U.S. patent application number 16/100040 was filed with the patent office on 2019-03-14 for methods and compositions for attenuating gene expression modulating anti-viral transfer vector immune responses.
This patent application is currently assigned to Selecta Biosciences, Inc.. The applicant listed for this patent is Selecta Biosciences, Inc.. Invention is credited to Takashi Kei Kishimoto.
Application Number | 20190076458 16/100040 |
Document ID | / |
Family ID | 54291579 |
Filed Date | 2019-03-14 |
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United States Patent
Application |
20190076458 |
Kind Code |
A1 |
Kishimoto; Takashi Kei |
March 14, 2019 |
METHODS AND COMPOSITIONS FOR ATTENUATING GENE EXPRESSION MODULATING
ANTI-VIRAL TRANSFER VECTOR IMMUNE RESPONSES
Abstract
Provided herein are methods and related compositions for
administering viral transfer vectors and antigen-presenting cell
targeted immunosuppressants.
Inventors: |
Kishimoto; Takashi Kei;
(Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Selecta Biosciences, Inc. |
Watertown |
MA |
US |
|
|
Assignee: |
Selecta Biosciences, Inc.
Watertown
MA
|
Family ID: |
54291579 |
Appl. No.: |
16/100040 |
Filed: |
August 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14846964 |
Sep 7, 2015 |
10071114 |
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16100040 |
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62101841 |
Jan 9, 2015 |
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62101861 |
Jan 9, 2015 |
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62101872 |
Jan 9, 2015 |
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62101882 |
Jan 9, 2015 |
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62051255 |
Sep 16, 2014 |
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62051258 |
Sep 16, 2014 |
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62051263 |
Sep 16, 2014 |
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62051267 |
Sep 16, 2014 |
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62047034 |
Sep 7, 2014 |
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62047044 |
Sep 7, 2014 |
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62047054 |
Sep 7, 2014 |
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62047051 |
Sep 7, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/436 20130101;
A61K 2039/577 20130101; C12N 2740/10041 20130101; C12N 2740/15032
20130101; A61P 37/06 20180101; A61P 37/00 20180101; A61K 47/6929
20170801; A61K 48/00 20130101; C12N 7/00 20130101; A61K 31/7088
20130101; A61P 37/02 20180101; A61K 48/005 20130101; A61P 43/00
20180101; C12N 2710/10032 20130101; A61K 9/1271 20130101; A61P
25/02 20180101; C12N 2750/14132 20130101; A61P 7/00 20180101; A61K
47/6923 20170801; A61K 9/5115 20130101; C12N 2710/00041 20130101;
A61K 9/5153 20130101; A61K 31/436 20130101; A61K 31/439 20130101;
A61K 47/6937 20170801; C12N 2750/14143 20130101; C12N 2740/16043
20130101; A61K 45/06 20130101; C12N 2750/14141 20130101; A61K
47/593 20170801; C12N 2740/15043 20130101; A61P 21/00 20180101;
A61K 47/6935 20170801; C12N 2710/10043 20130101; G06Q 99/00
20130101; A61K 39/001 20130101; A61K 31/00 20130101; A61K 2039/545
20130101; C12N 15/86 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; A61K 45/06 20060101 A61K045/06; A61K 31/439 20060101
A61K031/439; A61K 47/69 20170101 A61K047/69; A61K 47/59 20170101
A61K047/59; A61K 31/00 20060101 A61K031/00; A61K 9/51 20060101
A61K009/51; G06Q 99/00 20060101 G06Q099/00; A61K 39/00 20060101
A61K039/00; A61K 31/436 20060101 A61K031/436; C12N 7/00 20060101
C12N007/00; C12N 15/86 20060101 C12N015/86; A61K 48/00 20060101
A61K048/00 |
Claims
1. A method comprising: establishing an anti-gene expression
modulating viral transfer vector attenuated response in a subject
by concomitant administration of an antigen-presenting cell
targeted immunosuppressant and gene expression modulating viral
transfer vector to the subject, wherein the subject does not have
pre-existing immunity against the gene expression modulating viral
transfer vector.
2-3. (canceled)
4. A method comprising: establishing an anti-gene expression
modulating viral transfer vector attenuated response in a subject
by concomitant administration of an antigen-presenting cell
targeted immunosuppressant and gene expression modulating viral
transfer vector to the subject, and administering to the subject
one or more repeat doses of the gene expression modulating viral
transfer vector.
5-7. (canceled)
8. The method of claim 1, further comprising administering to the
subject one or more repeat doses of the viral transfer vector
subsequent to the concomitant administration of the viral transfer
vector and the antigen-presenting cell targeted immunosuppressant
to the subject.
9. A method comprising: determining a level of pre-existing
immunity to a gene expression modulating viral transfer vector in a
subject prior to administration of the gene expression modulating
viral transfer vector to the subject, concomitantly administering
to the subject an antigen-presenting cell targeted
immunosuppressant and gene expression modulating viral transfer
vector, and administering to the subject a dose of the gene
expression modulating viral transfer vector.
10. The method of claim 9, wherein the determining comprises
measuring a level of anti-viral transfer vector antibodies in the
subject prior to administration of the viral transfer vector to the
subject.
11-14. (canceled)
15. A method comprising: escalating transgene expression of a gene
expression modulating viral transfer vector in a subject by
repeatedly, concomitantly administering to the subject an
antigen-presenting cell targeted immunosuppressant and gene
expression modulating viral transfer vector.
16-30. (canceled)
31. A method comprising: determining the frequency and dosing of
concomitant administration of an antigen-presenting cell targeted
immunosuppressant and gene expression modulating viral transfer
vector in order to generate an anti-gene expression modulating
viral transfer vector attenuated response in a subject, and
directing the concomitant administration of the antigen-presenting
cell targeted immunosuppressant and gene expression modulating
viral transfer vector to a subject according to the determined
frequency and dosing.
32-37. (canceled)
38. The method of claim 31, wherein the subject does not have
pre-existing immunity against the viral transfer vector.
39. The method of claim 31, wherein the concomitant administration
is simultaneous administration.
40. The method of claim 1, wherein the subject is one to which the
viral transfer vector has not been previously administered.
41. The method of claim 1, wherein the viral transfer vector is a
retroviral transfer vector, an adenoviral transfer vector, a
lentiviral transfer vector or an adeno-associated viral transfer
vector.
42-46. (canceled)
47. The method of claim 1, wherein the gene expression modulating
transgene encodes a DNA-binding protein or a therapeutic RNA.
48-54. (canceled)
55. The method of claim 1, wherein the antigen-presenting cell
targeted immunosuppressant comprises a negatively-charged
particle.
56-64. (canceled)
65. The method of claim 1, wherein the antigen-presenting cell
targeted immunosuppressant comprises synthetic nanocarriers
comprising an immunosuppressant.
66. The method of claim 65, wherein the synthetic nanocarriers
further comprise a viral transfer vector antigen.
67. (canceled)
68. The method of claim 65, wherein the immunosuppressant and/or
the antigen, if present, are/is encapsulated in the synthetic
nanocarriers.
69. The method of claim 65, wherein the synthetic nanocarriers
comprise lipid nanoparticles, polymeric nanoparticles, metallic
nanoparticles, surfactant-based emulsions, dendrimers, buckyballs,
nanowires, virus-like particles or peptide or protein
particles.
70-75. (canceled)
76. The method of claim 65, wherein the mean of a particle size
distribution obtained using dynamic light scattering of a
population of the synthetic nanocarriers is a diameter greater than
110 nm.
77-89. (canceled)
90. The method of claim 65, wherein the load of immunosuppressant
comprised in the synthetic nanocarriers, on average across the
synthetic nanocarriers, is between 0.1% and 50%
(weight/weight).
91-94. (canceled)
95. The method of claim 65, wherein the immunosuppressant is
rapamycin.
96. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/846,964, filed Sep. 7, 2015, which claims
the benefit under 35 U.S.C. .sctn. 119 of U.S. provisional
application 62/047,034, filed Sep. 7, 2014; 62/051,255, filed Sep.
16, 2014; 62/101,841, filed Jan. 9, 2015; 62/047,044, filed Sep. 7,
2014, 62/051,258, filed Sep. 16, 2014; 62/101,861, filed Jan. 9,
2015; 62/047,054, filed Sep. 7, 2014; 62/051,263, filed Sep. 16,
2014; 62/101,872, filed Jan. 9, 2015; 62/047,051, filed Sep. 7,
2014, 62/051,267, filed Sep. 16, 2014; and 62/101,882, filed Jan.
9, 2015; the entire contents of each of which are incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to methods and compositions for
administering viral transfer vectors and antigen-presenting cell
targeted immunosuppressants.
SUMMARY OF THE INVENTION
[0003] Provided herein are methods and compositions for
administering gene expression modulating viral transfer vectors and
antigen-presenting cell targeted immunosuppressants. The viral
transfer vector comprises a gene expression modulating transgene
that encodes a protein, peptide or nucleic acid that may have a
therapeutic benefit for any one of the purposes provided herein in
any one of the methods or compositions provided herein.
[0004] In one aspect is a method comprising establishing an
anti-viral transfer vector attenuated response in a subject by
concomitant administration of an antigen-presenting cell targeted
immunosuppressant and viral transfer vector to the subject. In one
embodiment, the subject does not have pre-existing immunity against
the viral transfer vector.
[0005] In one embodiment of any one of the methods provided herein,
the anti-viral transfer vector attenuated response is a T cell
response against the viral transfer vector, and the method further
comprises administering the viral transfer vector to the subject
without an antigen-presenting cell targeted immunosuppressant prior
to the concomitant administration of the antigen-presenting cell
targeted immunosuppressant and viral transfer vector.
[0006] In one embodiment of any one of the methods provided herein,
the concomitant administration of the antigen-presenting cell
targeted immunosuppressant and viral transfer vector is repeated,
concomitant administration of the antigen-presenting cell targeted
immunosuppressant and viral transfer vector.
[0007] In another aspect is a method comprising establishing an
anti-viral transfer vector attenuated response in a subject by
concomitant administration of an antigen-presenting cell targeted
immunosuppressant and viral transfer vector to the subject, and
administering to the subject one or more repeat doses of the viral
transfer vector.
[0008] In one embodiment of any one of the methods provided herein,
the anti-viral transfer vector attenuated response is a T cell
response against the viral transfer vector, and the method further
comprises administering the viral transfer vector to the subject
without an antigen-presenting cell targeted immunosuppressant prior
to both the concomitant administration of the antigen-presenting
cell targeted immunosuppressant and viral transfer vector and the
one or more repeat doses of the viral transfer vector.
[0009] In one embodiment of any one of the methods provided herein,
the method further comprises providing or obtaining an
antigen-presenting cell targeted immunosuppressant alone or in
combination with a viral transfer vector.
[0010] In another aspect is a method comprising attenuating an
anti-viral transfer vector response, wherein the anti-viral
transfer vector response is a T cell response, by first
administering to a subject a viral transfer vector without an
antigen-presenting cell targeted immunosuppressant, and
subsequently concomitantly administering the viral transfer vector
and an antigen-presenting cell targeted immunosuppressant to the
subject.
[0011] In one embodiment of any one of the methods provided, the
method further comprises administering to the subject one or more
repeat doses of the viral transfer vector subsequent to the
concomitant administration of the viral transfer vector and the
antigen-presenting cell targeted immunosuppressant to the
subject.
[0012] In another aspect is a method comprising determining a level
of pre-existing immunity to a viral transfer vector in a subject
prior to administration of the viral transfer vector to the
subject, concomitantly administering to the subject an
antigen-presenting cell targeted immunosuppressant and viral
transfer vector, and administering to the subject a dose of the
viral transfer vector.
[0013] In one embodiment of any one of the methods provided, the
determining comprises measuring a level of anti-viral transfer
vector antibodies in the subject prior to administration of the
viral transfer vector to the subject. In another embodiment of any
one of the methods provided, the determining comprises measuring a
level of a T cell response against the viral transfer vector in the
subject prior to administration of the viral transfer vector to the
subject.
[0014] In one embodiment of any one of the methods provided, the
method further comprises one or more repeat doses of the viral
transfer vector.
[0015] In one embodiment of any one of the methods provided, the
level of pre-existing immunity is to a viral antigen of the viral
transfer vector. In one embodiment of any one of the methods
provided, the level of pre-existing immunity is to an antigen of a
protein transgene expression product of the viral transfer
vector.
[0016] In another aspect is a method comprising escalating
transgene expression of a viral transfer vector in a subject by
repeatedly, concomitantly administering to the subject an
antigen-presenting cell targeted immunosuppressant and viral
transfer vector.
[0017] In one embodiment of any one of the methods provided, the
method further comprises determining the frequency and dosing of
the repeated, concomitant administration of the antigen-presenting
cell targeted immunosuppressant and viral transfer vector that
increase the transgene expression in a subject.
[0018] In another aspect is a method comprising repeatedly,
concomitantly administering to a subject an antigen-presenting cell
targeted immunosuppressant and viral transfer vector, and selecting
one or more doses of the viral transfer vector to be less than the
dose of the viral transfer vector that would be selected for the
subject if the subject were expected to develop anti-viral transfer
vector immune responses due to the repeated administration of the
viral transfer vector.
[0019] In another aspect is a method comprising inducing an entity
to purchase or obtain an antigen-presenting cell targeted
immunosuppressant alone or in combination with a viral transfer
vector by communicating to the entity that concomitant
administration of the antigen-presenting cell targeted
immunosuppressant and viral transfer vector results in an
anti-viral transfer vector attenuated response in a subject.
[0020] In another aspect is a method comprising inducing an entity
to purchase or obtain an antigen-presenting cell targeted
immunosuppressant alone or in combination with a viral transfer
vector by communicating to the entity that efficacious repeated
viral transfer vector dosing is possible by concomitant
administration of the antigen-presenting cell targeted
immunosuppressant and viral transfer vector to a subject.
[0021] In one embodiment of any one of the methods provided herein,
the communicating further includes instructions for practicing any
one of the methods described herein or information describing the
benefits of concomitant administration of a viral transfer vector
with an antigen-presenting cell targeted immunosuppressant.
[0022] In one embodiment of any one of the methods provided herein,
the method further comprises distributing an antigen-presenting
cell targeted immunosuppressant or a viral transfer vector or both
to an entity.
[0023] In another aspect is a method comprising determining the
frequency and dosing of concomitant administration of an
antigen-presenting cell targeted immunosuppressant and viral
transfer vector in order to generate an anti-viral transfer vector
attenuated response in a subject.
[0024] In one embodiment of any one of the methods provided herein,
the method further comprises directing the concomitant
administration of the antigen-presenting cell targeted
immunosuppressant and viral transfer vector to a subject according
to the determined frequency and dosing.
[0025] In another aspect is a method comprising determining the
frequency and dosing of concomitant administration of an
antigen-presenting cell targeted immunosuppressant and viral
transfer vector in combination with one or more repeat doses of the
viral tranfer vector in order to generate an anti-viral transfer
vector attenuated response in a subject.
[0026] In one embodiment of any one of the methods provided herein,
the method further comprises directing both the concomitant
administration of the antigen-presenting cell targeted
immunosuppressant and viral transfer vector and administration of
the one or more repeat doses of the viral transfer vector to a
subject according to the determined frequency and dosing.
[0027] In one embodiment of any one of the methods provided herein,
the method further comprises directing the administration of a dose
of the viral transfer vector to the subject prior to both the
concomitant administration of the antigen-presenting cell targeted
immunosuppressant and viral transfer vector and administration of
the one or more repeat doses of the viral transfer vector to the
subject.
[0028] In one embodiment of any one of the methods provided herein,
the subject is one to which the viral transfer vector has not been
previously administered.
[0029] In one embodiment of any one of the methods provided herein,
the subject is one to which the viral transfer vector has been
previously administered no more than once.
[0030] In one embodiment of any one of the methods provided, the
amount of the viral transfer vector in the repeat dose(s) is at
least equal to the amount of the viral transfer vector in a prior
dose. In one embodiment of any one of the methods provided, the
amount of the viral transfer vector in the repeat dose(s) is less
than the amount of the viral transfer vector in a prior dose.
[0031] In one embodiment of any one of the methods provided, the
antigen-presenting cell targeted immunosuppressant is also
administered to the subject concomitantly with the one or more
repeat doses of the viral transfer vector. In one embodiment of any
one of the methods provided, the antigen-presenting cell targeted
immunosuppressant is not also administered to the subject
concomitantly with at least one of the one or more repeat doses of
the viral transfer vector.
[0032] In one embodiment of any one of the methods provided, the
subject does not have pre-existing immunity against the viral
transfer vector.
[0033] In one embodiment of any one of the methods provided, the
concomitant administration is simultaneous administration.
[0034] In one embodiment of any one of the methods provided, the
method further comprises determining a level of pre-existing
immunity to the viral transfer vector in the subject.
[0035] In one embodiment of any one of the methods provided herein,
the viral transfer vector is a retroviral transfer vector, an
adenoviral transfer vector, a lentiviral transfer vector or an
adeno-associated viral transfer vector.
[0036] In one embodiment of any one of the methods provided herein,
the viral transfer vector is an adenoviral transfer vector, and the
adenoviral transfer vector is a subgroup A, subgroup B, subgroup C,
subgroup D, subgroup E, or subgroup F adenoviral transfer
vector.
[0037] In one embodiment of any one of the methods provided herein,
the viral transfer vector is a lentiviral transfer vector, and the
lentiviral transfer vector is an HIV, SIV, FIV, EIAV or ovine
lentiviral vector.
[0038] In one embodiment of any one of the methods provided herein,
the viral transfer vector is an adeno-associated viral transfer
vector, and the adeno-associated viral transfer vector is an AAV1,
AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10 or AAV11
adeno-associated viral transfer vector.
[0039] In one embodiment of any one of the methods provided herein,
the viral transfer vector is a chimeric viral transfer vector. In
one embodiment of any one of the methods provided herein, the
chimeric viral transfer vector is an AAV-adenoviral transfer
vector.
