro Glu Ser Val Asp Val Pro 1 5 10 15 Ala Lys
Pro Ile Ser Thr Ser Ser Met Met Met Pro 20 25 1504PRTArtificial
sequenceSynthetic sequence 150Ala Ala Ala Ala 1 1517PRTArtificial
sequenceSynthetic sequence 151Pro Lys Lys Lys Arg Lys Val 1 5
15212PRTArtificial sequenceSynthetic sequence 152Pro Lys Lys Lys
Arg Lys Val Glu Asp Pro Tyr Cys 1 5 10 15333PRTArtificial
sequenceSynthetic sequence 153Pro Lys Lys Lys Arg Lys Val Gly Pro
Lys Lys Lys Arg Lys Val Gly 1 5 10 15 Pro Lys Lys Lys Arg Lys Val
Gly Pro Lys Lys Lys Arg Lys Val Gly 20 25 30 Cys 15412PRTArtificial
sequenceSynthetic sequence 154Cys Tyr Gly Arg Lys Lys Arg Arg Gln
Arg Arg Arg 1 5 10 15517PRTArtificial sequenceSynthetic sequence
155Cys Ser Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Tyr Leu
1 5 10 15 Ile 15617PRTArtificial sequenceSynthetic sequence 156Asp
Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys 1 5 10
15 Lys 15712PRTArtificial sequenceSynthetic sequence 157Pro Lys Lys
Lys Arg Lys Val Glu Asp Pro Tyr Cys 1 5 10 1589PRTArtificial
sequenceSynthetic sequence 158Pro Ala Ala Lys Arg Val Lys Leu Asp 1
5 1594PRTArtificial sequenceSynthetic sequence 159Ala Ala Ala Ala 1
16011PRTArtificial sequenceSynthetic sequence 160Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg 1 5 10 16112PRTArtificial
sequenceSynthetic sequence 161Arg Arg Gln Arg Arg Thr Ser Lys Leu
Met Lys Arg 1 5 10 16227PRTArtificial sequenceSynthetic sequence
162Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu
1 5
10 15 Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 25
16333PRTArtificial sequenceSynthetic sequence 163Lys Ala Leu Ala
Trp Glu Ala Lys Leu Ala Lys Ala Leu Ala Lys Ala 1 5 10 15 Leu Ala
Lys His Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Cys Glu 20 25 30
Ala 16416PRTArtificial sequenceSynthetic sequence 164Arg Gln Ile
Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15
1659PRTArtificial sequenceSynthetic sequence 165Arg Lys Lys Arg Arg
Gln Arg Arg Arg 1 5 1668PRTArtificial sequenceSynthetic sequence
166Arg Lys Lys Arg Arg Gln Arg Arg 1 5 16711PRTArtificial
sequenceSynthetic sequence 167Tyr Ala Arg Ala Ala Ala Arg Gln Ala
Arg Ala 1 5 10 16811PRTArtificial sequenceSynthetic sequence 168Thr
His Arg Leu Pro Arg Arg Arg Arg Arg Arg 1 5 10 16911PRTArtificial
sequenceSynthetic sequence 169Gly Gly Arg Arg Ala Arg Arg Arg Arg
Arg Arg 1 5 10 17010PRTArtificial sequenceSynthetic sequence 170Gln
Gln Gln Gln Gln Gln Gln Gln Gln Gln 1 5 10 171186PRTArtificial
sequenceSynthetic sequence 171Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg 20 25 30 Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 35 40 45 Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 50 55 60 Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 65 70
75 80 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg 85 90 95 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg 100 105 110 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg 115 120 125 Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg 130 135 140 Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg 145 150 155 160 Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 165 170 175 Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg 180 185 17264PRTArtificial
sequenceSynthetic sequence 172Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg 20 25 30 Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 35 40 45 Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 50 55 60
1734PRTArtificial sequenceSynthetic sequence 173Ala Ala Ala Ala 1
1744PRTArtificial sequenceSynthetic sequence 174Ala Ala Ala Ala 1
175116PRTArtificial sequenceSynthetic sequence 175Glu Glu Glu Glu
Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu 1 5 10 15 Glu Glu
Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu 20 25 30
Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu 35
40 45 Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu
Glu 50 55 60 Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu
Glu Glu Glu 65 70 75 80 Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu
Glu Glu Glu Glu Glu 85 90 95 Glu Glu Glu Glu Glu Glu Glu Glu Glu
Glu Glu Glu Glu Glu Glu Glu 100 105 110 Glu Glu Glu Glu 115
1764PRTArtificial sequenceSynthetic sequence 176Ala Ala Ala Ala 1
1774PRTArtificial sequenceSynthetic sequence 177Ala Ala Ala Ala 1
1784PRTArtificial sequenceSynthetic sequence 178Ala Ala Ala Ala 1
1794PRTArtificial sequenceSynthetic sequence 179Ala Ala Ala Ala 1
18041PRTArtificial sequenceSynthetic sequence 180Tyr Thr Ile Trp
Met Pro Glu Asn Pro Arg Pro Gly Thr Pro Cys Asp 1 5 10 15 Ile Phe
Thr Asn Ser Arg Gly Lys Arg Ala Ser Asn Gly Gly Gly Gly 20 25 30
Arg Arg Arg Arg Arg Arg Arg Arg Arg 35 40 1815PRTArtificial
sequenceSynthetic sequence 181Arg Gly Asp Gly Trp 1 5
18211PRTArtificial sequenceSynthetic sequence 182Gly Cys Gly Tyr
Gly Arg Gly Asp Ser Pro Gly 1 5 10 18332PRTArtificial
sequenceSynthetic sequence 183Tyr Thr Ile Trp Met Pro Glu Asn Pro
Arg Pro Gly Thr Pro Cys Asp 1 5 10 15 Ile Phe Thr Asn Ser Arg Gly
Lys Arg Ala Ser Asn Gly Gly Gly Gly 20 25 30 184164PRTArtificial
sequenceSynthetic sequence 184Glu Gly Ile Cys Arg Asn Arg Val Thr
Asn Asn Val Lys Asp Val Thr 1 5 10 15 Lys Leu Val Ala Asn Leu Pro
Lys Asp Tyr Met Ile Thr Leu Lys Tyr 20 25 30 Val Pro Gly Met Asp
Val Leu Pro Ser His Cys Trp Ile Ser Glu Met 35 40 45 Val Val Gln
Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser 50 55 60 Asn
Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val 65 70
75 80 Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser
Lys 85 90 95 Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu
Phe Thr Pro 100 105 110 Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile
Asp Ala Phe Lys Asp 115 120 125 Phe Val Val Ala Ser Glu Thr Ser Asp
Cys Val Val Ser Ser Thr Leu 130 135 140 Ser Pro Glu Lys Asp Ser Arg
Val Ser Val Thr Lys Pro Phe Met Leu 145 150 155 160 Pro Pro Val Ala
185192PRTArtificial sequenceSynthetic sequence 185Pro Glu Glu Gly
Ser Gly Cys Ser Val Arg Arg Arg Pro Tyr Gly Cys 1 5 10 15 Val Leu
Arg Ala Ala Leu Val Pro Leu Val Ala Gly Leu Val Ile Cys 20 25 30
Leu Val Val Cys Ile Gln Arg Phe Ala Gln Ala Gln Gln Gln Leu Pro 35
40 45 Leu Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His
Thr 50 55 60 Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly
Pro Ala Leu 65 70 75 80 Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp
Lys Gly Gln Leu Arg 85 90 95 Ile His Arg Asp Gly Ile Tyr Met Val
His Ile Gln Val Thr Leu Ala 100 105 110 Ile Cys Ser Ser Thr Thr Ala
Ser Arg His His Pro Thr Thr Leu Ala 115 120 125 Val Gly Ile Cys Ser
Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu 130 135 140 Ser Phe His
Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu 145 150 155 160
Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro 165
170 175 Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg
Pro 180 185 190 186138PRTArtificial sequenceSynthetic sequence
186Ser Ser Gly Leu Val Pro Arg Gly Ser His Met Asp Ala Val Ala Val
1 5 10 15 Tyr His Gly Lys Ile Ser Arg Glu Thr Gly Glu Lys Leu Leu
Leu Ala 20 25 30 Thr Gly Leu Asp Gly Ser Tyr Leu Leu Arg Asp Ser
Glu Ser Val Pro 35 40 45 Gly Val Tyr Cys Leu Cys Val Leu Tyr His
Gly Tyr Ile Tyr Thr Tyr 50 55 60 Arg Val Ser Gln Thr Glu Thr Gly
Ser Trp Ser Ala Glu Thr Ala Pro 65 70 75 80 Gly Val His Lys Arg Tyr
Phe Arg Lys Ile Lys Asn Leu Ile Ser Ala 85 90 95 Phe Gln Lys Pro
Asp Gln Gly Ile Val Ile Pro Leu Gln Tyr Pro Val 100 105 110 Glu Lys
Lys Ser Ser Ala Arg Ser Thr Gln Gly Thr Thr Gly Ile Arg 115 120 125
Glu Asp Pro Asp Val Cys Leu Lys Ala Pro 130 135 18713PRTArtificial
sequenceSynthetic sequence 187Asp Gly Ala Arg Tyr Cys Arg Gly Asp
Cys Phe Asp Gly 1 5 10 18811PRTArtificial sequenceSynthetic
sequence 188Gly Cys Gly Tyr Gly Arg Gly Asp Ser Pro Gly 1 5 10
189174PRTArtificial sequenceSynthetic sequence 189Arg Arg Arg Arg
Arg Arg Arg Arg Arg Met Glu Gly Ile Cys Arg Asn 1 5 10 15 Arg Val
Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu 20 25 30
Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val 35
40 45 Leu Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser
Asp 50 55 60 Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser
Glu Gly Leu 65 70 75 80 Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn
Ile Val Asp Asp Leu 85 90 95 Val Glu Cys Val Lys Glu Asn Ser Ser
Lys Asp Leu Lys Lys Ser Phe 100 105 110 Lys Ser Pro Glu Pro Arg Leu
Phe Thr Pro Glu Glu Phe Phe Arg Ile 115 120 125 Phe Asn Arg Ser Ile
Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu 130 135 140 Thr Ser Asp
Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser 145 150 155 160
Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala 165 170
190201PRTArtificial sequenceSynthetic sequence 190Arg Arg Arg Arg
Arg Arg Arg Arg Arg Pro Glu Glu Gly Ser Gly Cys 1 5 10 15 Ser Val
Arg Arg Arg Pro Tyr Gly Cys Val Leu Arg Ala Ala Leu Val 20 25 30
Pro Leu Val Ala Gly Leu Val Ile Cys Leu Val Val Cys Ile Gln Arg 35
40 45 Phe Ala Gln Ala Gln Gln Gln Leu Pro Leu Glu Ser Leu Gly Trp
Asp 50 55 60 Val Ala Glu Leu Gln Leu Asn His Thr Gly Pro Gln Gln
Asp Pro Arg 65 70 75 80 Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg
Ser Phe Leu His Gly 85 90 95 Pro Glu Leu Asp Lys Gly Gln Leu Arg
Ile His Arg Asp Gly Ile Tyr 100 105 110 Met Val His Ile Gln Val Thr
Leu Ala Ile Cys Ser Ser Thr Thr Ala 115 120 125 Ser Arg His His Pro
Thr Thr Leu Ala Val Gly Ile Cys Ser Pro Ala 130 135 140 Ser Arg Ser
Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly Cys Thr 145 150 155 160
Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys 165
170 175 Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu
Thr 180 185 190 Phe Phe Gly Val Gln Trp Val Arg Pro 195 200
191201PRTArtificial sequenceSynthetic sequence 191Pro Glu Glu Gly
Ser Gly Cys Ser Val Arg Arg Arg Pro Tyr Gly Cys 1 5 10 15 Val Leu
Arg Ala Ala Leu Val Pro Leu Val Ala Gly Leu Val Ile Cys 20 25 30
Leu Val Val Cys Ile Gln Arg Phe Ala Gln Ala Gln Gln Gln Leu Pro 35
40 45 Leu Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His
Thr 50 55 60 Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly
Pro Ala Leu 65 70 75 80 Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp
Lys Gly Gln Leu Arg 85 90 95 Ile His Arg Asp Gly Ile Tyr Met Val
His Ile Gln Val Thr Leu Ala 100 105 110 Ile Cys Ser Ser Thr Thr Ala
Ser Arg His His Pro Thr Thr Leu Ala 115 120 125 Val Gly Ile Cys Ser
Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu 130 135 140 Ser Phe His
Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu 145 150 155 160
Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro 165
170 175 Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg
Pro 180 185 190 Arg Arg Arg Arg Arg Arg Arg Arg Arg 195 200
192148PRTArtificial sequenceSynthetic sequence 192Met Gly Ser Ser
His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly
Ser His Met Asp Ala Val Ala Val Tyr His Gly Lys Ile Ser 20 25 30