[0040] In one embodiment of any one of the methods provided herein,
the gene expression modulating transgene encodes a DNA-binding
protein or a therapeutic RNA. In one embodiment of any one of the
methods provided herein, the DNA-binding protein is an artificial
transcription factor. In one embodiment of any one of the methods
provided herein, the therapeutic RNA is an inhibitor of mRNA
translation, agent of RNA interference (RNAi), catalytically active
RNA molecule (ribozyme), transfer RNA (tRNA) or a RNA that binds a
protein or other molecular ligand (aptamer). In one embodiment of
any one of the methods provided herein, the agent of RNAi is
double-stranded RNA, single-stranded RNA, micro RNA, short
interfering RNA, short hairpin RNA or a triplex-forming
oligonucleotide.
[0041] In one embodiment of any one of the methods provided herein,
the antigen-presenting cell targeted immunosuppressant comprises an
erythrocyte-binding therapeutic. In one embodiment of any one of
the methods provided herein, the erythrocyte-binding therapeutic
comprises ERY1, ERY19, ERY59, ERY64, ERY123, ERY141 and ERY162. In
one embodiment of any one of the methods provided herein, the
erythrocyte-binding therapeutic further comprises a viral transfer
vector antigen. In one embodiment of any one of the methods
provided herein, the viral transfer vector antigen is a viral
antigen.
[0042] In one embodiment of any one of the methods provided herein,
the antigen-presenting cell targeted immunosuppressant comprises a
negatively-charged particle. In one embodiment of any one of the
methods provided herein, the negatively-charged particle is a
polystyrene, PLGA, or diamond particle. In one embodiment of any
one of the methods provided herein, the zeta potential of the
particle is negative. In one embodiment of any one of the methods
provided herein, the zeta potential of the particle is less than
-50 mV. In one embodiment of any one of the methods provided
herein, the zeta potential of the particle is less than -100
mV.
[0043] In one embodiment of any one of the methods provided herein,
the antigen-presenting cell targeted immunosuppressant comprises an
apoptotic-body mimic and one or more viral transfer vector
antigens. In one embodiment of any one of the methods provided
herein, the apoptotic-body mimic is a particle that comprises the
one or more viral transfer vector antigens. In one embodiment of
any one of the methods provided herein, the one or more viral
transfer vector antigens comprise one or more viral antigens. In
one embodiment of any one of the methods provided herein, the
particle may also comprise an apoptotic signaling molecule. In one
embodiment of any one of the methods provided herein, the particle
comprises a polyglycolic acid polymer (PGA), polylactic acid
polymer (PLA), polysebacic acid polymer (PSA),
poly(lactic-co-glycolic) acid copolymer (PLGA),
poly(lactic-co-sebacic) acid copolymer (PLSA),
poly(glycolic-co-sebacic) acid copolymer (PGSA), polylactide
co-glycolide (PLG), or polyethylene glycol (PEG). In one embodiment
of any one of the methods provided herein, the average diameter of
the particle is between 0.1 and 5 .mu.m, between 0.1 and 4 .mu.m,
between 0.1 and 3 .mu.m, between 0.1 and 2 .mu.m, between 0.1 and 1
.mu.m or between 0.1 and 500 nm.
[0044] In one embodiment of any one of the methods provided herein,
the antigen-presenting cell targeted immunosuppressant comprises
synthetic nanocarriers comprising an immunosuppressant. In one
embodiment of any one of the methods provided herein, the synthetic
nanocarriers further comprise a viral transfer vector antigen. In
one embodiment of any one of the methods provided herein, the viral
transfer vector antigen is a viral antigen. In one embodiment of
any one of the methods provided herein, the immunosuppressant
and/or the antigen, if present, are/is encapsulated in the
synthetic nanocarriers.
[0045] In one embodiment of any one of the methods provided herein,
the synthetic nanocarriers comprise lipid nanoparticles, polymeric
nanoparticles, metallic nanoparticles, surfactant-based emulsions,
dendrimers, buckyballs, nanowires, virus-like particles or peptide
or protein particles. In one embodiment of any one of the methods
provided herein, the synthetic nanocarriers comprise polymeric
nanoparticles. In one embodiment of any one of the methods provided
herein, the polymeric nanoparticles comprise a polymer that is a
non-methoxy-terminated, pluronic polymer. In one embodiment of any
one of the methods provided herein, the polymeric nanoparticles
comprise a polyester, polyester attached to a polyether, polyamino
acid, polycarbonate, polyacetal, polyketal, polysaccharide,
polyethyloxazoline or polyethyleneimine. In one embodiment of any
one of the methods provided herein, the polyester comprises a
poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic
acid) or polycaprolactone. In one embodiment of any one of the
methods provided herein, the polymeric nanoparticles comprise a
polyester and a polyester attached to a polyether. In one
embodiment of any one of the methods provided herein, the polyether
comprises polyethylene glycol or polypropylene glycol.
[0046] In one embodiment of any one of the methods provided herein,
the mean of a particle size distribution obtained using dynamic
light scattering of a population of the synthetic nanocarriers is a
diameter greater than 110 nm. In one embodiment of any one of the
methods provided herein, the diameter is greater than 150 nm. In
one embodiment of any one of the methods provided herein, the
diameter is greater than 200 nm. In one embodiment of any one of
the methods provided herein, the diameter is greater than 250 nm.
In one embodiment of any one of the methods provided herein, the
diameter is less than 5 .mu.m. In one embodiment of any one of the
methods provided herein, the diameter is less than 4 .mu.m. In one
embodiment of any one of the methods provided herein, the diameter
is less than 3 .mu.m. In one embodiment of any one of the methods
provided herein, the diameter is less than 2 .mu.m. In one
embodiment of any one of the methods provided herein, the diameter
is less than 1 .mu.m. In one embodiment of any one of the methods
provided herein, the diameter is less than 500 nm. In one
embodiment of any one of the methods provided herein, the diameter
is less than 450 nm. In one embodiment of any one of the methods
provided herein, the diameter is less than 400 nm. In one
embodiment of any one of the methods provided herein, the diameter
is less than 350 nm. In one embodiment of any one of the methods
provided herein, the diameter is less than 300 nm.
[0047] In one embodiment of any one of the methods provided herein,
the load of immunosuppressant comprised in the synthetic
nanocarriers, on average across the synthetic nanocarriers, is
between 0.1% and 50% (weight/weight). In one embodiment of any one
of the methods provided herein, the load is between 0.1% and 25%.
In one embodiment of any one of the methods provided herein, the
load is between 1% and 25%. In one embodiment of any one of the
methods provided herein, the load is between 2% and 25%.
[0048] In one embodiment of any one of the methods provided herein,
the immunosuppressant is an inhibitor of the NF-kB pathway. In one
embodiment of any one of the methods provided herein, the
immunosuppressant is rapamycin.
[0049] In one embodiment of any one of the methods provided herein,
an aspect ratio of a population of the synthetic nanocarriers is
greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7 or 1:10.
[0050] In one embodiment of any one of the methods provided herein,
the method further comprises performing the method according to a
protocol that attenuates an anti-viral transfer vector response,
such as an antibody, T cell or B cell response, escalates transgene
expression or that establishes an anti-viral transfer vector
response. In one embodiment of any one of the methods provided
herein, the method further comprises determining a protocol that
attenuates an anti-viral transfer vector response, such as an
antibody, T cell or B cell response, escalates transgene expression
or that establishes an anti-viral transfer vector response.
[0051] In another embodiment of any one of the methods provided,
the method further comprises assessing an antibody immune response
against the viral transfer vector prior to, during or subsequent to
the administering to the subject.
[0052] In another aspect a method or composition as described in
any one of the Examples is provided.
[0053] In another aspect, any one of the compositions is for use in
any one of the methods provided.
[0054] In another aspect, any one of the methods is for use in
treating any one of the disease or disorders described herein. In
another aspect, any one of the methods is for use in attenuating an
anti-viral transfer vector response, establishing an attenuated
anti-viral transfer vector response, escalating transgene
expression or for repeated administration of a viral transfer
vector.
BRIEF DESCRIPTION OF THE FIGURES
[0055] FIG. 1. shows GFP expression in livers of mice injected with
AAV with or without synthetic nanocarriers comprising rapamycin at
prime or boost. All cells in suspension have been analyzed for GFP
expression with the exception of high side-scatter debris (2-3% of
total, a by-product of collagenase treatment) excluded by the first
`clean` gate. All the remaining cells were gated for relative GFP
strength (FL-1 channel). Numbers shown represent the percentage of
GFP-positive cells of the total parent population.
[0056] FIG. 2. shows GFP expression in livers of AAV-injected mice
as a function of boost with or without synthetic nanocarriers
comprising rapamycin. Data presented are the same as in FIG. 1, but
are grouped according to whether AAV boost employed
co-administration with the synthetic nanocarriers comprising
rapamycin or not (unboosted samples from gr. 5 and 6 are also shown
as a separate `supergroup`).
[0057] FIG. 3. demonstrates the GFP.sup.high cell share in livers
of animals injected with AAV with or without synthetic nanocarriers
comprising rapamycin. GFP-positive cells (as presented in FIG. 1)
were gated and then a population with an average GFP fluorescence
intensity of 10 times higher than average in the parent population
was gated again. Numbers presented are percentage from the parent
GFP-positive population as seen in FIG. 1.
[0058] FIG. 4. shows results from an experiment where mice were
bled at d14 after receiving a single AAV-GFP inoculation with or
without co-administration of synthetic nanocarriers comprising
rapamycin and their sera assayed for antibodies against AAV. Top
ODs for 1:40 serum dilutions are shown for all mice. Background
normal mouse serum had an OD of 0.227.
[0059] FIG. 5. shows results from an experiment where mice were
bled at days 14, 21 and 33 after receiving a single AAV-GFP
inoculation with or without co-administration of synthetic
nanocarriers comprising rapamycin (i.v.) and their sera assayed for
antibodies against AAV. Top ODs for 1:40 serum dilutions are shown
for all mice. Background normal mouse serum activity is shown.
Statistical significance is calculated using two-way ANOVA.
[0060] FIG. 6. shows results from an experiment where mice were
injected with AAV-GFP at days 0 and 21 with or without
co-administration of synthetic nanocarriers comprising rapamycin
(i.v.) at either or both injections, then bled at days 14 and 33
and their sera assayed for antibodies against AAV. Top ODs for 1:40
serum dilutions are shown for all mice. Background normal mouse
serum activity is shown. Statistical significance is calculated
using two-way ANOVA.
[0061] FIG. 7. provides data that are the same as in FIG. 6 with
the readings for individual mice shown. Two mice in the group
treated with synthetic nanocarriers comprising rapamycin only at
boost immunization (d21) did not show detectable antibodies at day
33 despite being positive at d14 (solid arrows). One of five mice
in both groups treated with synthetic nanocarriers comprising
rapamycin at the prime had a detectable antibody level at d33
(dashed arrows) with the mouse from the group treated with
synthetic nanocarriers comprising rapamycin at both prime and boost
having a lower antibody level (open diamonds).
[0062] FIG. 8. shows results from an experiment where mice mice
were bled at d14 after receiving a single AAV-GFP inoculation with
or without co-administration of synthetic nanocarriers comprising
rapamycin and their sera assayed for antibodies against AAV. Top
ODs for 1:40 serum dilutions are shown for all mice. Background
normal mouse serum had an OD of 0.227. N=15 mice per group.
[0063] FIG. 9. shows results from an experiment where mice were
bled at days 14, 21 and 33 after receiving a single AAV-GFP
inoculation with or without co-administration of synthetic
nanocarriers comprising rapamycin (i.v.) and their sera assayed for
antibodies against AAV. Top ODs for 1:40 serum dilutions are shown
for all mice. Background normal mouse serum levels are shown.
Statistical significance is calculated using two-way ANOVA. N=15
mice/group at day 14 and 5 mice/group at days 21 and 33.
[0064] FIG. 10 shows results from an experiment where mice were
injected with AAV8-GFP at days 0 and 21 with or without
co-administration of synthetic nanocarriers comprising rapamycin
(i.v.) at one or both injections, as indicated, and then bled at
days 14 and 33. Sera were assayed for antibodies against AAV8 by
ELISA. ODs for 1:40 serum dilutions are shown for all mice.
Background level of normal mouse serum is indicated by the dotted
line. Statistical significance is calculated using two-way
ANOVA.
[0065] FIG. 11 shows GFP expression in livers of mice injected with
AAV with or without synthetic nanocarriers comprising rapamycin at
prime or boost. All cells in suspension have been analyzed for GFP
expression with the exception of high side-scatter debris (2-3% of
total, a by-product of collagenase treatment) excluded by the first
`clean` gate. All the remaining cells were gated for relative GFP
strength (FL-1 channel). Numbers shown represent the percentage of
GFP-positive cells of the total parent population.
[0066] FIG. 12 shows RFP expression in livers of mice injected with
AAV with or without synthetic nanocarriers comprising rapamycin at
prime and/or boost. All cells in suspension have been analyzed for
RFP expression with the exception of high side-scatter debris.
Numbers shown represent the percentage of RFP-positive cells of the
total parent population of liver cells.
[0067] FIG. 13 shows cytotoxic activity in mice immunized with
AAV-GFP alone or in combination with synthetic nanocarriers
comprising rapamycin. Animals were injected with AAV8-GFP (i.v.) on
days 0 and 21 with or without synthetic nanocarriers comprising
rapamycin. Target cells pulsed with a combination of dominant
cytotoxic peptides from AAV capsid protein and the GFP transgene
were administered at 7 days after the last injection (day 28) and
their viability measured 18 hours later and compared to that of
non-peptide pulsed control cells.
[0068] FIG. 14 shows AAV-specific IFN-.gamma. production in mice
immunized with AAV-GFP alone or in combination with synthetic
nanocarriers comprising rapamycin. Animals were injected with
AAV-GFP (i.v.) on days 0 and 17 with or without NCS. Splenocytes
were isolated on day 25 and incubated in vitro with dominant MHC
class I-binding peptide from AAV capsid protein for 7 days and then
assayed by ELISpot with the same peptide. Each sample was run in
duplicate and presented with background subtracted.
[0069] FIG. 15 shows GFP-specific IFN-.gamma. production in mice
immunized with AAV-GFP alone or in combination with synthetic
nanocarriers comprising rapamycin. Animals were injected (i.v.)
with AAV8-GFP on days 0 and 17 with or without synthetic
nanocarriers comprising rapamycin. Splenocytes were isolated and
incubated in vitro with MHC class I-binding peptide from GFP for 7
days and then assayed by ELISpot with the same peptide. Each sample
was run in duplicate and presented with background subtracted.
[0070] FIG. 16 shows the design for an experiment.
[0071] FIG. 17 shows results from an experiment where mice were
injected with rAAV2/8-luciferase on day 0 with or without
co-administration of synthetic nanocarriers carrying 100 .mu.g of
rapamycin (i.v.) and then challenged with an i.v. injection of
AAV-hFIX on day 14. Sera was collected at various time points, as
indicated, and assayed for antibodies against AAV8 (left) and for
the levels of human factor IX protein (right).
[0072] FIG. 18 shows the experimental design for an experiment.
[0073] FIG. 19 shows results from an experiment where male C57BL/6
mice were injected (i.v.) with rAAV2/8-luciferase concomitantly
with synthetic nanocarriers carrying 100 .mu.g of rapamycin on day
0 and then injected with rAAV2/8-hFIX concomitantly with synthetic
nanocarriers carrying 100 .mu.g of rapamycin on day 21. Control
animals were treated similarly but with empty nanocarriers instead
of synthetic nanocarriers comprising rapamycin. Sera were collected
at various time points, as indicated, and assayed by ELISA for
antibodies against AAV (left) and for levels of human FIX protein
(right). AAV2/8-FIX vector copy number in the liver (middle) was
determined by PCR.
[0074] FIG. 20 shows the experimental design for an experiment.
[0075] FIG. 21 provides results that showed that concomitant i.v.
administration of synthetic nanocarriers carrying rapamycin with an
rAAV2/8 vector (AAV2/8-Luc) on day 0 did not have a profound impact
on the antibody response to an AAV5 vector (AAV5-hFIX) administered
on day 21. In contrast, the results also showed that mice
concomitantly treated with synthetic nanocarriers comprising
rapamycin and rAAV2/8-Luc on day 0 showed a robust response to
immunization with recombinant hFIX protein in complete Freund's
adjuvant (CFA) on day 21.
DETAILED DESCRIPTION OF THE INVENTION
[0076] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particularly
exemplified materials or process parameters 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 of the
invention only, and is not intended to be limiting of the use of
alternative terminology to describe the present invention.
[0077] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety for all purposes. Such incorporation by
reference is not intended to be an admission that any of the
incorporated publications, patents and patent applications cited
herein constitute prior art.
[0078] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the content clearly dictates otherwise. For example, reference to
"a polymer" includes a mixture of two or more such molecules or a
mixture of differing molecular weights of a single polymer species,
reference to "a synthetic nanocarrier" includes a mixture of two or
more such synthetic nanocarriers or a plurality of such synthetic
nanocarriers, reference to "a DNA molecule" includes a mixture of
two or more such DNA molecules or a plurality of such DNA
molecules, reference to "an immunosuppressant" includes a mixture
of two or more such immunosuppressant molecules or a plurality of
such immunosuppressant molecules, and the like.
[0079] As used herein, the term "comprise" or variations thereof
such as "comprises" or "comprising" are to be read to indicate the
inclusion of any recited integer (e.g. a feature, element,
characteristic, property, method/process step or limitation) or
group of integers (e.g. features, elements, characteristics,
properties, method/process steps or limitations) but not the
exclusion of any other integer or group of integers. Thus, as used
herein, the term "comprising" is inclusive and does not exclude
additional, unrecited integers or method/process steps.
[0080] In embodiments of any of the compositions and methods
provided herein, "comprising" may be replaced with "consisting
essentially of" or "consisting of". The phrase "consisting
essentially of" is used herein to require the specified integer(s)
or steps as well as those which do not materially affect the
character or function of the claimed invention. As used herein, the
term "consisting" is used to indicate the presence of the recited
integer (e.g. a feature, element, characteristic, property,
method/process step or limitation) or group of integers (e.g.
features, elements, characteristics, properties, method/process
steps or limitations) alone.
A. Introduction
[0081] Anti-viral transfer vectors are promising therapeutics for a
variety of applications such as gene expression modulation. Viral
transfer vectors, therefore, may comprise transgenes that encode
proteins or nucleic acids. Examples of such include microRNA
(miRNA), small interfering RNA (siRNA), as well as antisense
oligonucleotides that bind mutation sites in messenger RNA (such as
small nuclear RNA (snRNA)). Unfortunately, the promise of these
therapeutics has not yet been realized in the art in a large part
due to cellular and humoral immune responses against the viral
transfer vector. These immune responses include antibody, B cell
and T cell responses and can be specific to viral antigens of the
viral transfer vector, such as viral capsid or coat proteins or
peptides thereof.
[0082] Currently, many possible patients harbor some level of
pre-existing immunity against the viruses on which viral transfer
vectors are based. In fact, antibodies against viral antigens, such
as antibodies against adeno-associated viruses, are highly
prevalent in the human population. In addition, even if the level
of pre-existing immunity is low, for example due to the low
immunogenicity of the viral transfer vector, such low levels may
still prevent successful transduction (e.g., Jeune, et al., Human
Gene Therapy Methods, 24:59-67 (2013)). Thus, even low levels of
pre-existing immunity may hinder the use of a specific viral
transfer vector and may require a clinician to choose a viral
transfer vector based on a virus of a different serotype, that may
not be as efficacious, or even opt for a different type of therapy
if another viral transfer vector therapy is not available.
[0083] Additionally, viral vectors, such as adeno-associated
vectors, can be highly immunogenic and elicit humoral and
cell-mediated immunity that can compromise efficacy, particularly
with respect to re-administration. In fact, cellular and humoral
immune responses against a viral transfer vector can develop after
a single administration of the viral transfer vector. After viral
transfer vector administration, neutralizing antibody titers can
increase and remain high for several years and can reduce the
effectiveness of readministration of the viral transfer vector, as
repeated administration of a viral transfer vector generally
results in enhanced undesired immune responses. In addition, viral
transfer vector-specific CD8+ T cells may arise that eliminate
transduced cells expressing a desired transgene product, such as,
for example, on reexposure to a viral antigen, such as a capsid
protein. Indeed, it has been shown that AAV capsid antigen
triggered immune-mediated destruction of hepatocytes transduced
with an AAV viral transfer vector (e.g., Manno et al., Nature
Medicine, Vol. 12, No. 3, 2006). For many therapeutic applications,
it is anticipated that multiple rounds of administration of viral
transfer vectors will be needed for long-term benefits, and,
without the methods and compositions provided herein, the ability
to do so would be expected to be severely limited particularly if
readministration is needed.
[0084] The problems associated with the use of viral transfer
vectors for therapy is further compounded because viral transfer
vector antigens can persist for some time, such as for at least
several weeks, after a single administration (e.g., Nathawani et
al., N Engl J Med 365; 25, 2011; Nathwani, et al., N Engl J Med
371; 21, 2014). As an example, it has been found that long-lasting
capsid-specific humoral immunity developed in patients that
received a single infusion of an adeno-associated virus serotype 8
(AAV8) viral transfer vector (e.g., Nathwani, et al., N Engl J Med
371; 21, 2014). The persistence of antigen further hinders the
ability to use viral transfer vectors successfully. It is important
to evade immune responses against viral transfer vectors in order
for therapy with viral transfer vectors to be successful. Prior to
this invention, however, there was no way to do so and achieve
long-term immune response attenuation without the need for
long-term administration of an immunosuppressant.
[0085] The inventors have surprisingly and unexpectedly discovered
that the problems and limitations noted above can be overcome by
practicing the invention disclosed herein. Methods and compositions
are provided that offer solutions to the aformentioned obstacles to
effective use of viral transfer vectors for treatment. In
particular, it has been unexpectedly discovered that anti-viral
transfer vector immune responses can be attenuated with the methods
and related compositions provided herein. The methods and
compositions can increase the efficacy of treatment with viral
transfer vectors and provide for long-term immune attenuation even
if the administration of the viral transfer vector need be
repeated.
[0086] The invention will now be described in more detail
below.
B. Definitions
[0087] "Administering" or "administration" or "administer" means
giving or dispensing a material to a subject in a manner that is
pharmacologically useful. The term is intended to include "causing
to be administered". "Causing to be administered" means causing,
urging, encouraging, aiding, inducing or directing, directly or
indirectly, another party to administer the material. Any one of
the methods provided herein may comprise or further comprise a step
of administering concomitantly an antigen-presenting cell targeted
immunosuppressant and a viral transfer vector. In some embodiments,
the concomitant administration is performed repeatedly. In still
further embodiments, the concomitant administration is simultaneous
administration.
[0088] "Amount effective" in the context of a composition or dosage
form for administration to a subject as provided herein refers to
an amount of the composition or dosage form that produces one or
more desired results in the subject, for example, the reduction or
elimination of an immune response against a viral transfer vector
or the generation of an anti-viral transfer vector attenuated
response. The amount effective can be for in vitro or in vivo
purposes. For in vivo purposes, the amount can be one that a
clinician would believe may have a clinical benefit for a subject
that may experience undesired immune responses as a result of
administration of a viral transfer vector. In any one of the
methods provided herein, the composition(s) administered may be in
any one of the amounts effective as provided herein.
[0089] Amounts effective can involve reducing the level of an
undesired immune response, although in some embodiments, it
involves preventing an undesired immune response altogether.
Amounts effective can also involve delaying the occurrence of an
undesired immune response. An amount effective can also be an
amount that results in a desired therapeutic endpoint or a desired
therapeutic result. Amounts effective, preferably, result in a
tolerogenic immune response in a subject to an antigen, such as a
viral transfer vector antigen. Amounts effective, can also
preferably result in increased transgene expression (the transgene
being delivered by the viral transfer vector). This can be
determined by measuring transgene protein concentrations in various
tissues or systems of interest in the subject. This increased
expression may be measured locally or systemically. The achievement
of any of the foregoing can be monitored by routine methods.
[0090] In some embodiments of any one of the compositions and
methods provided, the amount effective is one in which the desired
immune response, such as the reduction or elimination of an immune
response against a viral transfer vector or the generation of an
anti-viral transfer vector attenuated response, persists in the
subject for at least 1 week, at least 2 weeks or at least 1 month.
In other embodiments of any one of the compositions and methods
provided, the amount effective is one which produces a measurable
desired immune response, such as the reduction or elimination of an
immune response against a viral transfer vector or the generation
of an anti-viral transfer vector attenuated response. In some
embodiments, the amount effective is one that produces a measurable
desired immune response (e.g., to a specific viral transfer vector
antigen), for at least 1 week, at least 2 weeks or at least 1
month.
[0091] Amounts effective will depend, of course, on the particular
subject being treated; the severity of a condition, disease or
disorder; the individual patient parameters including age, physical
condition, size and weight; the duration of the treatment; the
nature of concurrent therapy (if any); the specific route of
administration and like factors within the knowledge and expertise
of the health practitioner. These factors are well known to those
of ordinary skill in the art and can be addressed with no more than
routine experimentation.
[0092] "Anti-viral transfer vector immune response" or "immune
response against a viral transfer vector" or the like refers to any
undesired immune response against a viral transfer vector. In some
embodiments, the undesired immune response is an antigen-specific
immune response against the viral transfer vector or an antigen
thereof. In some embodiments, the immune response is specific to a
viral antigen of the viral transfer vector. In other embodiments,
the immune response is specific to a protein or peptide encoded by
the transgene of the viral transfer vector. In some embodiments,
the immune response is specific to a viral antigen of the viral
transfer vector and not to a protein or peptide that is encoded by
the transgene of the viral transfer vector. The immune response may
be an anti-viral transfer vector antibody response, an anti-viral
transfer vector T cell immune response, such as a CD4+ T cell or
CD8+ T cell immune response, or an anti-viral transfer vector B
cell immune response.
[0093] An anti-viral transfer vector immune response is said to be
an "anti-viral transfer vector attenuated response" when it is in
some manner reduced or eliminated in the subject or as compared to
an expected or measured response in the subject or another subject.
In some embodiments, the anti-viral transfer vector attenuated
response in a subject comprises a reduced anti-viral transfer
vector immune response (such as a T cell, B cell or antibody
response) measured using a biological sample obtained from the
subject following a concomitant administration as provided herein
as compared to an anti-viral transfer vector immune response
measured using a biological sample obtained from another subject,
such as a test subject, following administration to this other
subject of the viral transfer vector without concomitant
administration of the antigen-presenting cell targeted
immunosuppressant. In some embodiments, the biological sample is
obtained from the other subject following administration to this
other subject of the viral transfer vector without any
administration of the antigen-presenting cell targeted
immunosuppressant. In some embodiments, the anti-viral transfer
vector attenuated response is a reduced anti-viral transfer vector
immune response (such as a T cell, B cell or antibody response) in
a biological sample obtained from the subject following a
concomitant administration as provided herein upon a subsequent
viral transfer vector in vitro challenge performed on the subject's
biological sample as compared to the anti-viral transfer vector
immune response detected upon viral transfer vector in vitro
challenge performed on a biological sample obtained from another
subject, such as a test subject, following administration to this
other subject of the viral transfer vector without concomitant
administration of the antigen-presenting cell targeted
immunosuppressant. In some embodiments, the anti-viral transfer
vector attenuated response is a reduced anti-viral transfer vector
immune response (such as a T cell, B cell or antibody response) in
the subject following a concomitant administration as provided
herein upon a subsequent viral transfer vector challenge
administered to the subject as compared to the anti-viral transfer
vector immune response in another subject, such as a test subject,
upon a viral transfer vector challenge administered to this other
subject following administration to this other subject of the viral
transfer vector without concomitant administration of the
antigen-presenting cell targeted immunosuppressant. In some
embodiments, the viral transfer vector is administered without any
administration of the antigen-presenting cell targeted
immunosuppressant.
[0094] "Antigen" means a B cell antigen or T cell antigen. "Type(s)
of antigens" means molecules that share the same, or substantially
the same, antigenic characteristics. In some embodiments, antigens
may be proteins, polypeptides, peptides, lipoproteins, glycolipids,
polynucleotides, polysaccharides, etc.
[0095] "Antigen-presenting cell targeted immunosuppressant" means
an agent that results in antigen-presenting cells (APCs) having a
tolerogenic effect. Such an immunosuppressant can include
immunosuppressants coupled to a carrier that results in delivery to
APCs and a tolerogenic effect as well as agents that by virtue of
their form or characteristics can result in APC tolerogenic
effects. Examples of antigen-presenting cell targeted
immunosuppressants include, but are not limited to synthetic
nanocarriers that comprise an immunosuppressant as described
herein; immunosuppressants, as described herein, coupled to
antibodies or antigen-binding fragments thereof that target APCs
(or other ligand that targets an APC), erythrocyte-binding
therapeutics, as well as particles that by virtue of their
characteristics lead to APC tolerogenic immune responses, etc.
[0096] When the antigen-presenting cell targeted immunosuppressant
is a synthetic nanoarrier coupled to an immunosuppressant, in some
embodiments, the immunosuppressant is an element that is in
addition to the material that makes up the structure of the
synthetic nanocarrier. For example, in one embodiment, where the
synthetic nanocarrier is made up of one or more polymers, the
immunosuppressant is a compound that is in addition and, in some
embodiments, attached to the one or more polymers. As another
example, in one embodiment, where the synthetic nanocarrier is made
up of one or more lipids, the immunosuppressant is again in
addition to and, in some embodiments, attached to the one or more
lipids. In embodiments where the antigen-presenting cell targeted
immunosuppressant is a synthetic nanoarrier coupled to an
immunosuppressant, and the material of the synthetic nanocarrier
also results in a tolerogenic effect, the immunosuppressant is an
element present in addition to the material of the synthetic
nanocarrier that results in a tolerogenic effect.
[0097] "Antigen-specific" refers to an immune response that results
from the presence of an antigen of interest or that generates
molecules that specifically recognize or bind the antigen of
interest. Generally, while such responses are measurable against
the antigen of interest, the responses are reduced or negligible in
regard to other antigens. For example, where the immune response is
antigen-specific antibody production, antibodies are produced that
selectively bind the antigen of interest but not to other antigens.
As another example, where the immune response involves the
production of CD4+ or CD8+ T cells, antigen-specific CD4+ or CD8+ T
cells can bind to an antigen of interest or portion thereof when
presented in the context of MHC class I or II antigens,
respectively, by an antigen-presenting cell (APC) or, in case of
CD8+ T cells, by any other cell in which the antigen is produced
(e.g., a cell infected with a virus). In the case of immune
tolerance, antigen specificity refers to the selective prevention
or inhibition of a specific immune response to a target antigen
versus other unrelated or unassociated antigens (e.g. antigens that
are temporally or spatially dislocated from the target
antigen).
[0098] "Assessing an immune response" refers to any measurement or
determination of the level, presence or absence, reduction,
increase in, etc. of an immune response in vitro or in vivo. Such
measurements or determinations may be performed on one or more
samples obtained from a subject. Such assessing can be performed
with any one of the methods provided herein or otherwise known in
the art. The assessing may be assessing the number or percentage of
antibodies or T cells, such as those specific to a viral transfer
vector, such as in a sample from a subject. The assessing also may
be assessing any effect related to the immune response, such as
measuring the presence or absence of a cytokine, cell phenotype,
etc. Any one of the methods provided herein may comprise or further
comprise a step of assessing an immune response to a viral transfer
vector or antigen thereof. The assessing may be done directly or
indirectly. The term is intended to include actions that cause,
urge, encourage, aid, induce or direct another party to assess an
immune response.
[0099] "Attach" or "Attached" or "Couple" or "Coupled" (and the
like) means to chemically associate one entity (for example a
moiety) with another. In some embodiments, the attaching is
covalent, meaning that the attachment occurs in the context of the
presence of a covalent bond between the two entities. In
non-covalent embodiments, the non-covalent attaching is mediated by
non-covalent interactions including but not limited to charge
interactions, affinity interactions, metal coordination, physical
adsorption, host-guest interactions, hydrophobic interactions, TT
stacking interactions, hydrogen bonding interactions, van der Waals
interactions, magnetic interactions, electrostatic interactions,
dipole-dipole interactions, and/or combinations thereof. In
embodiments, encapsulation is a form of attaching.
[0100] "Average", as used herein, refers to the arithmetic mean
unless otherwise noted.
[0101] "Concomitantly" means administering two or more
materials/agents to a subject in a manner that is correlated in
time, preferably sufficiently correlated in time so as to provide a
modulation in an immune response, and even more preferably the two
or more materials/agents are administered in combination. In
embodiments, concomitant administration may encompass
administration of two or more materials/agents within a specified
period of time, preferably within 1 month, more preferably within 1
week, still more preferably within 1 day, and even more preferably
within 1 hour. In embodiments, the materials/agents may be
repeatedly administered concomitantly; that is concomitant
administration on more than one occasion, such as provided in the
Examples.
[0102] "Determining" means objectively ascertaining something, such
as a fact, relationship or quantity. In some embodiments, whether
or not a subject has a pre-existing immunity to a viral transfer
vector may be determined. The term is intended to include "causing
to be determined". "Causing to be determined" means causing,
urging, encouraging, aiding, inducing or directing another party to
perform a step of determining as provided herein. In some
embodiments, the step of determining may be determining whether or
not a subject has a pre-existing immunity to a viral transfer
vector. Any one of the methods provided herein may comprise or
further comprise a step of determining as described herein
including a step of determining whether or not a subject has a
pre-existing immunity to a viral transfer vector.
[0103] "Directing" means influencing, such as taking some action to
influence, in some manner the actions of another party, such as
causing or controlling the acts of the other party in such a manner
that they perform one or more steps as provided herein. In some
embodiments, the other party is an agent of the party that is doing
the directing. In other embodiments, the other party is not an
agent of the party that is doing the directing, but the step(s)
performed by the other party is/are attributable to or the result
of the directing. Accordingly, directing includes instructing or
providing instructions to perform one or more steps in order to
receive a benefit conditioned on the performance of the one or more
steps.
[0104] "Dosage form" means a pharmacologically and/or
immunologically active material in a medium, carrier, vehicle, or
device suitable for administration to a subject. Any one of the
compositions or doses provided herein may be in a dosage form.
[0105] "Dose" refers to a specific quantity of a pharmacologically
and/or immunologically active material for administration to a
subject for a given time. A "prior dose" refers to an earlier dose
of a material. In general, doses of the antigen-presenting cell
targeted immunosuppressants and/or viral transfer vectors in the
methods and compositions of the invention refer to the amount of
the antigen-presenting cell targeted immunosuppressants and/or
viral transfer vectors. Alternatively, the dose can be administered
based on the number of synthetic nanocarriers that provide the
desired amount of antigen-presenting cell targeted
immunosuppressant, in instances where the antigen-presenting cell
targeted immunosuppressant is a synthetic nanocarrier that
comprises an immunosuppressant. When dose is used in the context of
a repeated dosing, dose refers to the amount of each of the
repeated doses, which may be the same or different.
[0106] "Encapsulate" means to enclose at least a portion of a
substance within a synthetic nanocarrier. In some embodiments, a
substance is enclosed completely within a synthetic nanocarrier. In
other embodiments, most or all of a substance that is encapsulated
is not exposed to the local environment external to the synthetic
nanocarrier. In other embodiments, no more than 50%, 40%, 30%, 20%,
10% or 5% (weight/weight) is exposed to the local environment.
Encapsulation is distinct from absorption, which places most or all
of a substance on a surface of a synthetic nanocarrier, and leaves
the substance exposed to the local environment external to the
synthetic nanocarrier.
[0107] "Escalating transgene expression" refers to increasing the
level of the transgene expression product of a viral transfer
vector in a subject, the transgene being delivered by the viral
transfer vector. In some embodiments, the level of the transgene
expression product may be determined by measuring transgene protein
concentrations in various tissues or systems of interest in the
subject. Alternatively, when the transgene expression product is a
nucleic acid, the level of transgene expression may be measured by
transgene nucleic acid products. Escalating transgene expression
can be determined, for example, by measuring the amount of the
transgene expression product in a sample obtained from a subject
and comparing it to a prior sample. The sample may be a tissue
sample. In some embodiments, the transgene expression product can
be measured using flow cytometry.