Arg Glu Thr Gly Glu Lys Leu Leu Leu Ala Thr Gly Leu Asp Gly Ser 35
40 45 Tyr Leu Leu Arg Asp Ser Glu Ser Val Pro Gly Val Tyr Cys Leu
Cys 50 55 60 Val Leu Tyr His Gly Tyr Ile Tyr Thr Tyr Arg Val Ser
Gln Thr Glu 65 70 75 80 Thr Gly Ser Trp Ser Ala Glu Thr Ala Pro Gly
Val His Lys Arg Tyr 85 90 95 Phe Arg Lys Ile Lys Asn Leu Ile Ser
Ala Phe Gln Lys Pro Asp Gln 100 105 110 Gly Ile Val Ile Pro Leu Gln
Tyr Pro Val Glu Lys Lys Ser Ser Ala 115 120 125 Arg Ser Thr Gln Gly
Thr Thr Gly Ile Arg Glu Asp Pro Asp Val Cys 130 135 140 Leu Lys Ala
Pro 145 193147PRTArtificial sequenceSynthetic sequence 193Arg Arg
Arg Arg Arg Arg Arg Arg Arg Ser Ser Gly Leu Val Pro Arg 1 5 10 15
Gly Ser His Met Asp Ala Val Ala Val Tyr His Gly Lys Ile Ser Arg 20
25 30 Glu Thr Gly Glu Lys Leu Leu Leu Ala Thr Gly Leu Asp Gly Ser
Tyr 35 40 45 Leu Leu Arg Asp Ser Glu Ser Val Pro Gly Val Tyr Cys
Leu Cys Val 50 55 60 Leu Tyr His Gly Tyr Ile Tyr Thr Tyr Arg Val
Ser Gln Thr Glu Thr 65 70 75 80 Gly Ser Trp Ser Ala Glu Thr Ala Pro
Gly Val His Lys Arg Tyr Phe 85 90 95 Arg Lys Ile Lys Asn Leu Ile
Ser Ala Phe Gln Lys Pro Asp Gln Gly 100 105 110 Ile Val Ile Pro Leu
Gln Tyr Pro Val Glu Lys Lys Ser Ser Ala Arg 115 120 125 Ser Thr Gln
Gly Thr Thr Gly Ile Arg Glu Asp Pro Asp Val Cys Leu 130 135 140 Lys
Ala Pro 145 194167PRTArtificial sequenceSynthetic
sequencemisc_feature(1)..(1)Xaa can be any naturally occurring
amino acidmisc_feature(167)..(167)Xaa can be any naturally
occurring amino acid 194Xaa Met Glu Gly Ile Cys Arg Asn Arg Val Thr
Asn Asn Val Lys Asp 1 5 10 15 Val Thr Lys Leu Val Ala Asn Leu Pro
Lys Asp Tyr Met Ile Thr Leu 20 25 30 Lys Tyr Val Pro Gly Met Asp
Val Leu Pro Ser His Cys Trp Ile Ser 35 40 45 Glu Met Val Val Gln
Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys 50 55 60 Phe Ser Asn
Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys 65 70 75
80 Leu Val Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser
85 90 95 Ser Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg
Leu Phe 100 105 110 Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser
Ile Asp Ala Phe 115 120 125 Lys Asp Phe Val Val Ala Ser Glu Thr Ser
Asp Cys Val Val Ser Ser 130 135 140 Thr Leu Ser Pro Glu Lys Asp Ser
Arg Val Ser Val Thr Lys Pro Phe 145 150 155 160 Met Leu Pro Pro Val
Ala Xaa 165 195194PRTArtificial sequenceSynthetic
sequencemisc_feature(1)..(1)Xaa can be any naturally occurring
amino acidmisc_feature(194)..(194)Xaa can be any naturally
occurring amino acid 195Xaa Pro Glu Glu Gly Ser Gly Cys Ser Val Arg
Arg Arg Pro Tyr Gly 1 5 10 15 Cys Val Leu Arg Ala Ala Leu Val Pro
Leu Val Ala Gly Leu Val Ile 20 25 30 Cys Leu Val Val Cys Ile Gln
Arg Phe Ala Gln Ala Gln Gln Gln Leu 35 40 45 Pro Leu Glu Ser Leu
Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His 50 55 60 Thr Gly Pro
Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala 65 70 75 80 Leu
Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu 85 90
95 Arg Ile His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu
100 105 110 Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr
Thr Leu 115 120 125 Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile
Ser Leu Leu Arg 130 135 140 Leu Ser Phe His Gln Gly Cys Thr Ile Ala
Ser Gln Arg Leu Thr Pro 145 150 155 160 Leu Ala Arg Gly Asp Thr Leu
Cys Thr Asn Leu Thr Gly Thr Leu Leu 165 170 175 Pro Ser Arg Asn Thr
Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg 180 185 190 Pro Xaa
196139PRTArtificial sequenceSynthetic
sequencemisc_feature(1)..(1)Xaa can be any naturally occurring
amino acid 196Xaa Ser Ser Gly Leu Val Pro Arg Gly Ser His Met Asp
Ala Val Ala 1 5 10 15 Val Tyr His Gly Lys Ile Ser Arg Glu Thr Gly
Glu Lys Leu Leu Leu 20 25 30 Ala Thr Gly Leu Asp Gly Ser Tyr Leu
Leu Arg Asp Ser Glu Ser Val 35 40 45 Pro Gly Val Tyr Cys Leu Cys
Val Leu Tyr His Gly Tyr Ile Tyr Thr 50 55 60 Tyr Arg Val Ser Gln
Thr Glu Thr Gly Ser Trp Ser Ala Glu Thr Ala 65 70 75 80 Pro Gly Val
His Lys Arg Tyr Phe Arg Lys Ile Lys Asn Leu Ile Ser 85 90 95 Ala
Phe Gln Lys Pro Asp Gln Gly Ile Val Ile Pro Leu Gln Tyr Pro 100 105
110 Val Glu Lys Lys Ser Ser Ala Arg Ser Thr Gln Gly Thr Thr Gly Ile
115 120 125 Arg Glu Asp Pro Asp Val Cys Leu Lys Ala Pro 130 135
197143PRTArtificial sequenceSynthetic
sequencemisc_feature(5)..(5)Xaa can be any naturally occurring
amino acid 197Met Gly Ser Ser Xaa Ser Ser Gly Leu Val Pro Arg Gly
Ser His Met 1 5 10 15 Asp Ala Val Ala Val Tyr His Gly Lys Ile Ser
Arg Glu Thr Gly Glu 20 25 30 Lys Leu Leu Leu Ala Thr Gly Leu Asp
Gly Ser Tyr Leu Leu Arg Asp 35 40 45 Ser Glu Ser Val Pro Gly Val
Tyr Cys Leu Cys Val Leu Tyr His Gly 50 55 60 Tyr Ile Tyr Thr Tyr
Arg Val Ser Gln Thr Glu Thr Gly Ser Trp Ser 65 70 75 80 Ala Glu Thr
Ala Pro Gly Val His Lys Arg Tyr Phe Arg Lys Ile Lys 85 90 95 Asn
Leu Ile Ser Ala Phe Gln Lys Pro Asp Gln Gly Ile Val Ile Pro 100 105
110 Leu Gln Tyr Pro Val Glu Lys Lys Ser Ser Ala Arg Ser Thr Gln Gly
115 120 125 Thr Thr Gly Ile Arg Glu Asp Pro Asp Val Cys Leu Lys Ala
Pro 130 135 140 1984PRTArtificial sequenceSynthetic sequence 198Ala
Ala Ala Ala 1 1994PRTArtificial sequenceSynthetic sequence 199Ala
Ala Ala Ala 1 2004PRTArtificial sequenceSynthetic sequence 200Ala
Ala Ala Ala 1 20112PRTArtificial sequenceSynthetic sequence 201Leu
Pro Lys Lys Arg Lys Phe Ser Glu Ile Ser Ser 1 5 10
20210PRTArtificial sequenceSynthetic sequence 202Lys Arg Lys Arg
Trp Glu Asn Asp Ile Pro 1 5 10 20310PRTArtificial sequenceSynthetic
sequence 203Lys Arg Lys Arg Trp Glu Asn Asn Ile Pro 1 5 10
20412PRTArtificial sequenceSynthetic sequence 204Thr Gly Gly Val