[0108] "Establishing" or "establish" means to generate an outcome
or result or to deduce something, such as a fact or relationship.
Which use of this term will be apparent based on the context in
which it is used. For generating an outcome or result, the
establishing may be accomplished in a number of ways, including but
not limited to, taking steps to accomplish the outcome or result.
For example, in some embodiments, administration of material(s) as
provided herein can generate the outcome or result. For determining
something, such as a fact or relationship, the establishing may be
accomplished by performing experiments, making projections, etc.
For instance, establishing that administration of a viral transfer
vector is likely to generate an anti-viral transfer vector immune
response in a subject may be based on results of experiments on a
subject, including on one or more samples obtained therefrom.
Generally, the likelihood of generating an anti-viral transfer
vector immune response in a subject is the likelihood of generating
such a response with the administration (or repeated
administration, in some embodiments) of a viral transfer vector in
the absence of administration of an antigen-presenting cell
targeted immunosuppressant as provided herein. Likewise,
establishing that a subject has a pre-existing immunity to a viral
transfer vector may also be based on the result of experiments on a
subject, including on one or more samples obtained therefrom. In
another embodiment, such establishing may be determined by
assessing an immune response in the subject. In regard to
establishing a dose for administration, a dose of an
antigen-presenting cell targeted immunosuppressant or a viral
transfer vector may be determined by starting with a test dose and
using known scaling techniques (such as allometric or isometric
scaling) to determine the dose for administration. Such may also be
used to establish a protocol as provided herein. "Establishing" or
"establish" comprises "causing to be established." "Causing to be
established" means causing, urging, encouraging, aiding, inducing
or directing or acting in coordination with an entity for the
entity to perform a step of establishing as provided herein. In
some embodiments of any one of the methods provided herein, the
method may comprise or further comprise any one of the steps of
establishing as described herein.
[0109] "Frequency" refers to the interval of time at which the
antigen-presenting cell targeted immunosuppressant, the viral
transfer vector or both in combination (such as with concomitant
administration) are administered to a subject.
[0110] "Gene expression modulating transgene" refers to any nucleic
acid that encodes a gene expression modulator. "Gene expression
modulator" refers to a molecule that can enhance, inhibit or
modulate the expression of one or more endogenous genes. Gene
expression modulators, therefore, include DNA-binding proteins
(e.g., artificial transcription factors) as well as molecules that
mediate RNA interference. Gene expression modulators include RNAi
molecules (e.g., dsRNAs or ssRNAs), miRNA, and triplex-forming
oligonucleotides (TFOs). Gene expression modulators also may
include modified RNAs, including modified versions of any of the
foregoing RNA molecules.
[0111] "Immunosuppressant" means a compound that causes a
tolerogenic effect, preferably through its effects on APCs. A
tolerogenic effect generally refers to the modulation by the APC or
other immune cells systemically and/or locally, that reduces,
inhibits or prevents an undesired immune response to an antigen in
a durable fashion. In one embodiment, the immunosuppressant is one
that causes an APC to promote a regulatory phenotype in one or more
immune effector cells. For example, the regulatory phenotype may be
characterized by the inhibition of the production, induction,
stimulation or recruitment of antigen-specific CD4+ T cells or B
cells, the inhibition of the production of antigen-specific
antibodies, the production, induction, stimulation or recruitment
of Treg cells (e.g., CD4+CD25highFoxP3+ Treg cells), etc. This may
be the result of the conversion of CD4+ T cells or B cells to a
regulatory phenotype. This may also be the result of induction of
FoxP3 in other immune cells, such as CD8+ T cells, macrophages and
iNKT cells. In one embodiment, the immunosuppressant is one that
affects the response of the APC after it processes an antigen. In
another embodiment, the immunosuppressant is not one that
interferes with the processing of the antigen. In a further
embodiment, the immunosuppressant is not an apoptotic-signaling
molecule. In another embodiment, the immunosuppressant is not a
phospholipid.
[0112] Immunosuppressants include, but are not limited to, statins;
mTOR inhibitors, such as rapamycin or a rapamycin analog (i.e.,
rapalog); TGF-.beta. signaling agents; TGF-.beta. receptor
agonists; histone deacetylase inhibitors, such as Trichostatin A;
corticosteroids; inhibitors of mitochondrial function, such as
rotenone; P38 inhibitors; NF-.kappa..beta. inhibitors, such as
6Bio, Dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists;
prostaglandin E2 agonists (PGE2), such as Misoprostol;
phosphodiesterase inhibitors, such as phosphodiesterase 4 inhibitor
(PDE4), such as Rolipram; proteasome inhibitors; kinase inhibitors;
G-protein coupled receptor agonists; G-protein coupled receptor
antagonists; glucocorticoids; retinoids; cytokine inhibitors;
cytokine receptor inhibitors; cytokine receptor activators;
peroxisome proliferator-activated receptor antagonists; peroxisome
proliferator-activated receptor agonists; histone deacetylase
inhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3 KB
inhibitors, such as TGX-221; autophagy inhibitors, such as
3-Methyladenine; aryl hydrocarbon receptor inhibitors; proteasome
inhibitor I (PSI); and oxidized ATPs, such as P2X receptor
blockers. Immunosuppressants also include IDO, vitamin D3, retinoic
acid, cyclosporins, such as cyclosporine A, aryl hydrocarbon
receptor inhibitors, resveratrol, azathiopurine (Aza),
6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sanglifehrin
A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX
inhibitors, niflumic acid, estriol and triptolide. Other exemplary
immunosuppressants include, but are not limited, small molecule
drugs, natural products, antibodies (e.g., antibodies against CD20,
CD3, CD4), biologics-based drugs, carbohydrate-based drugs, RNAi,
antisense nucleic acids, aptamers, methotrexate, NSAIDs;
fingolimod; natalizumab; alemtuzumab; anti-CD3; tacrolimus (FK506),
abatacept, belatacept, etc. "Rapalog" refers to a molecule that is
structurally related to (an analog) of rapamycin (sirolimus).
Examples of rapalogs include, without limitation, temsirolimus
(CCI-779), everolimus (RAD001), ridaforolimus (AP-23573), and
zotarolimus (ABT-578). Additional examples of rapalogs may be
found, for example, in WO Publication WO 1998/002441 and U.S. Pat.
No. 8,455,510, the rapalogs of which are incorporated herein by
reference in their entirety.
[0113] The immunosuppressant can be a compound that directly
provides the tolerogenic effect on APCs or it can be a compound
that provides the tolerogenic effect indirectly (i.e., after being
processed in some way after administration). Immunosuppressants,
therefore, include prodrug forms of any of the compounds provided
herein. Further immunosuppressants, are known to those of skill in
the art, and the invention is not limited in this respect. In
embodiments, the immunosuppressant may comprise any one of the
agents provided herein.
[0114] "Inducing to purchase" refers to any act that suggests to an
entity to purchase an antigen-presenting cell targeted
immunosuppressant, a viral transfer vector or both to achieve a
beneficial effect as described herein or to perform any one of the
methods provided herein. Such acts includes packaging an
antigen-presenting cell targeted immunosuppressant, a viral
transfer vector or both that describes the benefits of concomitant
administration of an antigen-presenting cell targeted
immunosuppressant and a viral transfer vector in order to attenuate
an anti-viral transfer vector response, escalate transgene
expression or allow for repeated administration of a viral transfer
vector. Alternatively, the packaging may describe or suggest the
performance of any one of the methods provided herein. Acts that
induce an entity to purchase also include marketing an
antigen-presenting cell targeted immunosuppressant, a viral
transfer vector or an antigen-presenting cell targeted
immunosuppressant and a viral transfer vector product with
information describing or suggesting the use of such product for
carrying out any of the beneficial effects described herein or any
one of the methods provided herein. Alternatively, the marketing
includes materials that describe or suggest the use of such product
for attenuating an anti-viral transfer vector response, escalating
transgene expression or for repeated administration of a viral
transfer vector. As a further example, acts of inducing may also
comprise acts of communicating information describing or suggesting
any of the foregoing. The communicating is an action that can be
performed in any form whether written, oral, etc. If in written
form, the communicating may be performed via any medium including
an electronic or a paper-based medium. Further, acts of inducing
also include acts of distributing an antigen-presenting cell
targeted immunosuppressant, a viral transfer vector or both. Acts
of distributing include any action to make available the
antigen-presenting cell targeted immunosuppressant, viral transfer
vector or both to an entity with information, packaging, marketing
materials, etc. that describes, instructs or communicates any of
the benefits described herein or the steps of any one of the
methods provided herein or the ability to attenuate an anti-viral
transfer vector response, escalate transgene expression or allow
for repeated administration of a viral transfer vector. Acts of
distributing include selling, offering for sale, and transporting
for sale (e.g., transporting to pharmacies, hospitals, etc.)
[0115] "Load", when coupled to a synthetic nanocarrier, is the
amount of the immunosuppressant coupled to the synthetic
nanocarrier based on the total dry recipe weight of materials in an
entire synthetic nanocarrier (weight/weight). Generally, such a
load is calculated as an average across a population of synthetic
nanocarriers. In one embodiment, the load on average across the
synthetic nanocarriers is between 0.1% and 99%. In another
embodiment, the load is between 0.1% and 50%. In another
embodiment, the load is between 0.1% and 20%. In a further
embodiment, the load is between 0.1% and 10%. In still a further
embodiment, the load is between 1% and 10%. In still a further
embodiment, the load is between 7% and 20%. In yet another
embodiment, the load is at least 0.1%, at least 0.2%, at least
0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%,
at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least
3%, at least 4%, at least 5%, at least 6%, at least at least 7%, at
least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at
least 13%, at least 14%, at least 15%, at least 16%, at least 17%,
at least 18%, at least 19%, at least 20%, at least 25%, at least
30%, at least 40%, at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, at least 95%, at least 96%, at least 97%,
at least 98% or at least 99% on average across the population of
synthetic nanocarriers. In yet a further embodiment, the load is
0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%,
4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,
18%, 19% or 20% on average across the population of synthetic
nanocarriers. In some embodiments of the above embodiments, the
load is no more than 25% on average across a population of
synthetic nanocarriers. In embodiments, the load is calculated as
may be described in the Examples or as otherwise known in the art.
In some embodiments, when the form of the immunosuppressant is
itself a particle or particle-like, such as a nanocrystalline
immunosuppressant, the load of immunosuppressant is the amount of
the immunosuppressant in the particles or the like (weight/weight).
In such embodiments, the load can approach 97%, 98%, 99% or
more.
[0116] "Maximum dimension of a synthetic nanocarrier" means the
largest dimension of a nanocarrier measured along any axis of the
synthetic nanocarrier. "Minimum dimension of a synthetic
nanocarrier" means the smallest dimension of a synthetic
nanocarrier measured along any axis of the synthetic nanocarrier.
For example, for a spheroidal synthetic nanocarrier, the maximum
and minimum dimension of a synthetic nanocarrier would be
substantially identical, and would be the size of its diameter.
Similarly, for a cuboidal synthetic nanocarrier, the minimum
dimension of a synthetic nanocarrier would be the smallest of its
height, width or length, while the maximum dimension of a synthetic
nanocarrier would be the largest of its height, width or length. In
an embodiment, a minimum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is equal to or greater than 100 nm. In
an embodiment, a maximum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is equal to or less than 5 .mu.m.
Preferably, a minimum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is greater than 110 nm, more preferably
greater than 120 nm, more preferably greater than 130 nm, and more
preferably still greater than 150 nm. Aspects ratios of the maximum
and minimum dimensions of synthetic nanocarriers may vary depending
on the embodiment. For instance, aspect ratios of the maximum to
minimum dimensions of the synthetic nanocarriers may vary from 1:1
to 1,000,000:1, preferably from 1:1 to 100,000:1, more preferably
from 1:1 to 10,000:1, more preferably from 1:1 to 1000:1, still
more preferably from 1:1 to 100:1, and yet more preferably from 1:1
to 10:1. Preferably, a maximum dimension of at least 75%,
preferably at least 80%, more preferably at least 90%, of the
synthetic nanocarriers in a sample, based on the total number of
synthetic nanocarriers in the sample is equal to or less than 3
.mu.m, more preferably equal to or less than 2 .mu.m, more
preferably equal to or less than 1 .mu.m, more preferably equal to
or less than 800 nm, more preferably equal to or less than 600 nm,
and more preferably still equal to or less than 500 nm. In
preferred embodiments, a minimum dimension of at least 75%,
preferably at least 80%, more preferably at least 90%, of the
synthetic nanocarriers in a sample, based on the total number of
synthetic nanocarriers in the sample, is equal to or greater than
100 nm, more preferably equal to or greater than 120 nm, more
preferably equal to or greater than 130 nm, more preferably equal
to or greater than 140 nm, and more preferably still equal to or
greater than 150 nm. Measurement of synthetic nanocarrier
dimensions (e.g., effective diameter) may be obtained, in some
embodiments, by suspending the synthetic nanocarriers in a liquid
(usually aqueous) media and using dynamic light scattering (DLS)
(e.g. using a Brookhaven ZetaPALS instrument). For example, a
suspension of synthetic nanocarriers can be diluted from an aqueous
buffer into purified water to achieve a final synthetic nanocarrier
suspension concentration of approximately 0.01 to 0.1 mg/mL. The
diluted suspension may be prepared directly inside, or transferred
to, a suitable cuvette for DLS analysis. The cuvette may then be
placed in the DLS, allowed to equilibrate to the controlled
temperature, and then scanned for sufficient time to acquire a
stable and reproducible distribution based on appropriate inputs
for viscosity of the medium and refractive indicies of the sample.
The effective diameter, or mean of the distribution, is then
reported. Determining the effective sizes of high aspect ratio, or
non-spheroidal, synthetic nanocarriers may require augmentative
techniques, such as electron microscopy, to obtain more accurate
measurements. "Dimension" or "size" or "diameter" of synthetic
nanocarriers means the mean of a particle size distribution, for
example, obtained using dynamic light scattering.
[0117] "Measurable level" refers to any level that is above a
negative control level or would be considered to be above
background or signal noise. A measurable level is one that would be
considered to be a level indicating the presence of the molecule
being measured.
[0118] "Non-methoxy-terminated polymer" means a polymer that has at
least one terminus that ends with a moiety other than methoxy. In
some embodiments, the polymer has at least two termini that ends
with a moiety other than methoxy. In other embodiments, the polymer
has no termini that ends with methoxy. "Non-methoxy-terminated,
pluronic polymer" means a polymer other than a linear pluronic
polymer with methoxy at both termini. Polymeric nanoparticles as
provided herein can comprise non-methoxy-terminated polymers or
non-methoxy-terminated, pluronic polymers.
[0119] "Obtaining" means an act of acquiring a material(s) by any
means. The material may be acquired by producing it, purchasing it,
receiving it, etc. This term is intended to include "causing to
obtain". "Causing to obtain" means causing, urging, encouraging,
aiding, inducing or directing or acting in coordination with an
entity for the entity to obtain a material(s) as provided herein.
In some embodiments of any one of the methods provided herein, the
method may comprise or further comprise any one of the steps of
obtaining as described herein.
[0120] "Pharmaceutically acceptable excipient" or "pharmaceutically
acceptable carrier" means a pharmacologically inactive material
used together with a pharmacologically active material to formulate
the compositions. Pharmaceutically acceptable excipients comprise a
variety of materials known in the art, including but not limited to
saccharides (such as glucose, lactose, and the like), preservatives
such as antimicrobial agents, reconstitution aids, colorants,
saline (such as phosphate buffered saline), and buffers.
[0121] "Pre-existing immunity against the viral transfer vector"
refers to the presence of antibodies, T cells and/or B cells in a
subject, which cells have been previously primed by prior exposure
to antigens of the viral transfer vector or to crossreactive
antigens, including but not limited to other viruses. In some
embodiments, this pre-existing immunity is at a level that is
expected to result in anti-viral transfer vector immune response(s)
that interferes with the efficacy of the viral transfer vector. In
some embodiments, this pre-existing immunity is at a level that is
expected to result in anti-viral transfer vector immune response(s)
upon subsequent exposure to the viral transfer vector. Pre-existing
immunity can be assessed by determining the level of antibodies,
such as neutralizing antibodies, against a viral transfer vector
present in a sample, such as a blood sample, from the subject.
Assays for assessing the level of antibodies, such as neutralizing
antibodies, are described herein at least in the Examples and are
also known to those of ordinary skill in the art. Such an assay is
an ELISA assay. Pre-existing immunity can also be assessed by
determining antigen recall responses of immune cells, such as B or
T cells, stimulated in vivo or in vitro with viral transfer vector
antigens presented by APCs or viral antigen epitopes presented on
MHC class I or MHC class II molecules. Assays for antigen-specific
recall responses include, but are not limited to, ELISpot,
intracellular cytokine staining, cell proliferation, and cytokine
production assays. Generally, these and other assays are known to
those of ordinary skill in the art. In some embodiments, a subject
that does not exhibit pre-existing immunity against the viral
transfer vector is one with a level of anti-viral transfer vector
antibodies, such as neutralizing antibodies, or memory B or T cells
that would be considered to be negative. In other embodiments, a
subject that does not exhibit pre-existing immunity against the
viral transfer vector is one with a level of an anti-viral transfer
vector response that is no more than 3 standard deviations above a
mean negative control.
[0122] "Producing" refers to any action that results in a material
being made. An act of producing includes preparing the material or
processing it in some manner. In some embodiments, an act of
producing includes any act that makes that material available for
use by another. This term is intended to include "causing to
produce". "Causing to produce" means causing, urging, encouraging,
aiding, inducing or directing or acting in coordination with an
entity for the entity to make a material(s) as provided herein. In
some embodiments of any one of the methods provided herein, the
method may comprise or further comprise any one of the steps of
producing as described herein.
[0123] "Protocol" means a pattern of administering to a subject and
includes any dosing regimen of one or more substances to a subject.
Protocols are made up of elements (or variables); thus a protocol
comprises one or more elements. Such elements of the protocol can
comprise dosing amounts (doses), dosing frequency, routes of
administration, dosing duration, dosing rates, interval between
dosing, combinations of any of the foregoing, and the like. In some
embodiments, a protocol may be used to administer one or more
compositions of the invention to one or more test subjects. Immune
responses in these test subjects can then be assessed to determine
whether or not the protocol was effective in generating a desired
or desired level of an immune response or therapeutic effect. Any
therapeutic and/or immunologic effect may be assessed. One or more
of the elements of a protocol may have been previously demonstrated
in test subjects, such as non-human subjects, and then translated
into human protocols. For example, dosing amounts demonstrated in
non-human subjects can be scaled as an element of a human protocol
using established techniques such as alimetric scaling or other
scaling methods. Whether or not a protocol had a desired effect can
be determined using any of the methods provided herein or otherwise
known in the art. For example, a sample may be obtained from a
subject to which a composition provided herein has been
administered according to a specific protocol in order to determine
whether or not specific immune cells, cytokines, antibodies, etc.
were reduced, generated, activated, etc. An exemplary protocol is
one previously demonstrated to result in a tolerogenic immune
response against a viral transfer vector antigen or to achieve any
one of the beneficial results described herein. Useful methods for
detecting the presence and/or number of immune cells include, but
are not limited to, flow cytometric methods (e.g., FACS), ELISpot,
proliferation responses, cytokine production, and
immunohistochemistry methods. Antibodies and other binding agents
for specific staining of immune cell markers, are commercially
available. Such kits typically include staining reagents for
antigens that allow for FACS-based detection, separation and/or
quantitation of a desired cell population from a heterogeneous
population of cells. In embodiments, a composition as provided
herein is administered to a subject using one or more or all or
substantially all of the elements of which a protocol is comprised,
provided the selected element(s) are expected to achieve the
desired result in the subject. Such expectation may be based on
protocols determined in test subjects and scaling if needed. Any
one of the methods provided herein may comprise or further comprise
a step of administering a dose of the antigen-presenting cell
targeted immunosuppressant alone or in combination as described
herein with one or more doses of a viral transfer vector according
to a protocol that has been shown to attenuate an anti-viral
transfer vector immune response or allow for the repeated
administration of a viral transfer vector. Any one of the method
provided herein may comprise or further comprise determining such a
protocol that achieves any one of the beneficial results described
herein.
[0124] "Providing" means an action or set of actions that an
individual performs that supplies a material for practicing the
invention. Providing may include acts of producing, distributing,
selling, giving, making available, prescribing or administering the
material. The action or set of actions may be taken either directly
oneself or indirectly. Thus, this term is intended to include
"causing to provide". "Causing to provide" means causing, urging,
encouraging, aiding, inducing or directing or acting in
coordination with an entity for the entity to supply a material for
practicing of the present invention. In some embodiments of any one
of the methods provided herein, the method may comprise or further
comprise any one of the steps of providing as described herein.
[0125] "Repeat dose" or "repeat dosing" or the like means at least
one additional dose or dosing that is administered to a subject
subsequent to an earlier dose or dosing of the same material. For
example, a repeated dose of a viral transfer vector is at least one
additional dose of the viral transfer vector after a prior dose of
the same material. While the material may be the same, the amount
of the material in the repeated dose may be different from the
earlier dose. For example, in an embodiment of any one of the
methods or compositions provided herein, the amount of the viral
transfer vector in the repeated dose may be less than the amount of
the viral transfer vector of the earlier dose. Alternatively, in an
embodiment of any one of the methods or compositions provided
herein, the repeated dose may be in an amount that is at least
equal to the amount of the viral transfer vector in the earlier
dose. A repeat dose may be administered weeks, months or years
after the prior dose. In some embodiments of any one of the methods
provided herein, the repeat dose or dosing is administered at least
1 week after the dose or dosing that occurred just prior to the
repeat dose or dosing. Repeat dosing is considered to be
efficacious if it results in a beneficial effect for the subject.
Preferably, efficacious repeat dosing results in a beneficial
effect in conjunction with an attenuated anti-viral transfer vector
response.
[0126] "Selecting the doses of the viral transfer vector to be less
than" refers to the selection of the doses of the viral transfer
vector that is less than the amount of the viral transfer vector
that would be selected for administration to the subject if the
subject were to develop an anti-viral transfer vector immune
response to the viral transfer vector due to the repeated dosing of
the viral transfer vector. This term is intended to include
"causing to select". "Causing to select" means causing, urging,
encouraging, aiding, inducing or directing or acting in
coordination with an entity for the entity to select the
aforementioned lesser dosing. In some embodiments of any one of the
methods provided herein, the method may comprise or further
comprise any one of the steps of selecting as described herein.
[0127] "Simultaneous" means administration at the same time or
substantially at the same time where a clinician would consider any
time between administrations virtually nil or negligible as to the
impact on the desired therapeutic outcome. In some embodiments,
simultaneous means that the administrations occur with 5, 4, 3, 2,
1 or fewer minutes.
[0128] "Subject" means animals, including warm blooded mammals such
as humans and primates; avians; domestic household or farm animals
such as cats, dogs, sheep, goats, cattle, horses and pigs;
laboratory animals such as mice, rats and guinea pigs; fish;
reptiles; zoo and wild animals; and the like. As used herein, a
subject may be in one need of any one of the methods or
compositions provided herein.
[0129] "Synthetic nanocarrier(s)" means a discrete object that is
not found in nature, and that possesses at least one dimension that
is less than or equal to 5 microns in size. Albumin nanoparticles
are generally included as synthetic nanocarriers, however in
certain embodiments the synthetic nanocarriers do not comprise
albumin nanoparticles. In embodiments, synthetic nanocarriers do
not comprise chitosan. In other embodiments, synthetic nanocarriers
are not lipid-based nanoparticles. In further embodiments,
synthetic nanocarriers do not comprise a phospholipid.
[0130] A synthetic nanocarrier can be, but is not limited to, one
or a plurality of lipid-based nanoparticles (also referred to
herein as lipid nanoparticles, i.e., nanoparticles where the
majority of the material that makes up their structure are lipids),
polymeric nanoparticles, metallic nanoparticles, surfactant-based
emulsions, dendrimers, buckyballs, nanowires, virus-like particles
(i.e., particles that are primarily made up of viral structural
proteins but that are not infectious or have low infectivity),
peptide or protein-based particles (also referred to herein as
protein particles, i.e., particles where the majority of the
material that makes up their structure are peptides or proteins)
(such as albumin nanoparticles) and/or nanoparticles that are
developed using a combination of nanomaterials such as
lipid-polymer nanoparticles. Synthetic nanocarriers may be a
variety of different shapes, including but not limited to
spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and
the like. Synthetic nanocarriers according to the invention
comprise one or more surfaces. Exemplary synthetic nanocarriers
that can be adapted for use in the practice of the present
invention comprise: (1) the biodegradable nanoparticles disclosed
in U.S. Pat. No. 5,543,158 to Gref et al., (2) the polymeric
nanoparticles of Published US Patent Application 20060002852 to
Saltzman et al., (3) the lithographically constructed nanoparticles
of Published US Patent Application 20090028910 to DeSimone et al.,
(4) the disclosure of WO 2009/051837 to von Andrian et al., (5) the
nanoparticles disclosed in Published US Patent Application
2008/0145441 to Penades et al., (6) the protein nanoparticles
disclosed in Published US Patent Application 20090226525 to de los
Rios et al., (7) the virus-like particles disclosed in published US
Patent Application 20060222652 to Sebbel et al., (8) the nucleic
acid attached virus-like particles disclosed in published US Patent
Application 20060251677 to Bachmann et al., (9) the virus-like
particles disclosed in WO2010047839A1 or WO2009106999A2, (10) the
nanoprecipitated nanoparticles disclosed in P. Paolicelli et al.,
"Surface-modified PLGA-based Nanoparticles that can Efficiently
Associate and Deliver Virus-like Particles" Nanomedicine.
5(6):843-853 (2010), (11) apoptotic cells, apoptotic bodies or the
synthetic or semisynthetic mimics disclosed in U.S. Publication
2002/0086049, or (12) those of Look et al., Nanogel-based delivery
of mycophenolic acid ameliorates systemic lupus erythematosus in
mice" J. Clinical Investigation 123(4):1741-1749(2013).
[0131] Synthetic nanocarriers according to the invention that have
a minimum dimension of equal to or less than about 100 nm,
preferably equal to or less than 100 nm, do not comprise a surface
with hydroxyl groups that activate complement or alternatively
comprise a surface that consists essentially of moieties that are
not hydroxyl groups that activate complement. In a preferred
embodiment, synthetic nanocarriers according to the invention that
have a minimum dimension of equal to or less than about 100 nm,
preferably equal to or less than 100 nm, do not comprise a surface
that substantially activates complement or alternatively comprise a
surface that consists essentially of moieties that do not
substantially activate complement. In a more preferred embodiment,
synthetic nanocarriers according to the invention that have a
minimum dimension of equal to or less than about 100 nm, preferably
equal to or less than 100 nm, do not comprise a surface that
activates complement or alternatively comprise a surface that
consists essentially of moieties that do not activate complement.
In embodiments, synthetic nanocarriers exclude virus-like
particles. In embodiments, synthetic nanocarriers may possess an
aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or
greater than 1:10.
[0132] "Therapeutic protein" means any protein that may be used for
a therapeutic purpose. The therapeutic protein may be one used for
protein replacement or protein supplementation. Therapeutic
proteins include, but are not limited to, enzymes, enzyme
cofactors, hormones, blood clotting factors, cytokines, growth
factors, etc. Examples of other therapeutic proteins are provided
elsewhere herein.
[0133] "Transgene of the viral transfer vector" refers to the
nucleic acid material the viral transfer vector is used to
transport into a cell and, once in the cell, is expressed to
produce a protein or nucleic acid molecule, respectively, such as
for a therapeutic application as described herein. The transgene
may be a gene expression modulating transgene. "Expressed" or
"expression" or the like refers to the synthesis of a functional
(i.e., physiologically active for the desired purpose) gene product
after the transgene is transduced into a cell and processed by the
transduced cell. Such a gene product is also referred to herein as
a "transgene expression product". The expressed products are,
therefore, the resultant protein or nucleic acid, such as an
antisense oligonucleotide or a therapeutic RNA, encoded by the
transgene.
[0134] "Viral transfer vector" means a viral vector that has been
adapted to deliver a transgene as provided herein. "Viral vector"
refers to all of the viral components of a viral transfer vector
that delivers a transgene. Accordingly, "viral antigen" refers to
an antigen of the viral components of the viral transfer vector,
such as a capsid or coat protein, but not to the transgene or to
the product it encodes. "Viral transfer vector antigen" refers to
any antigen of the viral transfer vector including its viral
components as well as a protein transgene expression product. Viral
vectors are engineered to transduce one or more desired nucleic
acids into a cell. The transgene may be a gene expression
modulating transgene. In some embodiments, the transgene is one
that encodes a protein provided herein, such as a therapeutic
protein, a DNA-binding protein, etc. In other embodiments, the
transgene is one that encodes an antisense nucleic acid, snRNA, an
RNAi molecule (e.g., dsRNAs or ssRNAs), miRNA, or triplex-forming
oligonucleotides (TFOs), etc. Viral vectors can be based on,
without limitation, retroviruses (e.g., murine retrovirus, avian
retrovirus, Moloney murine leukemia virus (MoMuLV), Harvey murine
sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon
ape leukemia virus (GaLV) and Rous Sarcoma Virus (RSV)),
lentiviruses, herpes viruses, adenoviruses, adeno-associated
viruses, alphaviruses, etc. Other examples are provided elsewhere
herein or are known in the art. The viral vectors may be based on
natural variants, strains, or serotypes of viruses, such as any one
of those provided herein. The viral vectors may also be based on
viruses selected through molecular evolution. The viral vectors may
also be engineered vectors, recombinant vectors, mutant vectors, or
hybrid vectors. In some embodiments, the viral vector is a
"chimeric viral vector". In such embodiments, this means that the
viral vector is made up of viral components that are derived from
more than one virus or viral vector.
C. Compositions for Use in the Inventive Methods
[0135] As mentioned above, cellular and humoral immune responses
against the viral transfer vector can adversely effect the efficacy
of viral transfer vector therapeutics and can also interfere with
their readministration, making long-term treatment impossible for
many patients. As evidenced in the art, treatment with viral
transfer vectors would not be expected to be successful for some
patients due to prior exposure to a virus on which the viral
transfer is based. In addition, even if a patient did not have a
pre-existing immunity against a viral transfer vector, a single
administration of the viral transfer vector is likely to result in
cellular and humoral immune responses, such as neutralizing
antibody titers and/or the activation of memory T cells, that would
not allow for successful readministration. Further compounding
these issues is the long-term persistence of viral transfer vector
antigens.
[0136] Importantly, the methods and compositions provided herein
have been found to overcome the aforementioned obstacles by
attenuating immune responses against viral transfer vectors. The
methods and compositions provided herein have also been found to
allow for the readministration of viral transfer vectors and
provide for long-lasting tolerance against the viral transfer
vector without the need for long-term immunosuppression.
Accordingly, the methods and compositions provided herein are
useful for the treatment of subjects with a viral transfer vector.
Viral transfer vectors can be used to deliver transgenes for a
variety of purposes, including for gene expression modulation, the
methods and compositions provided herein are also so
applicable.
Subjects
[0137] The subject as provided herein may be one with any one of
the diseases or disorders as provided herein, and the transgene is
one that encodes a gene expression modulator that may be used to
control expression of any one of the proteins as provided herein.
The protein may be an extracellular, intracellular or
membrane-bound protein. In some embodiments, the subject has a
disease or disorder whereby the subject's endogenous version of the
protein is defective or produced in limited amounts or not at all,
and the gene expression modulator can control expression of such a
protein. Thus, the gene expression modulator can, in some
embodiments, control the expression of any one of the proteins as
provided herein, or an endogenous version thereof (such as an
endogenous version of a therapeutic protein as provided
herein).
[0138] Examples of therapeutic proteins include, but are not
limited to, infusible or injectable therapeutic proteins, enzymes,
enzyme cofactors, hormones, blood or blood coagulation factors,
cytokines and interferons, growth factors, adipokines, etc.
[0139] Examples of infusible or injectable therapeutic proteins
include, for example, Tocilizumab (Roche/Actemra.RTM.), alpha-1
antitrypsin (Kamada/AAT), Hematide.RTM. (Affymax and Takeda,
synthetic peptide), albinterferon alfa-2b (Novartis/Zalbin.TM.),
Rhucin.RTM. (Pharming Group, C1 inhibitor replacement therapy),
tesamorelin (Theratechnologies/Egrifta, synthetic growth
hormone-releasing factor), ocrelizumab (Genentech, Roche and
Biogen), belimumab (GlaxoSmithKline/Benlysta.RTM.), pegloticase
(Savient Pharmaceuticals/Krystexxa.TM.), taliglucerase alfa
(Protalix/Uplyso), agalsidase alfa (Shire/Replagal.RTM.), and
velaglucerase alfa (Shire).
[0140] Examples of enzymes include lysozyme, oxidoreductases,
transferases, hydrolases, lyases, isomerases, asparaginases,
uricases, glycosidases, proteases, nucleases, collagenases,
hyaluronidases, heparinases, heparanases, kinases, phosphatases,
lysins and ligases. Other examples of enzymes include those that
used for enzyme replacement therapy including, but not limited to,
imiglucerase (e.g., CEREZYME.TM.), a-galactosidase A (a-gal A)
(e.g., agalsidase beta, FABRYZYME.TM.), acid a-glucosidase (GAA)
(e.g., alglucosidase alfa, LUMIZYME.TM., MYOZYME.TM.), and
arylsulfatase B (e.g., laronidase, ALDURAZYME.TM., idursulfase,
ELAPRASE.TM., arylsulfatase B, NAGLAZYME.TM.).
[0141] Examples of hormones include Melatonin
(N-acetyl-5-methoxytryptamine), Serotonin, Thyroxine (or
tetraiodothyronine) (a thyroid hormone), Triiodothyronine (a
thyroid hormone), Epinephrine (or adrenaline), Norepinephrine (or
noradrenaline), Dopamine (or prolactin inhibiting hormone),
Antimullerian hormone (or mullerian inhibiting factor or hormone),
Adiponectin, Adrenocorticotropic hormone (or corticotropin),
Angiotensinogen and angiotensin, Antidiuretic hormone (or
vasopressin, arginine vasopressin), Atrial-natriuretic peptide (or
atriopeptin), Calcitonin, Cholecystokinin, Corticotropin-releasing
hormone, Erythropoietin, Follicle-stimulating hormone, Gastrin,
Ghrelin, Glucagon, Glucagon-like peptide (GLP-1), GIP,
Gonadotropin-releasing hormone, Growth hormone-releasing hormone,
Human chorionic gonadotropin, Human placental lactogen, Growth
hormone, Inhibin, Insulin, Insulin-like growth factor (or
somatomedin), Leptin, Luteinizing hormone, Melanocyte stimulating
hormone, Orexin, Oxytocin, Parathyroid hormone, Prolactin, Relaxin,
Secretin, Somatostatin, Thrombopoietin, Thyroid-stimulating hormone
(or thyrotropin), Thyrotropin-releasing hormone, Cortisol,
Aldosterone, Testosterone, Dehydroepiandrosterone, Androstenedione,
Dihydrotestosterone, Estradiol, Estrone, Estriol, Progesterone,
Calcitriol (1,25-dihydroxyvitamin D3), Calcidiol (25-hydroxyvitamin
D3), Prostaglandins, Leukotrienes, Prostacyclin, Thromboxane,
Prolactin releasing hormone, Lipotropin, Brain natriuretic peptide,
Neuropeptide Y, Histamine, Endothelin, Pancreatic polypeptide,
Renin, and Enkephalin.
[0142] Examples of blood or blood coagulation factors include
Factor I (fibrinogen), Factor II (prothrombin), tissue factor,
Factor V (proaccelerin, labile factor), Factor VII (stable factor,
proconvertin), Factor VIII (antihemophilic globulin), Factor IX
(Christmas factor or plasma thromboplastin component), Factor X
(Stuart-Prower factor), Factor Xa, Factor XI, Factor XII (Hageman
factor), Factor XIII (fibrin-stabilizing factor), von Willebrand
factor, von Heldebrant Factor, prekallikrein (Fletcher factor),
high-molecular weight kininogen (HMWK) (Fitzgerald factor),
fibronectin, fibrin, thrombin, antithrombin, such as antithrombin
III, heparin cofactor II, protein C, protein S, protein Z, protein
Z-related protease inhibitot (ZPI), plasminogen, alpha
2-antiplasmin, tissue plasminogen activator (tPA), urokinase,
plasminogen activator inhibitor-1 (PAI1), plasminogen activator
inhibitor-2 (PAI2), cancer procoagulant, and epoetin alfa (Epogen,
Procrit).