Met Lys Arg Lys Arg Gly Ser Val 1 5 10 20513PRTArtificial
sequenceSynthetic sequence 205Pro Ile Leu Pro Leu Ile Cys Arg Arg
Arg Gly Ser Pro 1 5 10 20612PRTArtificial sequenceSynthetic
sequence 206Thr Tyr Ser Gly Val Lys Arg Lys Arg Asn Val Val 1 5 10
20712PRTArtificial sequenceSynthetic sequence 207Thr His Ile Gly
Tyr Lys Arg Lys Arg Asp Ser Val 1 5 10 20812PRTArtificial
sequenceSynthetic sequence 208Leu Ser Gly Thr Lys Arg Lys Arg Ala
Tyr Phe Ile 1 5 10 20912PRTArtificial sequenceSynthetic sequence
209Gln Arg Arg Leu Leu Lys Arg Lys Arg Gly Ser Leu 1 5 10
21012PRTArtificial sequenceSynthetic sequence 210Gln Ile Gly Lys
Lys Arg Lys Arg Asp Tyr Leu Asp 1 5 10 21112PRTArtificial
sequenceSynthetic sequence 211Lys Arg Gly Lys Arg Lys Arg Leu Val
Arg Pro Trp 1 5 10 21212PRTArtificial sequenceSynthetic sequence
212Lys Lys Gly Lys Arg Lys Arg Leu Val Arg Pro Trp 1 5 10
21312PRTArtificial sequenceSynthetic sequence 213Pro Ser Arg Lys
Arg Lys Arg Glu Ser Asp His Ile 1 5 10 21412PRTArtificial
sequenceSynthetic sequence 214Pro Ser Arg Lys Arg Lys Arg Asp His
Tyr Ala Val 1 5 10 21512PRTArtificial sequenceSynthetic sequence
215Ile Ser Arg Lys Arg Lys Arg Asp Leu Glu Phe Val 1 5 10
21612PRTArtificial sequenceSynthetic sequence 216Ile Thr Arg Lys
Arg Lys Arg Asp Leu Val Phe Thr 1 5 10 21712PRTArtificial
sequenceSynthetic sequence 217Glu Pro Asn Pro Arg Lys Arg Lys Arg
Ser Glu Leu 1 5 10 21812PRTArtificial sequenceSynthetic sequence
218Thr Ser Pro Ser Arg Lys Arg Lys Trp Asp Gln Val 1 5 10
21912PRTArtificial sequenceSynthetic sequence 219Thr Leu Glu Arg
Lys Arg Lys Leu Ala Val Leu Tyr 1 5 10 22012PRTArtificial
sequenceSynthetic sequence 220Arg Arg Arg Lys Arg Arg Arg Glu Trp
Glu Asp Phe 1 5 10 22112PRTArtificial sequenceSynthetic sequence
221His Arg Tyr Cys Gly Lys Arg Arg Arg Arg Thr Arg 1 5 10
22211PRTArtificial sequenceSynthetic sequence 222Ser Val Leu Gly
Lys Arg Ser Arg Thr Trp Glu 1 5 10 22312PRTArtificial
sequenceSynthetic sequence 223Tyr Gly Arg Val Ser Lys Arg Pro Arg
Tyr Gln Phe 1 5 10 22411PRTArtificial sequenceSynthetic sequence
224Arg Lys Arg Gly Arg Lys Arg Phe Arg Ser Val 1 5 10
22512PRTArtificial sequenceSynthetic sequence 225Lys Arg Lys Tyr
Ala Val Phe Leu Glu Ser Gln Asn 1 5 10 22612PRTArtificial
sequenceSynthetic sequence 226Lys Arg Lys Tyr Ser Ile Tyr Leu Gly
Ser Gln Ser 1 5 10 22712PRTArtificial sequenceSynthetic sequence
227Lys Arg Lys Trp Met Ala Phe Val Met Gly Asp Pro 1 5 10
22812PRTArtificial sequenceSynthetic sequence 228Lys Arg Lys Cys
Ala Val Phe Leu Glu Gly Gln Asn 1 5 10 22912PRTArtificial
sequenceSynthetic sequence 229Ile Pro Arg Lys Arg Ser Phe Ala Glu
Leu Tyr Asp 1 5 10 23012PRTArtificial sequenceSynthetic sequence
230Arg Leu Thr Pro Arg Lys Arg Ala Phe Ser Glu Val 1 5 10
2319PRTArtificial sequenceSynthetic sequence 231Lys Arg Ser Trp Ser
Met Ala Phe Cys 1 5 23210PRTArtificial sequenceSynthetic sequence
232Lys Arg Thr Asn Ala Gln Ala Phe Thr Glu 1 5 10
23312PRTArtificial sequenceSynthetic sequence 233Lys Arg Pro Tyr
Ser Ile Ala Phe Pro Leu Gly Gln 1 5 10 23414PRTArtificial
sequenceSynthetic sequence 234Arg Arg Arg Ser Val Leu Lys Arg Ser
Trp Ser Val Ala Phe 1 5 10 23512PRTArtificial sequenceSynthetic
sequence 235Lys Arg Arg Tyr Ser Asp Ala Phe Arg Leu Pro Val 1 5 10
23612PRTArtificial sequenceSynthetic sequence 236Lys Arg Lys Tyr
Ser Asp Ala Phe Gly Leu Pro Val 1 5 10 23712PRTArtificial
sequenceSynthetic sequence 237Ile Gly Arg Lys Arg Gly Tyr Ser Val
Ala Phe Gly 1 5 10 23812PRTArtificial sequenceSynthetic sequence
238Ile Gly Arg Lys Arg Val Asn Ala Val Ala Phe Tyr 1 5 10
23912PRTArtificial sequenceSynthetic sequence 239Trp Ala Gly Arg
Lys Arg Thr Trp Arg Asp Ala Phe 1 5 10 24012PRTArtificial
sequenceSynthetic sequence 240Ser Ser His Arg Lys Arg Lys Phe Ser
Asp Ala Phe 1 5 10 24112PRTArtificial sequenceSynthetic sequence
241Pro Ser His Arg Lys Arg Lys Phe Ser Asp Ala Phe 1 5 10
24212PRTArtificial sequenceSynthetic sequence 242Thr Ala His Arg
Lys Arg Lys Phe Ser Asp Ala Phe 1 5 10 24312PRTArtificial
sequenceSynthetic sequence 243Arg Val Gln Arg Lys Arg Lys Trp Ser
Glu Ala Phe 1 5 10 24412PRTArtificial sequenceSynthetic sequence
244Arg Leu Thr Arg Lys Arg Lys Tyr Asp Cys Ala Phe 1 5 10
24512PRTArtificial sequenceSynthetic sequence 245Leu Val Asn Arg
Lys Arg Arg Tyr Trp Glu Ala Phe 1 5 10 24612PRTArtificial
sequenceSynthetic sequence 246Leu Gly Lys Arg Tyr Asp Arg Asp Trp
Asp Tyr Lys 1 5 10 24712PRTArtificial sequenceSynthetic sequence
247Arg Ser Ser Gly Ile Leu Gly Lys Arg Lys Phe Glu 1 5 10
24812PRTArtificial sequenceSynthetic sequence 248Val His Lys Thr
Val Leu Gly Lys Arg Lys Tyr Trp 1 5 10 24912PRTArtificial
sequenceSynthetic sequence 249Ser Ile Leu Gly Lys Arg Lys Asn Arg
Asp Pro Ser 1 5 10 25012PRTArtificial sequenceSynthetic sequence
250Gln Ser Val Leu Gly Lys Arg Lys Ser Arg Pro Phe 1 5 10
25112PRTArtificial sequenceSynthetic sequence 251Thr Val His Leu
Gly Lys Arg Arg Leu Arg Pro Trp 1 5 10 25212PRTArtificial
sequenceSynthetic sequence 252Arg Val Leu Gly Lys Arg Lys Thr Gly
Arg Ser Pro 1 5 10 25311PRTArtificial sequenceSynthetic sequence
253Val Leu Gly Lys Arg Lys Arg Asp Asp Cys Trp 1 5 10
25411PRTArtificial sequenceSynthetic sequence 254His Gly Arg Gln
Val Leu Gly Lys Arg Lys Arg 1 5 10 25512PRTArtificial
sequenceSynthetic sequence 255Ser Val Leu Gly Lys Arg Lys Arg His
Pro Lys Val 1 5 10 25612PRTArtificial sequenceSynthetic sequence
256Ser Val Leu Gly Lys Arg Lys Arg His His Leu Asp 1 5 10