[0143] Examples of cytokines include lymphokines, interleukins, and
chemokines, type 1 cytokines, such as IFN-.gamma., TGF-.beta., and
type 2 cytokines, such as IL-4, IL-10, and IL-13.
[0144] Examples of growth factors include Adrenomedullin (AM),
Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic
proteins (BMPs), Brain-derived neurotrophic factor (BDNF),
Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast
growth factor (FGF), Glial cell line-derived neurotrophic factor
(GDNF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte
macrophage colony-stimulating factor (GM-CSF), Growth
differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF),
Hepatoma-derived growth factor (HDGF), Insulin-like growth factor
(IGF), Migration-stimulating factor, Myostatin (GDF-8), Nerve
growth factor (NGF) and other neurotrophins, Platelet-derived
growth factor (PDGF), Thrombopoietin (TPO), Transforming growth
factor alpha(TGF-.alpha.), Transforming growth factor
beta(TGF-.beta.), Tumour necrosis factor-alpha(TNF-.alpha.),
Vascular endothelial growth factor (VEGF), Wnt Signaling Pathway,
placental growth factor (PlGF), [(Foetal Bovine Somatotrophin)]
(FBS), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7.
[0145] Examples of adipokines, include leptin and adiponectin.
[0146] Additional examples of proteins include, but are not limited
to, receptors, signaling proteins, cytoskeletal proteins, scaffold
proteins, transcription factors, structural proteins, membrane
proteins, cytosolic proteins, binding proteins, nuclear proteins,
secreted proteins, golgi proteins, endoplasmic reticulum proteins,
mitochondrial proteins, and vesicular proteins, etc.
[0147] Examples of diseases or disorders include, but are not
limited to, lysosomal storage diseases/disorders, such as
Santavuori-Haltia disease (Infantile Neuronal Ceroid Lipofuscinosis
Type 1), Jansky-Bielschowsky Disease (late infantile neuronal
ceroid lipofuscinosis, Type 2), Batten disease (juvenile neuronal
ceroid lipofuscinosis, Type 3), Kufs disease (neuronal ceroid
lipofuscinosis, Type 4), Von Gierke disease (glycogen storage
disease, Type Ia), glycogen storage disease, Type Ib, Pompe disease
(glycogen storage disease, Type II), Forbes or Cori disease
(glycogen storage disease, Type III), mucolipidosis II (I-Cell
disease), mucolipidosis III (Pseudo-Hurler polydystrophy),
mucolipdosis IV (sialolipidosis), cystinosis (adult nonnephropathic
type), cystinosis (infantile nephropathic type), cystinosis
(juvenile or adolescent nephropathic), Salla disease/infantile
sialic acid storage disorder, and saposin deficiencies; disorders
of lipid and sphingolipid degradation, such as GM1 gangliosidosis
(infantile, late infantile/juvenile, and adult/chronic), Tay-Sachs
disease, Sandhoff disease, GM2 gangliodisosis, Ab variant, Fabry
disease, Gaucher disease, Types I, II and III, metachromatic
leukidystrophy, Krabbe disease (early and late onset), Neimann-Pick
disease, Types A, B, C1, and C2, Farber disease, and Wolman disease
(cholesteryl esther storage disease); disorders of
mucopolysaccharide degradation, such as Hurler syndrome (MPSI),
Scheie syndrome (MPS IS), Hurler-Scheie syndrome (MPS IH/S), Hunter
syndrome (MPS II), Sanfillippo A syndrome (MPS IIIA), Sanfillippo B
syndrome (MPS IIIB), Sanfillippo C syndrome (MPS IIIC), Sanfillippo
D syndrome (MPS IIID), Morquio A syndrome (MPS IVA), Morquio B
syndrome (MPS IVB), Maroteaux-Lamy syndrome (MPS VI), and Sly
syndrome (MPS VII); disorders of glycoprotein degradation, such as
alpha mannosidosis, beta mannosidosis, fucosidosis,
asparylglucosaminuria, mucolipidosis I (sialidosis),
galactosialidosis, Schindler disease, and Schindler disease, Type
II/Kanzaki disease; and leukodystrophy diseases/disorders, such as
abetalipoproteinemia, neonatal adrenoleukodystrophy, Canavan
disease, cerebrotendinous xanthromatosis, Pelizaeus Merzbacher
disease, Tangier disease, Refum disease, infantile, and Refum
disease, classic.
[0148] Additional examples of such diseases/disorders of a subject
as provided herein include, but are not limited to, acid maltase
deficiency (e.g., Pompe disease, glycogenosis type 2, lysosomal
storage disease); carnitine deficiency; carnitine palmityl
transferase deficiency; debrancher enzyme deficiency (e.g., Cori or
Forbes disease, glycogenosis type 3); lactate dehydrogenase
deficiency (e.g., glycogenosis type 11); myoadenylate deaminase
deficiency; phosphofructokinase deficiency (e.g., Tarui disease,
glycogenosis type 7); phosphogylcerate kinase deficiency (e.g.,
glycogenosis type 9); phosphogylcerate mutase deficiency (e.g.,
glycogenosis type 10); phosphorylase deficiency (e.g., McArdle
disease, myophosphorylase deficiency, glycogenosis type 5);
Gaucher's Disease (e.g., chromosome 1, enzyme glucocerebrosidase
affected); Achondroplasia (e.g., chromosome 4, fibroblast growth
factor receptor 3 affected); Huntington's Disease (e.g., chromosome
4, huntingtin); Hemochromatosis (e.g., chromosome 6, HFE protein);
Cystic Fibrosis (e.g., chromosome 7, CFTR); Friedreich's Ataxia
(chromosome 9, frataxin); Best Disease (chromosome 11, VMD2);
Sickle Cell Disease (chromosome 11, hemoglobin); Phenylketoniuria
(chromosome 12, phenylalanine hydroxylase); Marfan's Syndrome
(chromosome 15, fibrillin); Myotonic Dystophy (chromosome 19,
dystophia myotonica protein kinase); Adrenoleukodystrophy
(x-chromosome, lignoceroyl-CoA ligase in peroxisomes); Duchene's
Muscular Dystrophy (x-chromosome, dystrophin); Rett Syndrome
(x-chromosome, methylCpG-binding protein 2); Leber's Hereditary
Optic Neuropathy (mitochondria, respiratory proteins); Mitochondria
Encephalopathy, Lactic Acidosis and Stroke (MELAS) (mitochondria,
transfer RNA); and Enzyme deficiencies of the Urea Cycle.
[0149] Still additional examples of such diseases or disorders
include, but are not limited to, Sickle Cell Anemia, Myotubular
Myopathy, Hemophilia B, Lipoprotein lipase deficiency, Ornithine
Transcarbamylase Deficiency, Crigler-Najjar Syndrome, Mucolipidosis
IV, Niemann-Pick A, Sanfilippo A, Sanfilippo B, Sanfilippo C,
Sanfilippo D, b-thalassaemia and Duchenne Muscular Dystrophy. Still
further examples of diseases or disorders include those that are
the result of defects in lipid and sphingolipid degradation,
mucopolysaccharide degradation, glycoprotein degradation,
leukodystrophies, etc.
[0150] It follows that therapeutic proteins also include
Myophosphorylase, glucocerebrosidase, fibroblast growth factor
receptor 3, huntingtin, HFE protein, CFTR, frataxin, VMD2,
hemoglobin, phenylalanine hydroxylase, fibrillin, dystophia
myotonica protein kinase, lignoceroyl-CoA ligase, dystrophin,
methylCpG-binding protein 2, Beta hemoglobin, Myotubularin,
Cathepsin A, Factor IX, Lipoprotein lipase, Beta galactosidase,
Ornithine Transcarbamylase, Iduronate-2-Sulfatase, Acid-Alpha
Glucosidase, UDP-glucuronosyltransferase 1-1,
GlcNAc-1-phosphotransferase, GlcNAc-1-phosphotransferase,
Mucolipin-1, Microsomal triglyceride transfer protein,
Sphingomyelinase, Acid ceramidase, Lysosomal acid lipase,
Alpha-L-iduronidase, Heparan N-sulfatase,
alpha-N-acetylglucosaminidase, acetyl-CoA alpha-glucosaminide
acetyltransferase, N-acetylglucosamine 6-sulfatase,
N-acetylgalactosamine-6 sulfatase, Alpha-mannosidase,
Alpha-galactosidase A, Cystic fibrosis conductance transmembrane
regulator, and respiratory proteins.
[0151] As further examples, the gene expression modulator may
control the expression of proteins associated with disorders of
lipid and sphingolipid degradation (e.g., .beta.-Galactosidase-1,
.beta.-Hexosaminidase A, .beta.-Hexosaminidases A and B, GM2
Activator Protein, 8-Galactosidase A, Glucocerebrosidase,
Glucocerebrosidase, Glucocerebrosidase, Arylsulfatase A,
Galactosylceramidase, Sphingomyelinase, Sphingomyelinase, NPC1, HE1
protein (Cholesterol Trafficking Defect), Acid Ceramidase,
Lysosomal Acid Lipase); disorders of mucopolysaccharide degradation
(e.g., L-Iduronidase, L-Iduronidase, L-Iduronidase, Iduronate
Sulfatase, Heparan N-Sulfatase, N-Acetylglucosaminidase,
Acetyl-CoA-Glucosaminidase, Acetyltransferase,
Acetylglucosamine-6-Sulfatase, Galactosamine-6-Sulfatase,
Arylsulfatase B, Glucuronidase); disorders of glycoprotein
degradation (e.g., Mannosidase, mannosidase, 1-fucosidase,
Aspartylglycosaminidase, Neuraminidase, Lysosomal protective
protein, Lysosomal 8-N-acetylgalactosaminidase, Lysosomal
8-N-acetylgalactosaminidase); lysosomal storage disorders (e.g.,
Palmitoyl-protein thioesterase, at least 4 subtypes, Lysosomal
membrane protein, Unknown, Glucose-6-phosphatase,
Glucose-6-phosphate translocase, Acid maltase, Debrancher enzyme
amylo-1,6 glucosidase, N-acetylglucosamine-1-phosphotransferase,
N-acetylglucosamine-1-phosphotransferase, Ganglioside sialidase
(neuraminidase), Lysosomal cystine transport protein, Lysosomal
cystine transport protein, Lysosomal cystine transport protein,
Sialic acid transport protein Saposins, A, B, C, D) and
leukodystrophies (e.g., Microsomal triglyceride transfer
protein/apolipoprotein B, Peroxisomal membrane transfer protein,
Peroxins, Aspartoacylase, Sterol-27-hydroxlase, Proteolipid
protein, ABC1 transporter, Peroxisome membrane protein 3 or
Peroxisome biogenesis factor 1, Phytanic acid oxidase).
[0152] The transgene of the viral transfer vectors as provided
herein is a gene expression modulating transgene. Such a transgene
encodes a gene expression modulator that can enhance, inhibit or
modulate the expression of one or more endogenous genes. The
endogenous gene may encode any one of the proteins as provided
herein provided the protein is an endogenous protein of the
subject. Accordingly, the subject may be one with any one of the
diseases or disorders provided herein where there would be a
benefit provided by gene expression modulation.
[0153] Gene expression modulators include DNA-binding proteins
(e.g., artificial transcription factors, such as those of U.S.
Publication No. 20140296129, the artificial transcription factors
of which are incorporated herein by reference; and transcriptional
silencer protein NRF of U.S. Publication No. 20030125286, the
transcriptional silencer protein NRF of which is incorporated
herein by reference) as well as therapeutic RNAs. Therapeutic RNAs
include, but are not limited to, inhibitors of mRNA translation
(antisense), agents of RNA interference (RNAi), catalytically
active RNA molecules (ribozymes), transfer RNA (tRNA) and RNAs that
bind proteins and other molecular ligands (aptamers). Gene
expression modulators are any agents of the foregoing and include
antisense nucleic acids, RNAi molecules (e.g., double-stranded RNAs
(dsRNAs), single-stranded RNAs (ssRNAs), micro RNAs (miRNAs), short
interfering RNAs (siRNAs), short hairpin RNAs (shRNAs)) and
triplex-forming oligonucleotides (TFOs). Gene expression modulators
also may include modified versions of any of the foregoing RNA
molecules and, thus, include modified mRNAs, such as synthetic
chemically modified RNAs.
[0154] The gene expression modulator may be an antisense nucleic
acid. Antisense nucleic acids can provide for the targeted
inhibition of gene expression (e.g., the expression of mutant
protein, a dominantly active gene product, a protein associated
with toxicity or gene products that are introduced into a cell by
an infectious agent, such as a virus). Thus, gene expression
modulating viral transfer vectors can be used for treating diseases
or disorders associated with dominant-negative or gain-of-function
pathogenetic mechanisms, cancer, or infection. The subject of any
one of the methods provided herein may be a subject that has a
viral infection, inflammatory disorder, cardiovascular disease,
cancer, genetic disorder or autoimmune disease. Antisense nucleic
acids may also interfere with mRNA splicing machinery and disrupt
normal cellular mRNA processing. Accordingly, the gene expression
modulating transgene may encode elements that interact with
spliceosome proteins. Examples of antisense nucleic acids (and
related constructs) can be found in, for example, U.S. Publication
Nos. 20050020529 and 20050271733, the antisense nucleic acids and
constructs of which are incorporated herein by reference.
[0155] The gene expression modulator may also be a ribozyme (i.e.,
a RNA molecule that can cleave other RNAs, such as single-stranded
RNA). Such molecules may be engineered to recognize specific
nucleotide sequences in a RNA molecule and cleave it (Cech, J.
Amer. Med. Assn., 260:3030, 1988). For example, ribozymes can be
engineered so that only mRNAs with sequences complementary to a
construct containing the ribozyme are inactivated. Types of
ribozymes and how to prepare related constructs are known in the
art (Hasselhoff, et al., Nature, 334:585, 1988; and U.S.
Publication No. 20050020529, the teachings of which pertaining to
such ribozymes and methods are incorporated herein by
reference).
[0156] The gene expression modulator may be an interfering RNA
(RNAi). RNA interference refers to the process of sequence-specific
post-transcriptional gene silencing mediated by interfering RNAs.
Generally, the presence of dsRNA can trigger an RNAi response. RNAi
has been studied in a variety of systems. Fire et al., 1998,
Nature, 391, 806, RNAi in C. elegans; Bahramian and Zarbl, 1999,
Molecular and Cellular Biology, 19, 274-283 and Wianny and Goetz,
1999, Nature Cell Biol., 2, 70, RNAi mediated by dsRNA in mammalian
systems; Hammond et al., 2000, Nature, 404, 293, RNAi in Drosophila
cells; Elbashir et al., 2001, Nature, 411, 494, RNAi induced by
introduction of duplexes of synthetic 21-nucleotide RNAs in
cultured mammalian cells. Such work, along with others, has
provided guidance as to the length, structure, chemical
composition, and sequence that are helpful in the construction of
RNAi molecules in order to mediate RNAi activity. Various
publications provide examples of RNAi molecules that can be used as
gene expression modulators. Such publications include, U.S. Pat.
Nos. 8,993,530, 8,877,917, 8,293,719, 7,947,659, 7,919,473,
7,790,878, 7,737,265, 7,592,322; and U.S. Publication Nos.
20150197746, 20140350071, 20140315835, 20130156845 and 20100267805,
the teaching related to the types of RNAi molecules as well as
their production are incorporated herein by reference.
[0157] Aptamers can bind various protein targets and disrupt the
interactions of those proteins with other proteins. Accordingly,
the gene expression modulator may be an aptamer, and the gene
expression modulating transgene can encode such an aptamer.
Aptamers may be selected for their ability to prevent transcription
of a gene by specifically binding the DNA-binding sites of
regulatory proteins. PCT Publication Nos. WO 98/29430 and WO
00/20040 provides examples of aptamers that were used to modulate
gene expression; and U.S. Publication No. 20060128649 also provide
examples of such aptamers, the aptamers of each of which are
incorporated herein by reference.
[0158] As a further example, the gene expression modulatory may be
a triplex oligomer. Such a molecule can stall transcription.
Generally, this is known as the triplex strategy as the oligomer
winds around double-helical DNA, forming a three-strand helix. Such
molecules can be designed to recognize a unique site on a chosen
gene (Maher, et al., Antisense Res. and Dev., 1(3):227, 1991;
Helene, C., Anticancer Drug Design, 6(6):569, 1991).
[0159] The sequence of a transgene may also include an expression
control sequence. Expression control DNA sequences include
promoters, enhancers, and operators, and are generally selected
based on the expression systems in which the expression construct
is to be utilized. In some embodiments, promoter and enhancer
sequences are selected for the ability to increase gene expression,
while operator sequences may be selected for the ability to
regulate gene expression. The transgene may also include sequences
that facilitate, and preferably promote, homologous recombination
in a host cell. The transgene may also include sequences that are
necessary for replication in a host cell.
[0160] Exemplary expression control sequences include promoter
sequences, e.g., cytomegalovirus promoter; Rous sarcoma virus
promoter; and simian virus 40 promoter; as well as any other types
of promoters that are disclosed elsewhere herein or are otherwise
known in the art. Generally, promoters are operatively linked
upstream (i.e., 5') of the sequence coding for a desired expression
product. The transgene also may include a suitable polyadenylation
sequence (e.g., the SV40 or human growth hormone gene
polyadenylation sequence) operably linked downstream (i.e., 3') of
the coding sequence.
Viral Vectors
[0161] Viruses have evolved specialized mechanisms to transport
their genomes inside the cells that they infect; viral vectors
based on such viruses can be tailored to transduce cells to
specific applications. Examples of viral vectors that may be used
as provided herein are known in the art or described herein.