25712PRTArtificial sequenceSynthetic sequence 257Pro Val Leu Gly
Lys Arg Lys Arg Ser Leu Ser Ser 1 5 10 25812PRTArtificial
sequenceSynthetic sequence 258Arg Val Leu Gly Lys Arg Lys Arg Glu
Asp Arg Pro 1 5 10 25912PRTArtificial sequenceSynthetic sequence
259Ile Leu Gly Lys Arg Lys Arg Ser His His Pro Tyr 1 5 10
26012PRTArtificial sequenceSynthetic sequence 260Pro Ile Leu Gly
Lys Arg Lys Arg His Leu Phe Leu 1 5 10 26112PRTArtificial
sequenceSynthetic sequence 261Leu Leu Gly Lys Arg Lys Arg Pro Ser
Ile Glu His 1 5 10 26212PRTArtificial sequenceSynthetic sequence
262Ser Met Leu Gly Lys Arg Lys Arg Cys Ile Ile Ser 1 5 10
26312PRTArtificial sequenceSynthetic sequence 263Thr Leu Gly Lys
Arg Lys Arg Ile Ser Cys Val Thr 1 5 10 26412PRTArtificial
sequenceSynthetic sequence 264Asp Thr Arg Leu Gly Lys Arg Lys Arg
Arg Pro Trp 1 5 10 2654PRTArtificial sequenceSynthetic sequence
265Ala Ala Ala Ala 1 2664PRTArtificial sequenceSynthetic sequence
266Ala Ala Ala Ala 1 2674PRTArtificial sequenceSynthetic sequence
267Ala Ala Ala Ala 1 2684PRTArtificial sequenceSynthetic sequence
268Ala Ala Ala Ala 1 2694PRTArtificial sequenceSynthetic sequence
269Ala Ala Ala Ala 1 2704PRTArtificial sequenceSynthetic sequence
270Ala Ala Ala Ala 1 271428PRTArtificial sequenceSynthetic sequence
271Met Ala Val Gly Ala Ser Gly Leu Glu Gly Asp Lys Met Ala Gly Ala
1 5 10 15 Met Pro Leu Gln Leu Leu Leu Leu Leu Ile Leu Leu Gly Pro
Gly Asn 20 25 30 Ser Leu Gln Leu Trp Asp Thr Trp Ala Asp Glu Ala
Glu Lys Ala Leu 35 40 45 Gly Pro Leu Leu Ala Arg Asp Arg Arg Gln
Ala Thr Glu Tyr Glu Tyr 50 55 60 Leu Asp Tyr Asp Phe Leu Pro Glu
Thr Glu Pro Pro Glu Met Leu Arg 65 70 75 80 Asn Ser Thr Asp Thr Thr
Pro Leu Thr Gly Pro Gly Thr Pro Glu Ser 85 90 95 Thr Thr Val Glu
Pro Ala Ala Arg Arg Ser Thr Gly Leu Asp Ala Gly 100 105 110 Gly Ala
Val Thr Glu Leu Thr Thr Glu Leu Ala Asn Met Gly Asn Leu 115 120 125
Ser Thr Asp Ser Ala Ala Met Glu Ile Gln Thr Thr Gln Pro Ala Ala 130
135 140 Thr Glu Ala Gln Thr Thr Gln Pro Val Pro Thr Glu Ala Gln Thr
Thr 145 150 155 160 Pro Leu Ala Ala Thr Glu Ala Gln Thr Thr Arg Leu
Thr Ala Thr Glu 165 170 175 Ala Gln Thr Thr Pro Leu Ala Ala Thr Glu
Ala Gln Thr Thr Pro Pro 180 185 190 Ala Ala Thr Glu Ala Gln Thr Thr
Gln Pro Thr Gly Leu Glu Ala Gln 195 200 205 Thr Thr Ala Pro Ala Ala
Met Glu Ala Gln Thr Thr Ala Pro Ala Ala 210 215 220 Met Glu Ala Gln
Thr Thr Pro Pro Ala Ala Met Glu Ala Gln Thr Thr 225 230 235 240 Gln
Thr Thr Ala Met Glu Ala Gln Thr Thr Ala Pro Glu Ala Thr Glu 245 250
255 Ala Gln Thr Thr Gln Pro Thr Ala Thr Glu Ala Gln Thr Thr Pro Leu
260 265 270 Ala Ala Met Glu Ala Leu Ser Thr Glu Pro Ser Ala Thr Glu
Ala Leu 275 280 285 Ser Met Glu Pro Thr Thr Lys Arg Gly Leu Phe Ile
Pro Phe Ser Val 290 295 300 Ser Ser Val Thr His Lys Gly Ile Pro Met
Ala Ala Ser Asn Leu Ser 305 310 315 320 Val Asn Tyr Pro Val Gly Ala
Pro Asp His Ile Ser Val Lys Gln Cys 325 330 335 Leu Leu Ala Ile Leu
Ile Leu Ala Leu Val Ala Thr Ile Phe Phe Val 340 345 350 Cys Thr Val
Val Leu Ala Val Arg Leu Ser Arg Lys Gly His Met Tyr 355 360 365 Pro
Val Arg Asn Tyr Ser Pro Thr Glu Met Val Cys Ile Ser Ser Leu 370 375
380 Leu Pro Asp Gly Gly Glu Gly Pro Ser Ala Thr Ala Asn Gly Gly Leu
385 390 395 400 Ser Lys Ala Lys Ser Pro Gly Leu Thr Pro Glu Pro Arg
Glu Asp Arg 405
410 415 Glu Gly Asp Asp Leu Thr Leu His Ser Phe Leu Pro 420 425
2721203PRTArtificial sequenceSynthetic sequence 272Met Ala Ala Cys
Gly Arg Val Arg Arg Met Phe Arg Leu Ser Ala Ala 1 5 10 15 Leu His
Leu Leu Leu Leu Phe Ala Ala Gly Ala Glu Lys Leu Pro Gly 20 25 30
Gln Gly Val His Ser Gln Gly Gln Gly Pro Gly Ala Asn Phe Val Ser 35
40 45 Phe Val Gly Gln Ala Gly Gly Gly Gly Pro Ala Gly Gln Gln Leu
Pro 50 55 60 Gln Leu Pro Gln Ser Ser Gln Leu Gln Gln Gln Gln Gln
Gln Gln Gln 65 70 75 80 Gln Gln Gln Gln Pro Gln Pro Pro Gln Pro Pro
Phe Pro Ala Gly Gly 85 90 95 Pro Pro Ala Arg Arg Gly Gly Ala Gly
Ala Gly Gly Gly Trp Lys Leu 100 105 110 Ala Glu Glu Glu Ser Cys Arg
Glu Asp Val Thr Arg Val Cys Pro Lys 115 120 125 His Thr Trp Ser Asn
Asn Leu Ala Val Leu Glu Cys Leu Gln Asp Val 130 135 140 Arg Glu Pro
Glu Asn Glu Ile Ser Ser Asp Cys Asn His Leu Leu Trp 145 150 155 160
Asn Tyr Lys Leu Asn Leu Thr Thr Asp Pro Lys Phe Glu Ser Val Ala 165
170 175 Arg Glu Val Cys Lys Ser Thr Ile Thr Glu Ile Lys Glu Cys Ala
Asp 180 185 190 Glu Pro Val Gly Lys Gly Tyr Met Val Ser Cys Leu Val
Asp His Arg 195 200 205 Gly Asn Ile Thr Glu Tyr Gln Cys His Gln Tyr
Ile Thr Lys Met Thr 210 215 220 Ala Ile Ile Phe Ser Asp Tyr Arg Leu
Ile Cys Gly Phe Met Asp Asp 225 230 235 240 Cys Lys Asn Asp Ile Asn
Ile Leu Lys Cys Gly Ser Ile Arg Leu Gly 245 250 255 Glu Lys Asp Ala
His Ser Gln Gly Glu Val Val Ser Cys Leu Glu Lys 260 265 270 Gly Leu
Val Lys Glu Ala Glu Glu Arg Glu Pro Lys Ile Gln Val Ser 275 280 285
Glu Leu Cys Lys Lys Ala Ile Leu Arg Val Ala Glu Leu Ser Ser Asp 290
295 300 Asp Phe His Leu Asp Arg His Leu Tyr Phe Ala Cys Arg Asp Asp
Arg 305 310 315 320 Glu Arg Phe Cys Glu Asn Thr Gln Ala Gly Glu Gly
Arg Val Tyr Lys 325 330 335 Cys Leu Phe Asn His Lys Phe Glu Glu Ser
Met Ser Glu Lys Cys Arg 340 345 350 Glu Ala Leu Thr Thr Arg Gln Lys
Leu Ile Ala Gln Asp Tyr Lys Val 355 360 365 Ser Tyr Ser Leu Ala Lys
Ser Cys Lys Ser Asp Leu Lys Lys Tyr Arg 370 375 380 Cys Asn Val Glu
Asn Leu Pro Arg Ser Arg Glu Ala Arg Leu Ser Tyr 385 390 395 400 Leu
Leu Met Cys Leu Glu Ser Ala Val His Arg Gly Arg Gln Val Ser 405 410
415 Ser Glu Cys Gln Gly Glu Met Leu Asp Tyr Arg Arg Met Leu Met Glu
420 425 430 Asp Phe Ser Leu Ser Pro Glu Ile Ile Leu Ser Cys Arg Gly
Glu Ile 435 440 445 Glu His His Cys Ser Gly Leu His Arg Lys Gly Arg
Thr Leu His Cys 450 455 460 Leu Met Lys Val Val Arg Gly Glu Lys Gly
Asn Leu Gly Met Asn Cys 465 470 475 480 Gln Gln Ala Leu Gln Thr Leu
Ile Gln Glu Thr Asp Pro Gly Ala Asp 485 490 495 Tyr Arg Ile Asp Arg
Ala Leu Asn Glu Ala Cys Glu Ser Val Ile Gln 500 505 510 Thr Ala Cys
Lys His Ile Arg Ser Gly Asp Pro Met Ile Leu Ser Cys 515 520 525 Leu
Met Glu His Leu Tyr Thr Glu Lys Met Val Glu Asp Cys Glu His 530 535
540 Arg Leu Leu Glu Leu Gln Tyr Phe Ile Ser Arg Asp Trp Lys Leu Asp
545 550 555 560 Pro Val Leu Tyr Arg Lys Cys Gln Gly Asp Ala Ser Arg
Leu Cys His 565 570 575 Thr His Gly Trp Asn Glu Thr Ser Glu Phe Met
Pro Gln Gly Ala Val 580 585 590 Phe Ser Cys Leu Tyr Arg His Ala Tyr
Arg Thr Glu Glu Gln Gly Arg 595 600 605 Arg Leu Ser Arg Glu Cys Arg
Ala Glu Val Gln Arg Ile Leu His Gln 610 615 620 Arg Ala Met Asp Val
Lys Leu Asp Pro Ala Leu Gln Asp Lys Cys Leu 625 630 635 640 Ile Asp
Leu Gly Lys Trp Cys Ser Glu Lys Thr Glu Thr Gly Gln Glu 645 650 655
Leu Glu Cys Leu Gln Asp His Leu Asp Asp Leu Val Val Glu Cys Arg 660
665 670 Asp Ile Val Gly Asn Leu Thr Glu Leu Glu Ser Glu Asp Ile Gln
Ile 675 680 685 Glu Ala Leu Leu Met Arg Ala Cys Glu Pro Ile Ile Gln
Asn Phe Cys 690 695 700 His Asp Val Ala Asp Asn Gln Ile Asp Ser Gly
Asp Leu Met Glu Cys 705 710 715 720 Leu Ile Gln Asn Lys His Gln Lys
Asp Met Asn Glu Lys Cys Ala Ile 725 730 735 Gly Val Thr His Phe Gln
Leu Val Gln Met Lys Asp Phe Arg Phe Ser 740 745 750 Tyr Lys Phe Lys
Met Ala Cys Lys Glu Asp Val Leu Lys Leu Cys Pro 755 760 765 Asn Ile
Lys Lys Lys Val Asp Val Val Ile Cys Leu Ser Thr Thr Val 770 775 780
Arg Asn Asp Thr Leu Gln Glu Ala Lys Glu His Arg Val Ser Leu Lys 785
790 795 800 Cys Arg Arg Gln Leu Arg Val Glu Glu Leu Glu Met Thr Glu
Asp Ile 805 810 815 Arg Leu Glu Pro Asp Leu Tyr Glu Ala Cys Lys Ser
Asp Ile Lys Asn 820 825 830 Phe Cys Ser Ala Val Gln Tyr Gly Asn Ala
Gln Ile Ile Glu Cys Leu 835 840 845 Lys Glu Asn Lys Lys Gln Leu Ser
Thr Arg Cys His Gln Lys Val Phe 850 855 860 Lys Leu Gln Glu Thr Glu
Met Met Asp Pro Glu Leu Asp Tyr Thr Leu 865 870 875 880 Met Arg Val
Cys Lys Gln Met Ile Lys Arg Phe Cys Pro Glu Ala Asp 885 890 895 Ser
Lys Thr Met Leu Gln Cys Leu Lys Gln Asn Lys Asn Ser Glu Leu 900 905
910 Met Asp Pro Lys Cys Lys Gln Met Ile Thr Lys Arg Gln Ile Thr Gln
915 920 925 Asn Thr Asp Tyr Arg Leu Asn Pro Met Leu Arg Lys Ala Cys
Lys Ala 930 935 940 Asp Ile Pro Lys Phe Cys His Gly Ile Leu Thr Lys
Ala Lys Asp Asp 945 950 955 960 Ser Glu Leu Glu Gly Gln Val Ile Ser
Cys Leu Lys Leu Arg Tyr Ala 965 970 975 Asp Gln Arg Leu Ser Ser Asp
Cys Glu Asp Gln Ile Arg Ile Ile Ile 980 985 990 Gln Glu Ser Ala Leu
Asp Tyr Arg Leu Asp Pro Gln Leu Gln Leu His 995 1000 1005 Cys Ser
Asp Glu Ile Ser Ser Leu Cys Ala Glu Glu Ala Ala Ala 1010 1015 1020
Gln Glu Gln Thr Gly Gln Val Glu Glu Cys Leu Lys Val Asn Leu 1025
1030 1035 Leu Lys Ile Lys Thr Glu Leu Cys Lys Lys Glu Val Leu Asn
Met 1040 1045 1050 Leu Lys Glu Ser Lys Ala Asp Ile Phe Val Asp Pro
Val Leu His 1055 1060 1065 Thr Ala Cys Ala Leu Asp Ile Lys His His
Cys Ala Ala Ile Thr 1070 1075 1080 Pro Gly Arg Gly Arg Gln Met Ser
Cys Leu Met Glu Ala Leu Glu 1085 1090 1095 Asp Lys Arg Val Arg Leu
Gln Pro Glu Cys Lys Lys Arg Leu Asn 1100 1105 1110 Asp Arg Ile Glu
Met Trp Ser Tyr Ala Ala Lys Val Ala Pro Ala 1115 1120 1125 Asp Gly
Phe Ser Asp Leu Ala Met Gln Val Met Thr Ser Pro Ser 1130 1135 1140
Lys Asn Tyr Ile Leu Ser Val Ile Ser Gly Ser Ile Cys Ile Leu 1145
1150 1155 Phe Leu Ile Gly Leu Met Cys Gly Arg Ile Thr Lys Arg Val
Thr 1160 1165 1170 Arg Glu Leu Lys Asp Arg Leu Gln Tyr Arg Ser Glu
Thr Met Ala 1175 1180 1185 Tyr Lys Gly Leu Val Trp Ser Gln Asp Val
Thr Gly Ser Pro Ala 1190 1195 1200 273742PRTArtificial
sequenceSynthetic sequence 273Met Asp Lys Phe Trp Trp His Ala Ala
Trp Gly Leu Cys Leu Val Pro 1 5 10 15 Leu Ser Leu Ala Gln Ile Asp
Leu Asn Ile Thr Cys Arg Phe Ala Gly 20 25 30 Val Phe His Val Glu
Lys Asn Gly Arg Tyr Ser Ile Ser Arg Thr Glu 35 40 45 Ala Ala Asp
Leu Cys Lys Ala Phe Asn Ser Thr Leu Pro Thr Met Ala 50 55 60 Gln
Met Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr Cys Arg Tyr Gly 65 70
75 80 Phe Ile Glu Gly His Val Val Ile Pro Arg Ile His Pro Asn Ser
Ile 85 90 95 Cys Ala Ala Asn Asn Thr Gly Val Tyr Ile Leu Thr Ser
Asn Thr Ser 100 105 110 Gln Tyr Asp Thr Tyr Cys Phe Asn Ala Ser Ala
Pro Pro Glu Glu Asp 115 120 125 Cys Thr Ser Val Thr Asp Leu Pro Asn
Ala Phe Asp Gly Pro Ile Thr 130 135 140 Ile Thr Ile Val Asn Arg Asp
Gly Thr Arg Tyr Val Gln Lys Gly Glu 145 150 155 160 Tyr Arg Thr Asn
Pro Glu Asp Ile Tyr Pro Ser Asn Pro Thr Asp Asp 165 170 175 Asp Val
Ser Ser Gly Ser Ser Ser Glu Arg Ser Ser Thr Ser Gly Gly 180 185 190
Tyr Ile Phe Tyr Thr Phe Ser Thr Val His Pro Ile Pro Asp Glu Asp 195
200 205 Ser Pro Trp Ile Thr Asp Ser Thr Asp Arg Ile Pro Ala Thr Thr
Leu 210 215 220 Met Ser Thr Ser Ala Thr Ala Thr Glu Thr Ala Thr Lys
Arg Gln Glu 225 230 235 240 Thr Trp Asp Trp Phe Ser Trp Leu Phe Leu
Pro Ser Glu Ser Lys Asn 245 250 255 His Leu His Thr Thr Thr Gln Met
Ala Gly Thr Ser Ser Asn Thr Ile 260 265 270 Ser Ala Gly Trp Glu Pro
Asn Glu Glu Asn Glu Asp Glu Arg Asp Arg 275 280 285 His Leu Ser Phe
Ser Gly Ser Gly Ile Asp Asp Asp Glu Asp Phe Ile 290 295 300 Ser Ser
Thr Ile Ser Thr Thr Pro Arg Ala Phe Asp His Thr Lys Gln 305 310 315
320 Asn Gln Asp Trp Thr Gln Trp Asn Pro Ser His Ser Asn Pro Glu Val
325 330 335 Leu Leu Gln Thr Thr Thr Arg Met Thr Asp Val Asp Arg Asn