Suitable viral vectors include, for instance, retroviral vectors,
lentiviral vectors, herpes simplex virus (HSV)-based vectors,
adenovirus-based vectors, adeno-associated virus (AAV)-based
vectors, and AAV-adenoviral chimeric vectors.
[0162] The viral transfer vectors provided herein may be based on a
retrovirus. Retrovirus is a single-stranded positive sense RNA
virus capable of infecting a wide variety of host cells. Upon
infection, the retroviral genome integrates into the genome of its
host cell, using its own reverse transcriptase enzyme to produce
DNA from its RNA genome. The viral DNA is then replicated along
with host cell DNA, which translates and transcribes the viral and
host genes. A retroviral vector can be manipulated to render the
virus replication-incompetent. As such, retroviral vectors are
thought to be particularly useful for stable gene transfer in vivo.
Examples of retroviral vectors can be found, for example, in U.S.
Publication Nos. 20120009161, 20090118212, and 20090017543, the
viral vectors and methods of their making being incorporated by
reference herein in their entirety.
[0163] Lentiviral vectors are examples of retroviral vectors that
can be used for the production of a viral transfer vector as
provided herein. Lentiviruses have the ability to infect
non-dividing cells, a property that constitute a more efficient
method of a gene delivery vector (see, e.g., Durand et al.,
Viruses. 2011 February; 3(2): 132-159). Examples of lentiviruses
include HIV (humans), simian immunodeficiency virus (SIV), feline
immunodeficiency virus (FIV), equine infectious anemia virus (EIAV)
and visna virus (ovine lentivirus). Unlike other retroviruses,
HIV-based vectors are known to incorporate their passenger genes
into non-dividing cells. Examples of lentiviral vectors can be
found, for example, in U.S. Publication Nos. 20150224209,
20150203870, 20140335607, 20140248306, 20090148936, and
20080254008, the viral vectors and methods of their making being
incorporated by reference herein in their entirety.
[0164] Herpes simplex virus (HSV)-based viral vectors are also
suitable for use as provided herein. Many replication-deficient HSV
vectors contain a deletion to remove one or more intermediate-early
genes to prevent replication. Advantages of the herpes vector are
its ability to enter a latent stage that can result in long-term
DNA expression, and its large viral DNA genome that can accommodate
exogenous DNA up to 25 kb. For a description of HSV-based vectors,
see, for example, U.S. Pat. Nos. 5,837,532, 5,846,782, 5,849,572,
and 5,804,413, and International Patent Applications WO 91/02788,
WO 96/04394, WO 98/15637, and WO 99/06583, the description of which
viral vectors and methods of their making being incorporated by
reference in its entirety.
[0165] Adenoviruses (Ads) are nonenveloped viruses that can
transfer DNA in vivo to a variety of different target cell types.
The virus can be made replication-deficient by deleting select
genes required for viral replication. The expendable
non-replication-essential E3 region is also frequently deleted to
allow additional room for a larger DNA insert. Viral transfer
vectors can be based on adenoviruses. Adenoviral transfer vectors
can be produced in high titers and can efficiently transfer DNA to
replicating and non-replicating cells. Unlike lentivirus,
adenoviral DNA does not integrate into the genome and therefore is
not replicated during cell division, instead they replicate in the
nucleus of the host cell using the host's replication
machinery.
[0166] The adenovirus on which a viral transfer vector may be based
may be from any origin, any subgroup, any subtype, mixture of
subtypes, or any serotype. For instance, an adenovirus can be of
subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g.,
serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g.,
serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10,
13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-48), subgroup E
(e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), an
unclassified serogroup (e.g., serotypes 49 and 51), or any other
adenoviral serotype. Adenoviral serotypes 1 through 51 are
available from the American Type Culture Collection (ATCC,
Manassas, Va.). Non-group C adenoviruses, and even non-human
adenoviruses, can be used to prepare replication-deficient
adenoviral vectors. Non-group C adenoviral vectors, methods of
producing non-group C adenoviral vectors, and methods of using
non-group C adenoviral vectors are disclosed in, for example, U.S.
Pat. Nos. 5,801,030, 5,837,511, and 5,849,561, and International
Patent Applications WO 97/12986 and WO 98/53087. Any adenovirus,
even a chimeric adenovirus, can be used as the source of the viral
genome for an adenoviral vector. For example, a human adenovirus
can be used as the source of the viral genome for a
replication-deficient adenoviral vector. Further examples of
adenoviral vectors can be found in U.S. Publication Nos.
20150093831, 20140248305, 20120283318, 20100008889, 20090175897 and
20090088398, the description of which viral vectors and methods of
their making being incorporated by reference in its entirety.
[0167] The viral transfer vectors provided herein can also be based
on adeno-associated viruses (AAVs). AAV vectors have been of
particular interest for use in therapeutic applications such as
those described herein. AAV is a DNA virus, which is not known to
cause human disease. Generally, AAV requires co-infection with a
helper virus (e.g., an adenovirus or a herpes virus), or expression
of helper genes, for efficient replication. AAVs have the ability
to stably infect host cell genomes at specific sites, making them
more predictable than retroviruses; however, generally, the cloning
capacity of the vector is 4.9 kb. AAV vectors that have been used
in gene therapy applications generally have had approximately 96%
of the parental genome deleted, such that only the terminal repeats
(ITRs), which contain recognition signals for DNA replication and
packaging, remain. For a description of AAV-based vectors, see, for
example, U.S. Pat. Nos. 8,679,837, 8,637,255, 8,409,842, 7,803,622,
and 7,790,449, and U.S. Publication Nos. 20150065562, 20140155469,
20140037585, 20130096182, 20120100606, and 20070036757, the viral
vectors of which and methods or their making being incorporated
herein by reference in their entirety. The AAV vectors may be
recombinant AAV vectors. The AAV vectors may also be
self-complementary (sc) AAV vectors, which are described, for
example, in U.S. Patent Publications 2007/01110724 and
2004/0029106, and U.S. Pat. Nos. 7,465,583 and 7,186,699, the
vectors and methods of production of which are herein incorporated
by reference.
[0168] The adeno-associated virus on which a viral transfer vector
may be of any serotype or a mixture of serotypes. AAV serotypes
include AAV1, AAV 2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
AAV10, and AAV11. For example, when the viral transfer vector is
based on a mixture of serotypes, the viral transfer vector may
contain the capsid signal sequences taken from one AAV serotype
(for example selected from any one of AAV serotypes 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, and 11) and packaging sequences from a different
serotype (for example selected from any one of AAV serotypes 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, and 11). In some embodiments of any one of
the methods or compositions provided herein, therefore, the AAV
vector is an AAV 2/8 vector. In other embodiments of any one of the
methods or compositions provided herein, the AAV vector is an AAV
2/5 vector.
[0169] The viral transfer vectors provided herein may also be based
on an alphavirus. Alphaviruses include Sindbis (and VEEV) virus,
Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus,
Cabassou virus, Chikungunya virus, Eastern equine encephalitis
virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands
J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg
virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus,
O'nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River
virus, Salmon pancreas disease virus, Semliki Forest virus,
Southern elephant seal virus, Tonate virus, Trocara virus, Una
virus, Venezuelan equine encephalitis virus, Western equine
encephalitis virus, and Whataroa virus. Generally, the genome of
such viruses encode nonstructural (e.g., replicon) and structural
proteins (e.g., capsid and envelope) that can be translated in the
cytoplasm of the host cell. Ross River virus, Sindbis virus,
Semliki Forest virus (SFV), and Venezuelan equine encephalitis
virus (VEEV) have all been used to develop viral transfer vectors
for transgene delivery. Pseudotyped viruses may be formed by
combining alphaviral envelope glycoproteins and retroviral capsids.
Examples of alphaviral vectors can be found in U.S. Publication
Nos. 20150050243, 20090305344, and 20060177819; the vectors and
methods of their making are incorporated herein by reference in
their entirety.
Antigen-Presenting Cell Targeted Immunosuppressants
[0170] Antigen-presenting cell targeted immunosuppressant can
include agents that by virtue of their form or characteristics can
result in APC tolerogenic effects. Antigen-presenting cell targeted
immunosuppressant also include agents that comprise a carrier to
which an immunosuppressant is conjugated.
[0171] Antigen-presenting cell targeted immunosuppressants include
negatively-charged particles, such as polystyrene, PLGA, or diamond
particles of a certain size and zeta potential, such as those
described in U.S. Publication No. 20150010631, the description of
such particles and methods of their production being incorporated
herein by reference. Such particles may have any particle shape or
conformation. However, in some embodiments it is preferred to use
particles that are less likely to clump in vivo. In one embodiment,
these particles have a spherical shape. Generally, it is not
necessary for such particles to be uniform in size, although such
particles must generally be of a size sufficient to trigger
phagocytosis in an antigen-presenting cell or other MPS cell.
Preferably, these particles are microscopic or nanoscopic in size,
in order to enhance solubility, avoid possible complications caused
by aggregation in vivo and to facilitate pinocytosis.
[0172] These particles may an average diameter of from about 0.1
.mu.m to about 10 .mu.m, about 0.2 .mu.m to about 2 .mu.m, about
0.3 .mu.m to about 5 .mu.m, or about 0.5 .mu.m to about 3 .mu.m. In
some embodiments, these particles may have an average diameter of
about 0.1 .mu.m, about 0.2 .mu.m, about 0.3 .mu.m, about 0.4 .mu.m,
about 0.5 .mu.m, about 1.0 .mu.m, about 1.5 .mu.m, about 2.0 .mu.m,
about 2.5 .mu.m, about 3.0 .mu.m, about 3.5 .mu.m, about 4.0 .mu.m,
about 4.5 .mu.m, or about 5.0 .mu.m. These particles need not be of
uniform diameter, and a pharmaceutical formulation may contain a
plurality of particles with a mixture of particle sizes.
[0173] In some embodiments, these particles are non-metallic. In
these embodiments, these particles may be formed from a polymer. In
a preferred embodiment, these particles are biodegradable. Examples
of suitable particles include polystyrene particles, PLGA
particles, PLURONICS stabilized polypropylene sulfide particles,
and diamond particles. Additionally, these particles can be formed
from a wide range of other materials. For example, these particles
may be composed of glass, silica, polyesters of hydroxy carboxylic
acids, polyanhydrides of dicarboxylic acids, or copolymers of
hydroxy carboxylic acids and dicarboxylic acids. More generally,
these particles may be composed of other materials as described in
U.S. Publication No. 20150010631.
[0174] The particles generally possess a particular zeta potential.
In certain embodiments, the zeta potential is negative. The zeta
potential may be less than about -100 mV or less than about -50 mV.
In certain embodiments, the particles possess a zeta potential
between -100 mV and 0 mV, between -75 mV and 0 mV, between -60 mV
and 0 mV, between -50 mV and 0 mV, between -40 mV and 0 mV, between
-30 mV and 0 mV, between -20 mV and +0 mV, between -10 mV and -0
mV, between -100 mV and -50 mV, between -75 mV and -50 mV, or
between -50 mV and -40 mV.
[0175] In another embodiment, these particles also comprise one or
more antigens as provided herein. In some of these embodiments, the
one or more antigens are encapsulated in the particles.
[0176] Another example of an antigen-presenting cell targeted
immunosuppressant is an immunosuppressants in nanocrystalline form,
whereby the form of the immunosuppressant itself is a particle or
particle-like. In these embodiments, such forms mimic a virus or
other foreign pathogen. Many drugs have been nanosized and
appropriate methods for producing such drug forms would be known to
one of ordinary skill in the art. Drug nanocrystals, such as
nanocrystalline rapamycin, are known to those of ordinary skill in
the art (Katteboinaa, et al. 2009, International Journal of
PharmTech Resesarch; Vol. 1, No. 3; pp 682-694. As used herein, a
"drug nanocrystal" refers to a form of a drug (e.g., an
immunosuppressant) that does not include a carrier or matrix
material. In some embodiments, drug nanocrystals comprise 90%, 95%,
98%, or 99% or more drug. Methods for producing drug nanocrystals
include, without limitation, milling, high pressure homogenization,
precipitation, spray drying, rapid expansion of supercritical
solution (RESS), Nanoedge.RTM. technology (Baxter Healthcare), and
Nanocrystal Technology.TM. (Elan Corporation). In some embodiments,
a surfactant or a stabilizer may be used for steric or
electrostatic stability of the drug nanocrystal. In some
embodiments, the nanocrystal or nanocrytalline form of an
immunosuppressant may be used to increase the solubility,
stability, and/or bioavailability of the immunosuppressant,
particularly immunosuppressants that are insoluble or labile.
[0177] Antigen-presenting call targeted immunosuppressants also may
be an apoptotic-body mimic and cause an associated antigen(s) to be
tolerized. Such mimics are described in U.S. Publication No.
20120076831, which mimics and methods of their making are
incorporated herein by reference. The apoptotic-body mimics may be
particles, beads, branched polymers, dendrimers, or liposomes.
Preferably the mimic is particulate, and generally spherical,
ellipsoidal, rod-shaped, globular, or polyhedral in shape.
Alternatively, however, the mimic may be of an irregular or
branched shape. In preferred embodiments, the mimic is composed of
material which is biodegradable. It is further preferred that the
mimic have a net neutral or negative charge, in order to reduce
non-specific binding to cell surfaces which, in general, bear a net
negative charge. Preferably the mimic surface is composed of a
material that minimizes non-specific or unwanted biological
interactions. When a particle, the mimic surface may be coated with
a material to prevent or decrease non-specific interactions. Steric
stabilization by coating particles with hydrophilic layers such as
poly(ethylene glycol) (PEG) and its copolymers such as PLURONICS
(including copolymers of poly(ethylene glycol)-bl-poly(propylene
glycol)-bl-poly(ethylene glycol)) may reduce the non-specific
interactions with proteins of the interstitium.
[0178] When particles, the mimics may be particles composed of
glass, silica, polyesters of hydroxy carboxylic acids,
polyanhydrides of dicarboxylic acids, or copolymers of hydroxy
carboxylic acids and dicarboxylic acids. These mimics may be
quantum dots, or composed of quantum dots, such as quantum dot
polystyrene particles. These mimics may comprise materials
including polyglycolic acid polymers (PGA), polylactic acid
polymers (PLA), polysebacic acid polymers (PSA),
poly(lactic-co-glycolic) acid copolymers (PLGA),
poly(lactic-co-sebacic) acid copolymers (PLSA),
poly(glycolic-co-sebacic) acid copolymers (PGSA), etc. The mimics
may also be polystyrene beads.
[0179] These mimics may comprise one or more antigens. The mimics
may be capable of being conjugated, either directly or indirectly,
to one or more antigens to which tolerance is desired. In some
instances, the mimic will have multiple binding sites in order to
have multiple copies of the antigen exposed and increase the
likelihood of a tolerogenic response. The mimic may have one
antigen on its surface or multiple different antigens on the
surface. Alternatively, however, the mimic may have a surface to
which conjugating moieties may be adsorbed without chemical bond
formation.
[0180] In some embodiments, the mimics may also comprise an
apoptotic signaling molecule, although this is not necessarily
required, such as with polystyrene beads. Apoptotic signaling
molecules include, but are not limited to, the apoptosis signaling
molecules described in U.S. Publication No. 20050113297, which
apoptosis signaling molecules are herein incorporated by reference.
Molecules suitable for use in these particles include molecules
that target phagocytes, which include macrophages, dendritic cells,
monocytes and neutrophils. Such molecules may be thrombospondins or
Annexin I.
[0181] Antigen-presenting cell targeted immunosuppressants may also
be erythrocyte-binding therapeutics, such as those described in
U.S. Publication No. 20120039989, which therapeutics and methods of
their making are incorporated herein by reference. As described,
peptides that specifically bind to erythrocytes (also known as red
blood cells) were discovered. These peptides bind specifically to
erythrocytes even in the presence of other factors present in blood
and can be used to create immunotolerance. Accordingly, an
erythrocyte-binding therapeutic comprises one or more antigens to
which tolerance is desired and an erythrocyte affinity ligand. The
one or more antigens may be viral transfer vector antigens as
described herein, such as one or more viral antigens (e.g., of one
or more viral capsid proteins). Also, the one or more antigens may
also be or include one or more antigens of an expressed transgene
as provided. The antigens may form a mixture to which tolerance is
desired.
[0182] Examples of peptides that specifically bind erythrocytes
include ERY1, ERY19, ERY59, ERY64, ERY123, ERY141 and ERY162. In
addition to peptides that bind erythrocytes, proteins, such as
antibodies, for example single chain antibodies, and antigen
binding fragments thereof may also be used as the affinity ligands.
The affinity ligands may also include nucleotide aptamer ligands
for erythrocyte surface components. Accordingly, aptamers can be
made and used in place of other erythrocyte affinity ligands. DNA
and RNA aptamers may be used to provide non-covalent erythrocyte
binding. Aptamers can be classified as DNA aptamers, RNA aptamers,
or peptide aptamers. Additionally, the affinity ligands may be a
fusion of two or more affinity ligands, such as erythrocyte-binding
peptides. Further, the components of the erythrocyte-binding
therapeutics may be associated with a carrier such as a
polymersome, a liposome or micelle or some types of nanoparticles.
In some embodiments, the components are encapsulated in such a
carrier. In some embodiments, the carrier comprises an affinity
ligand as described herein and one or more antigens. In such an
embodiment, the affinity ligand and one or more antigens do not
necessarily need to be conjugated to each other.