Gly Thr 340 345 350 Thr Ala Tyr Glu Gly Asn Trp Asn Pro Glu Ala His
Pro Pro Leu Ile 355 360 365 His His Glu His His Glu Glu Glu Glu Thr
Pro His Ser Thr Ser Thr 370 375 380 Ile Gln Ala Thr Pro Ser Ser Thr
Thr Glu Glu Thr Ala Thr Gln Lys 385 390 395 400 Glu Gln Trp Phe Gly
Asn Arg Trp His Glu Gly Tyr Arg Gln Thr Pro 405 410 415 Lys Glu Asp
Ser His Ser Thr Thr Gly Thr Ala Ala Ala Ser Ala His 420 425 430 Thr
Ser His Pro Met Gln Gly Arg Thr Thr Pro Ser Pro Glu Asp Ser 435 440
445 Ser Trp Thr Asp Phe Phe Asn Pro Ile Ser His Pro Met Gly Arg Gly
450 455 460 His Gln Ala Gly Arg Arg Met Asp Met Asp Ser Ser His Ser
Ile Thr 465 470 475 480 Leu Gln Pro Thr Ala Asn Pro Asn Thr Gly Leu
Val Glu Asp Leu Asp 485 490 495 Arg Thr Gly Pro Leu Ser Met Thr Thr
Gln Gln Ser Asn Ser Gln Ser 500 505 510 Phe Ser Thr Ser His Glu Gly
Leu Glu Glu Asp Lys Asp His Pro Thr 515 520 525 Thr Ser Thr Leu Thr
Ser Ser Asn Arg Asn Asp Val Thr Gly Gly Arg 530 535 540 Arg Asp Pro
Asn His Ser Glu Gly Ser Thr Thr Leu Leu Glu Gly Tyr 545 550 555 560
Thr Ser His Tyr Pro His Thr Lys Glu Ser Arg Thr Phe Ile Pro Val 565
570 575 Thr Ser Ala Lys Thr Gly Ser Phe Gly Val Thr Ala Val Thr Val
Gly 580 585 590 Asp Ser Asn Ser Asn Val Asn Arg Ser Leu Ser Gly Asp
Gln Asp Thr 595 600 605 Phe His Pro Ser Gly Gly Ser His Thr Thr His
Gly Ser Glu Ser Asp 610 615 620 Gly His Ser His Gly Ser Gln Glu Gly
Gly Ala Asn Thr Thr Ser Gly 625 630 635 640 Pro Ile Arg Thr Pro Gln
Ile Pro Glu Trp Leu Ile Ile Leu Ala Ser 645 650 655 Leu Leu Ala Leu
Ala Leu Ile Leu Ala Val Cys Ile Ala Val Asn Ser 660 665 670 Arg Arg
Arg Cys Gly Gln Lys Lys Lys Leu Val Ile Asn Ser Gly Asn 675 680 685
Gly Ala Val Glu Asp Arg Lys Pro Ser Gly Leu Asn Gly Glu Ala Ser 690
695 700 Lys Ser Gln Glu Met Val His Leu Val Asn Lys Glu Ser Ser Glu
Thr 705 710 715 720 Pro Asp Gln Phe Met Thr Ala Asp Glu Thr Arg Asn
Leu Gln Asn Val 725 730 735 Asp Met Lys Ile Gly Val 740
274426PRTArtificial sequenceSynthetic sequence 274Met Glu Gln Arg
Pro Arg Gly Cys Ala Ala Val Ala Ala Ala Leu Leu 1 5 10 15 Leu Val
Leu Leu Gly Ala Arg Ala Gln Gly Gly Thr Arg Ser Pro Arg 20 25 30
Cys Asp Cys Ala Gly Asp Phe His Lys Lys Ile Gly Leu Phe Cys Cys 35
40 45 Arg Gly Cys Pro Ala Gly His Tyr Leu Lys Ala Pro Cys Thr Glu
Pro 50 55 60 Cys Gly Asn Ser Thr Cys Leu Val Cys Pro Gln Asp Thr
Phe Leu Ala 65 70 75 80 Trp Glu Asn His His Asn Ser Glu Cys Ala Arg
Cys Gln Ala Cys Asp 85 90 95 Glu Gln Ala Ser Gln Val Ala Leu Glu
Asn Cys Ser Ala Val Ala Asp 100 105 110 Thr Arg Cys Gly Cys Lys Pro
Gly Trp Phe Val Glu Cys Gln Val Ser 115 120 125 Gln Cys Val Ser Ser
Ser Pro Phe Tyr Cys Gln Pro Cys Leu Asp Cys 130 135 140 Gly Ala Leu
His Arg His Thr Arg Leu Leu Cys Ser Arg Arg Asp Thr 145 150 155 160
Asp Cys Gly Thr Cys Leu Pro Gly Phe Tyr Glu His Gly Asp Gly Cys 165
170 175 Val Ser Cys Pro Thr Pro Pro Pro Ser Leu Ala Gly Ala Pro Trp
Gly 180 185 190 Ala Val Gln Ser Ala Val Pro Leu Ser Val Ala Gly Gly
Arg Val Gly 195 200 205 Val Phe Trp Val Gln Val Leu Leu Ala Gly Leu
Val Val Pro Leu Leu 210 215 220 Leu Gly Ala Thr Leu Thr Tyr Thr Tyr
Arg His Cys Trp Pro His Lys 225 230 235 240 Pro Leu Val Thr Ala Asp
Glu Ala Gly Met Glu Ala Leu Thr Pro Pro 245 250 255 Pro Ala Thr His
Leu Ser Pro Leu Asp Ser Ala His Thr Leu Leu Ala 260 265 270 Pro Pro
Asp Ser Ser Glu Lys Ile Cys Thr Val Gln Leu Val Gly Asn 275 280 285
Ser Trp Thr Pro Gly Tyr Pro Glu Thr Gln Glu Ala Leu Cys Pro Gln 290
295 300 Val Thr Trp Ser Trp Asp Gln Leu Pro Ser Arg Ala Leu Gly Pro
Ala 305 310
315 320 Ala Ala Pro Thr Leu Ser Pro Glu Ser Pro Ala Gly Ser Pro Ala
Met 325 330 335 Met Leu Gln Pro Gly Pro Gln Leu Tyr Asp Val Met Asp
Ala Val Pro 340 345 350 Ala Arg Arg Trp Lys Glu Phe Val Arg Thr Leu
Gly Leu Arg Glu Ala 355 360 365 Glu Ile Glu Ala Val Glu Val Glu Ile
Gly Arg Phe Arg Asp Gln Gln 370 375 380 Tyr Glu Met Leu Lys Arg Trp
Arg Gln Gln Gln Pro Ala Gly Leu Gly 385 390 395 400 Ala Val Tyr Ala
Ala Leu Glu Arg Met Gly Leu Asp Gly Cys Val Glu 405 410 415 Asp Leu
Arg Ser Arg Leu Gln Arg Gly Pro 420 425 275417PRTArtificial
sequenceSynthetic sequence 275Met Ala Ala Pro Gly Ser Ala Arg Arg
Pro Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Gly Leu Met
His Cys Ala Ser Ala Ala Met Phe Met 20 25 30 Val Lys Asn Gly Asn
Gly Thr Ala Cys Ile Met Ala Asn Phe Ser Ala 35 40 45 Ala Phe Ser
Val Asn Tyr Asp Thr Lys Ser Gly Pro Lys Asn Met Thr 50 55 60 Phe
Asp Leu Pro Ser Asp Ala Thr Val Val Leu Asn Arg Ser Ser Cys 65 70
75 80 Gly Lys Glu Asn Thr Ser Asp Pro Ser Leu Val Ile Ala Phe Gly
Arg 85 90 95 Gly His Thr Leu Thr Leu Asn Phe Thr Arg Asn Ala Thr
Arg Tyr Ser 100 105 110 Val Gln Leu Met Ser Phe Val Tyr Asn Leu Ser
Asp Thr His Leu Phe 115 120 125 Pro Asn Ala Ser Ser Lys Glu Ile Lys
Thr Val Glu Ser Ile Thr Asp 130 135 140 Ile Arg Ala Asp Ile Asp Lys
Lys Tyr Arg Cys Val Ser Gly Thr Gln 145 150 155 160 Val His Met Asn
Asn Val Thr Val Thr Leu His Asp Ala Thr Ile Gln 165 170 175 Ala Tyr
Leu Ser Asn Ser Ser Phe Ser Arg Gly Glu Thr Arg Cys Glu 180 185 190
Gln Asp Arg Pro Ser Pro Thr Thr Ala Pro Pro Ala Pro Pro Ser Pro 195
200 205 Ser Pro Ser Pro Val Pro Lys Ser Pro Ser Val Asp Lys Tyr Asn
Val 210 215 220 Ser Gly Thr Asn Gly Thr Cys Leu Leu Ala Ser Met Gly
Leu Gln Leu 225 230 235 240 Asn Leu Thr Tyr Glu Arg Lys Asp Asn Thr
Thr Val Thr Arg Leu Leu 245 250 255 Asn Ile Asn Pro Asn Lys Thr Ser
Ala Ser Gly Ser Cys Gly Ala His 260 265 270 Leu Val Thr Leu Glu Leu
His Ser Glu Gly Thr Thr Val Leu Leu Phe 275 280 285 Gln Phe Gly Met
Asn Ala Ser Ser Ser Arg Phe Phe Leu Gln Gly Ile 290 295 300 Gln Leu
Asn Thr Ile Leu Pro Asp Ala Arg Asp Pro Ala Phe Lys Ala 305 310 315
320 Ala Asn Gly Ser Leu Arg Ala Leu Gln Ala Thr Val Gly Asn Ser Tyr
325 330 335 Lys Cys Asn Ala Glu Glu His Val Arg Val Thr Lys Ala Phe
Ser Val 340 345 350 Asn Ile Phe Lys Val Trp Val Gln Ala Phe Lys Val
Glu Gly Gly Gln 355 360 365 Phe Gly Ser Val Glu Glu Cys Leu Leu Asp
Glu Asn Ser Met Leu Ile 370 375 380 Pro Ile Ala Val Gly Gly Ala Leu
Ala Gly Leu Val Leu Ile Val Leu 385 390 395 400 Ile Ala Tyr Leu Val
Gly Arg Lys Arg Ser His Ala Gly Tyr Gln Thr 405 410 415 Ile
276410PRTArtificial sequenceSynthetic sequence 276Met Val Cys Phe
Arg Leu Phe Pro Val Pro Gly Ser Gly Leu Val Leu 1 5 10 15 Val Cys
Leu Val Leu Gly Ala Val Arg Ser Tyr Ala Leu Glu Leu Asn 20 25 30
Leu Thr Asp Ser Glu Asn Ala Thr Cys Leu Tyr Ala Lys Trp Gln Met 35
40 45 Asn Phe Thr Val Arg Tyr Glu Thr Thr Asn Lys Thr Tyr Lys Thr
Val 50 55 60 Thr Ile Ser Asp His Gly Thr Val Thr Tyr Asn Gly Ser
Ile Cys Gly 65 70 75 80 Asp Asp Gln Asn Gly Pro Lys Ile Ala Val Gln
Phe Gly Pro Gly Phe 85 90 95 Ser Trp Ile Ala Asn Phe Thr Lys Ala
Ala Ser Thr Tyr Ser Ile Asp 100 105 110 Ser Val Ser Phe Ser Tyr Asn
Thr Gly Asp Asn Thr Thr Phe Pro Asp 115 120 125 Ala Glu Asp Lys Gly
Ile Leu Thr Val Asp Glu Leu Leu Ala Ile Arg 130 135 140 Ile Pro Leu
Asn Asp Leu Phe Arg Cys Asn Ser Leu Ser Thr Leu Glu 145 150 155 160
Lys Asn Asp Val Val Gln His Tyr Trp Asp Val Leu Val Gln Ala Phe 165
170 175 Val Gln Asn Gly Thr Val Ser Thr Asn Glu Phe Leu Cys Asp Lys
Asp 180 185 190 Lys Thr Ser Thr Val Ala Pro Thr Ile His Thr Thr Val
Pro Ser Pro 195 200 205 Thr Thr Thr Pro Thr Pro Lys Glu Lys Pro Glu
Ala Gly Thr Tyr Ser 210 215 220 Val Asn Asn Gly Asn Asp Thr Cys Leu
Leu Ala Thr Met Gly Leu Gln 225 230 235 240 Leu Asn Ile Thr Gln Asp
Lys Val Ala Ser Val Ile Asn Ile Asn Pro 245 250 255 Asn Thr Thr His
Ser Thr Gly Ser Cys Arg Ser His Thr Ala Leu Leu 260 265 270 Arg Leu
Asn Ser Ser Thr Ile Lys Tyr Leu Asp Phe Val Phe Ala Val 275 280 285
Lys Asn Glu Asn Arg Phe Tyr Leu Lys Glu Val Asn Ile Ser Met Tyr 290
295 300 Leu Val Asn Gly Ser Val Phe Ser Ile Ala Asn Asn Asn Leu Ser
Tyr 305 310 315 320 Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys Asn
Lys Glu Gln Thr 325 330 335 Val Ser Val Ser Gly Ala Phe Gln Ile Asn
Thr Phe Asp Leu Arg Val 340 345 350 Gln Pro Phe Asn Val Thr Gln Gly
Lys Tyr Ser Thr Ala Gln Asp Cys 355 360 365 Ser Ala Asp Asp Asp Asn
Phe Leu Val Pro Ile Ala Val Gly Ala Ala 370 375 380 Leu Ala Gly Val
Leu Ile Leu Val Leu Leu Ala Tyr Phe Ile Gly Leu 385 390 395 400 Lys
His His His Ala Gly Tyr Glu Gln Phe 405 410 277585PRTArtificial
sequenceSynthetic sequence 277Met Thr Pro Pro Arg Leu Phe Trp Val
Trp Leu Leu Val Ala Gly Thr 1 5 10 15 Gln Gly Val Asn Asp Gly Asp
Met Arg Leu Ala Asp Gly Gly Ala Thr 20 25 30 Asn Gln Gly Arg Val
Glu Ile Phe Tyr Arg Gly Gln Trp Gly Thr Val 35 40 45 Cys Asp Asn
Leu Trp Asp Leu Thr Asp Ala Ser Val Val Cys Arg Ala 50 55 60 Leu
Gly Phe Glu Asn Ala Thr Gln Ala Leu Gly Arg Ala Ala Phe Gly 65 70
75 80 Gln Gly Ser Gly Pro Ile Met Leu Asp Glu Val Gln Cys Thr Gly
Thr 85 90 95 Glu Ala Ser Leu Ala Asp Cys Lys Ser Leu Gly Trp Leu
Lys Ser Asn 100 105 110 Cys Arg His Glu Arg Asp Ala Gly Val Val Cys
Thr Asn Glu Thr Arg 115 120 125 Ser Thr His Thr Leu Asp Leu Ser Arg
Glu Leu Ser Glu Ala Leu Gly 130 135 140 Gln Ile Phe Asp Ser Gln Arg
Gly Cys Asp Leu Ser Ile Ser Val Asn 145 150 155 160 Val Gln Gly Glu
Asp Ala Leu Gly Phe Cys Gly His Thr Val Ile Leu 165 170 175 Thr Ala
Asn Leu Glu Ala Gln Ala Leu Trp Lys Glu Pro Gly Ser Asn 180 185 190
Val Thr Met Ser Val Asp Ala Glu Cys Val Pro Met Val Arg Asp Leu 195
200 205 Leu Arg Tyr Phe Tyr Ser Arg Arg Ile Asp Ile Thr Leu Ser Ser
Val 210 215 220 Lys Cys Phe His Lys Leu Ala Ser Ala Tyr Gly Ala Arg
Gln Leu Gln 225 230 235 240 Gly Tyr Cys Ala Ser Leu Phe Ala Ile Leu
Leu Pro Gln Asp Pro Ser 245 250 255 Phe Gln Met Pro Leu Asp Leu Tyr
Ala Tyr Ala Val Ala Thr Gly Asp 260 265 270 Ala Leu Leu Glu Lys Leu
Cys Leu Gln Phe Leu Ala Trp Asn Phe Glu 275 280 285 Ala Leu Thr Gln
Ala Glu Ala Trp Pro Ser Val Pro Thr Asp Leu Leu 290 295 300 Gln Leu
Leu Leu Pro Arg Ser Asp Leu Ala Val Pro Ser Glu Leu Ala 305 310 315
320 Leu Leu Lys Ala Val Asp Thr Trp Ser Trp Gly Glu Arg Ala Ser His
325 330 335 Glu Glu Val Glu Gly Leu Val Glu Lys Ile Arg Phe Pro Met
Met Leu 340 345 350 Pro Glu Glu Leu Phe Glu Leu Gln Phe Asn Leu Ser
Leu Tyr Trp Ser 355 360 365 His Glu Ala Leu Phe Gln Lys Lys Thr Leu
Gln Ala Leu Glu Phe His 370 375 380 Thr Val Pro Phe Gln Leu Leu Ala
Arg Tyr Lys Gly Leu Asn Leu Thr 385 390 395 400 Glu Asp Thr Tyr Lys
Pro Arg Ile Tyr Thr Ser Pro Thr Trp Ser Ala 405 410 415 Phe Val Thr
Asp Ser Ser Trp Ser Ala Arg Lys Ser Gln Leu Val Tyr 420 425 430 Gln
Ser Arg Arg Gly Pro Leu Val Lys Tyr Ser Ser Asp Tyr Phe Gln 435 440
445 Ala Pro Ser Asp Tyr Arg Tyr Tyr Pro Tyr Gln Ser Phe Gln Thr Pro
450 455 460 Gln His Pro Ser Phe Leu Phe Gln Asp Lys Arg Val Ser Trp
Ser Leu 465 470 475 480 Val Tyr Leu Pro Thr Ile Gln Ser Cys Trp Asn
Tyr Gly Phe Ser Cys 485 490 495 Ser Ser Asp Glu Leu Pro Val Leu Gly
Leu Thr Lys Ser Gly Gly Ser 500 505 510 Asp Arg Thr Ile Ala Tyr Glu
Asn Lys Ala Leu Met Leu Cys Glu Gly 515 520 525 Leu Phe Val Ala Asp
Val Thr Asp Phe Glu Gly Trp Lys Ala Ala Ile 530 535 540 Pro Ser Ala
Leu Asp Thr Asn Ser Ser Lys Ser Thr Ser Ser Phe Pro 545 550 555 560
Cys Pro Ala Gly His Phe Asn Gly Phe Arg Thr Val Ile Arg Pro Phe 565
570 575 Tyr Leu Thr Asn Ser Ser Gly Val Asp 580 585
* * * * *