[0183] Antigen-presenting cell targeted immunosuppressants also
include any one of the immunosuppressants provided herein coupled
to a carrier that targets APCs. The carrier in some embodiments may
be an antibody or antigen binding fragment thereof (or some other
ligand) that is specific to an APC marker. Such markers include,
but are not limited to, CD1a (R4, T6, HTA-1); CD1b (R1); CD1c
(M241, R7); CD1d (R3); CD1e (R2); CD11b (.alpha.M Integrin chain,
CR3, Mo1, C3niR, Mac-1); CD11c (.alpha.X Integrin, p150, 95, AXb2);
CDw117 (Lactosylceramide, LacCer); CD19 (B4); CD33 (gp67); CD 35
(CR1, C3b/C4b receptor); CD 36 (GpIIIb, GPIV, PASIV); CD39
(ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD45 (LCA,
T200, B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); CD49d
(VLA-4.alpha., .alpha.4 Integrin); CD49e (VLA-5.alpha., .alpha.5
Integrin); CD58 (LFA-3); CD64 (Fc.gamma.RI); CD72 (Ly-19.2,
Ly-32.2, Lyb-2); CD73 (Ecto-5'nucloticlase); CD74 (Ii, invariant
chain); CD80 (B7, B7-1, BB1); CD81 (TAPA-1); CD83 (HB15); CD85a
(ILT5, LIR3, HL9); CD85d (ILT4, LIR2, MIR10); CD85j (ILT2, LIR1,
MIR7); CD85k (ILT3, LIR5, HM18); CD86 (B7-2/B70); CD88 (C5aB); CD97
(BL-KDD/F12); CD101 (IGSF2, P126, V7); CD116 (GM-CSFR.alpha.);
CD120a (TMFRI, p55); CD120b (TNFRII, p'75, TNFR p80); CD123
(IL-3R.alpha.); CD139; CD148 (HPTP-.eta., p260, DEP-1); CD150
(SLAM, IPO-3); CD156b (TACE, ADAM17, cSVP); CD157 (Mo5, BST-1);
CD167a (DDR1, trkE, cak); CD168 (RHAMM, IHABP, HMMR); CD169
(Sialoadhesin, Siglec-1); CD170 (Siglec-5); CD171 (L1CAM, NILE);
CD172 (SIRP-1.alpha., MyD-1); CD172b (SIRP.beta.); CD180 (RP105,
Bgp95, Ly64); CD184 (CXCR4, NPY3R); CD193 (CCR3); CD196 (CCR6);
CD197 (CCR7 (ws CDw197)); CDw197 (CCR7, EBI1, BLR2); CD200 (OX2);
CD205 (DEC-205); CD206 (MMR); CD207 (Langerin); CD208 (DC-LAMP);
CD209 (DC-SIGN); CDw218a (IL18R.alpha.); CDw218b (IL8R.beta.);
CD227 (MUC1, PUM, PEM, EMA); CD230 (Prion Protein (PrP)); CD252
(OX40L, TNF (ligand) superfamily, member 4); CD258 (LIGHT, TNF
(ligand) superfamily, member 14); CD265 (TRANCE-R, TNF-R
superfamily, member 11a); CD271 (NGFR, p75, TNFR superfamily,
member 16); CD273 (B7DC, PDL2); CD274 (B7H1, PDL1); CD275 (B7H2,
ICOSL); CD276 (B7H3); CD277 (BT3.1, B7 family: Butyrophilin 3);
CD283 (TLR3, TOLL-like receptor 3); CD289 (TLR9, TOLL-like receptor
9); CD295 (LEPR); CD298 (ATP1B3, Na K ATPase .beta.3 submit);
CD300a (CMRF-35H); CD300c (CMRF-35A); CD301 (MGL1, CLECSF14); CD302
(DCL1); CD303 (BDCA2); CD304 (BDCA4); CD312 (EMR2); CD317 (BST2);
CD319 (CRACC, SLAMF7); CD320 (8D6); and CD68 (gp110, Macrosialin);
class II MHC; BDCA-1; and Siglec-H. Methods for preparing
antibody-drug conjugates can be found in U.S. Publication No.
20150231241, which methods are herein incorporated by reference.
Other methods are known to those in the art.
[0184] The antigen-presenting cell targeted immunosuppressant may
also be synthetic nanocarriers that comprise any one of the
immunosuppressants as described herein. Such synthetic nanocarriers
include those of U.S. Publication No. 20100151000, the synthetic
nanocarriers of which, and methods of their making, are
incorporated herein by reference. As described, it was found that
tolerogenic responses can be generated in vivo by administering
particles (e.g., liposomes or polymeric particles) comprising both
a NF-.kappa.B inhibitor and an antigen. Accordingly, particles that
comprise an inhibitor of the NF-.kappa.B pathway and one or more
viral transfer vector antigens can be used as antigen-presenting
cell targeted immunosuppressants as provided herein. In some
embodiments, the particle is liposomal. In other embodiments, the
particle comprises a carrier particle, such as a metal particle
(e.g., a tungsten, gold, platinum or iridium particle). In still
other embodiments, the particle comprises a polymeric matrix or
carrier, illustrative examples of which include biocompatible
polymeric particles (e.g., particles fabricated with
poly(lactide-co-glycolide)). In still other embodiments, the
particle comprises a ceramic or inorganic matrix or carrier.
[0185] The inhibitor of the NF-.kappa.B pathway can decrease the
level or functional activity of a member of the NF-.kappa.B
pathway, and can be selected from BTK, LYN, BCR Ig.alpha., BCR
Ig.beta., Syk, Blnk, PLC.gamma.2, PKC.beta., DAG, CARMA1, BCL10,
MALT1, PI3K, PIPS, AKT, p38 MAPK, ERK, COT, IKK.alpha., IKK.beta.,
IKK.gamma., NIK, RelA/p65, P105/p50, c-Rel, RelB, p52, NIK, Leu13,
CD81, CD19, CD21 and its ligands in the complement and coagulation
cascade, TRAF6, ubiquitin ligase, Tab2, TAK1, NEMO, NOD2, RIP2,
Lck, fyn, Zap70, LAT, GRB2, SOS, CD3 zeta, Slp-76, GADS, ITK,
PLC.gamma.1, PKC.theta., ICOS, CD28, SHP2, SAP, SLAM and 2B4. In
some embodiments, the NF-.kappa.B pathway inhibitor decreases the
level or functional activity of any one or more of RelA/p65,
P105/p50, c-Rel, RelB or p52.
[0186] A wide variety of other synthetic nanocarriers can be used
according to the invention, and in some embodiments, coupled to an
immunosuppressant to provide still other antigen-presenting cell
targeted immunosuppressants. In some embodiments, synthetic
nanocarriers are spheres or spheroids. In some embodiments,
synthetic nanocarriers are flat or plate-shaped. In some
embodiments, synthetic nanocarriers are cubes or cubic. In some
embodiments, synthetic nanocarriers are ovals or ellipses. In some
embodiments, synthetic nanocarriers are cylinders, cones, or
pyramids.
[0187] In some embodiments, it is desirable to use a population of
synthetic nanocarriers that is relatively uniform in terms of size
or shape so that each synthetic nanocarrier has similar properties.
For example, at least 80%, at least 90%, or at least 95% of the
synthetic nanocarriers of any one of the compositions or methods
provided, based on the total number of synthetic nanocarriers, may
have a minimum dimension or maximum dimension that falls within 5%,
10%, or 20% of the average diameter or average dimension of the
synthetic nanocarriers.
[0188] Synthetic nanocarriers can be solid or hollow and can
comprise one or more layers. In some embodiments, each layer has a
unique composition and unique properties relative to the other
layer(s). To give but one example, synthetic nanocarriers may have
a core/shell structure, wherein the core is one layer (e.g. a
polymeric core) and the shell is a second layer (e.g. a lipid
bilayer or monolayer). Synthetic nanocarriers may comprise a
plurality of different layers.
[0189] In some embodiments, synthetic nanocarriers may optionally
comprise one or more lipids. In some embodiments, a synthetic
nanocarrier may comprise a liposome. In some embodiments, a
synthetic nanocarrier may comprise a lipid bilayer. In some
embodiments, a synthetic nanocarrier may comprise a lipid
monolayer. In some embodiments, a synthetic nanocarrier may
comprise a micelle. In some embodiments, a synthetic nanocarrier
may comprise a core comprising a polymeric matrix surrounded by a
lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In some
embodiments, a synthetic nanocarrier may comprise a non-polymeric
core (e.g., metal particle, quantum dot, ceramic particle, bone
particle, viral particle, proteins, nucleic acids, carbohydrates,
etc.) surrounded by a lipid layer (e.g., lipid bilayer, lipid
monolayer, etc.).
[0190] In other embodiments, synthetic nanocarriers may comprise
metal particles, quantum dots, ceramic particles, etc. In some
embodiments, a non-polymeric synthetic nanocarrier is an aggregate
of non-polymeric components, such as an aggregate of metal atoms
(e.g., gold atoms).
[0191] In some embodiments, synthetic nanocarriers may optionally
comprise one or more amphiphilic entities. In some embodiments, an
amphiphilic entity can promote the production of synthetic
nanocarriers with increased stability, improved uniformity, or
increased viscosity. In some embodiments, amphiphilic entities can
be associated with the interior surface of a lipid membrane (e.g.,
lipid bilayer, lipid monolayer, etc.). Many amphiphilic entities
known in the art are suitable for use in making synthetic
nanocarriers in accordance with the present invention. Such
amphiphilic entities include, but are not limited to,
phosphoglycerides; phosphatidylcholines; dipalmitoyl
phosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine
(DOPE); dioleyloxypropyltriethylammonium (DOTMA);
dioleoylphosphatidylcholine; cholesterol; cholesterol ester;
diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol
(DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol
(PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid,
such as palmitic acid or oleic acid; fatty acids; fatty acid
monoglycerides; fatty acid diglycerides; fatty acid amides;
sorbitan trioleate (Span.RTM.85) glycocholate; sorbitan monolaurate
(Span.RTM.20); polysorbate 20 (Tween.RTM.20); polysorbate 60
(Tween.RTM.60); polysorbate 65 (Tween.RTM.65); polysorbate 80
(Tween.RTM.80); polysorbate 85 (Tween.RTM.85); polyoxyethylene
monostearate; surfactin; a poloxomer; a sorbitan fatty acid ester
such as sorbitan trioleate; lecithin; lysolecithin;
phosphatidylserine; phosphatidylinositol; sphingomyelin;
phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic
acid; cerebrosides; dicetylphosphate;
dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine;
hexadecyl-amine; acetyl palmitate; glycerol ricinoleate; hexadecyl
sterate; isopropyl myristate; tyloxapol; poly(ethylene
glycol)5000-phosphatidylethanolamine; poly(ethylene
glycol)400-monostearate; phospholipids; synthetic and/or natural
detergents having high surfactant properties; deoxycholates;
cyclodextrins; chaotropic salts; ion pairing agents; and
combinations thereof. An amphiphilic entity component may be a
mixture of different amphiphilic entities. Those skilled in the art
will recognize that this is an exemplary, not comprehensive, list
of substances with surfactant activity. Any amphiphilic entity may
be used in the production of synthetic nanocarriers to be used in
accordance with the present invention.
[0192] In some embodiments, synthetic nanocarriers may optionally
comprise one or more carbohydrates. Carbohydrates may be natural or
synthetic. A carbohydrate may be a derivatized natural
carbohydrate. In certain embodiments, a carbohydrate comprises
monosaccharide or disaccharide, including but not limited to
glucose, fructose, galactose, ribose, lactose, sucrose, maltose,
trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid,
galactoronic acid, mannuronic acid, glucosamine, galatosamine, and
neuramic acid. In certain embodiments, a carbohydrate is a
polysaccharide, including but not limited to pullulan, cellulose,
microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC),
hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran,
glycogen, hydroxyethylstarch, carageenan, glycon, amylose,
chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,
starch, chitin, inulin, konjac, glucommannan, pustulan, heparin,
hyaluronic acid, curdlan, and xanthan. In embodiments, the
synthetic nanocarriers do not comprise (or specifically exclude)
carbohydrates, such as a polysaccharide. In certain embodiments,
the carbohydrate may comprise a carbohydrate derivative such as a
sugar alcohol, including but not limited to mannitol, sorbitol,
xylitol, erythritol, maltitol, and lactitol.
[0193] In some embodiments, synthetic nanocarriers can comprise one
or more polymers. In some embodiments, the synthetic nanocarriers
comprise one or more polymers that is a non-methoxy-terminated,
pluronic polymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of the
polymers that make up the synthetic nanocarriers are
non-methoxy-terminated, pluronic polymers. In some embodiments, all
of the polymers that make up the synthetic nanocarriers are
non-methoxy-terminated, pluronic polymers. In some embodiments, the
synthetic nanocarriers comprise one or more polymers that is a
non-methoxy-terminated polymer. In some embodiments, at least 1%,
2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight)
of the polymers that make up the synthetic nanocarriers are
non-methoxy-terminated polymers. In some embodiments, all of the
polymers that make up the synthetic nanocarriers are
non-methoxy-terminated polymers. In some embodiments, the synthetic
nanocarriers comprise one or more polymers that do not comprise
pluronic polymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of the
polymers that make up the synthetic nanocarriers do not comprise
pluronic polymer. In some embodiments, all of the polymers that
make up the synthetic nanocarriers do not comprise pluronic
polymer. In some embodiments, such a polymer can be surrounded by a
coating layer (e.g., liposome, lipid monolayer, micelle, etc.). In
some embodiments, elements of the synthetic nanocarriers can be
attached to the polymer.
[0194] Immunosuppressants can be coupled to the synthetic
nanocarriers by any of a number of methods. Generally, the
attaching can be a result of bonding between the immunosuppressants
and the synthetic nanocarriers. This bonding can result in the
immunosuppressants being attached to the surface of the synthetic
nanocarriers and/or contained (encapsulated) within the synthetic
nanocarriers. In some embodiments, however, the immunosuppressants
are encapsulated by the synthetic nanocarriers as a result of the
structure of the synthetic nanocarriers rather than bonding to the
synthetic nanocarriers. In preferable embodiments, the synthetic
nanocarrier comprises a polymer as provided herein, and the
immunosuppressants are attached to the polymer.
[0195] When attaching occurs as a result of bonding between the
immunosuppressants and synthetic nanocarriers, the attaching may
occur via a coupling moiety. A coupling moiety can be any moiety
through which an immunosuppressant is bonded to a synthetic
nanocarrier. Such moieties include covalent bonds, such as an amide
bond or ester bond, as well as separate molecules that bond
(covalently or non-covalently) the immunosuppressant to the
synthetic nanocarrier. Such molecules include linkers or polymers
or a unit thereof. For example, the coupling moiety can comprise a
charged polymer to which an immunosuppressant electrostatically
binds. As another example, the coupling moiety can comprise a
polymer or unit thereof to which it is covalently bonded.
[0196] In preferred embodiments, the synthetic nanocarriers
comprise a polymer as provided herein. These synthetic nanocarriers
can be completely polymeric or they can be a mix of polymers and
other materials.
[0197] In some embodiments, the polymers of a synthetic nanocarrier
associate to form a polymeric matrix. In some of these embodiments,
a component, such as an immunosuppressant, can be covalently
associated with one or more polymers of the polymeric matrix. In
some embodiments, covalent association is mediated by a linker. In
some embodiments, a component can be noncovalently associated with
one or more polymers of the polymeric matrix. For example, in some
embodiments, a component can be encapsulated within, surrounded by,
and/or dispersed throughout a polymeric matrix. Alternatively or
additionally, a component can be associated with one or more
polymers of a polymeric matrix by hydrophobic interactions, charge
interactions, van der Waals forces, etc. A wide variety of polymers
and methods for forming polymeric matrices therefrom are known
conventionally.
[0198] Polymers may be natural or unnatural (synthetic) polymers.
Polymers may be homopolymers or copolymers comprising two or more
monomers. In terms of sequence, copolymers may be random, block, or
comprise a combination of random and block sequences. Typically,
polymers in accordance with the present invention are organic
polymers.
[0199] In some embodiments, the polymer comprises a polyester,
polycarbonate, polyamide, or polyether, or unit thereof. In other
embodiments, the polymer comprises poly(ethylene glycol) (PEG),
polypropylene glycol, poly(lactic acid), poly(glycolic acid),
poly(lactic-co-glycolic acid), or a polycaprolactone, or unit
thereof. In some embodiments, it is preferred that the polymer is
biodegradable. Therefore, in these embodiments, it is preferred
that if the polymer comprises a polyether, such as poly(ethylene
glycol) or polypropylene glycol or unit thereof, the polymer
comprises a block-co-polymer of a polyether and a biodegradable
polymer such that the polymer is biodegradable. In other
embodiments, the polymer does not solely comprise a polyether or
unit thereof, such as poly(ethylene glycol) or polypropylene glycol
or unit thereof.
[0200] Other examples of polymers suitable for use in the present
invention include, but are not limited to polyethylenes,
polycarbonates (e.g. poly(1,3-dioxan-2one)), polyanhydrides (e.g.
poly(sebacic anhydride)), polypropylfumerates, polyamides (e.g.
polycaprolactam), polyacetals, polyethers, polyesters (e.g.,
polylactide, polyglycolide, polylactide-co-glycolide,
polycaprolactone, polyhydroxyacid (e.g.
poly(.beta.-hydroxyalkanoate))), poly(orthoesters),
polycyanoacrylates, polyvinyl alcohols, polyurethanes,
polyphosphazenes, polyacrylates, polymethacrylates, polyureas,
polystyrenes, and polyamines, polylysine, polylysine-PEG
copolymers, and poly(ethyleneimine), poly(ethylene imine)-PEG
copolymers.
[0201] In some embodiments, polymers in accordance with the present
invention include polymers which have been approved for use in
humans by the U.S. Food and Drug Administration (FDA) under 21
C.F.R. .sctn. 177.2600, including but not limited to polyesters
(e.g., polylactic acid, poly(lactic-co-glycolic acid),
polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2one));
polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g.,
polyethylene glycol); polyurethanes; polymethacrylates;
polyacrylates; and polycyanoacrylates.
[0202] In some embodiments, polymers can be hydrophilic. For
example, polymers may comprise anionic groups (e.g., phosphate
group, sulphate group, carboxylate group); cationic groups (e.g.,
quaternary amine group); or polar groups (e.g., hydroxyl group,
thiol group, amine group). In some embodiments, a synthetic
nanocarrier comprising a hydrophilic polymeric matrix generates a
hydrophilic environment within the synthetic nanocarrier. In some
embodiments, polymers can be hydrophobic. In some embodiments, a
synthetic nanocarrier comprising a hydrophobic polymeric matrix
generates a hydrophobic environment within the synthetic
nanocarrier. Selection of the hydrophilicity or hydrophobicity of
the polymer may have an impact on the nature of materials that are
incorporated within the synthetic nanocarrier.
[0203] In some embodiments, polymers may be modified with one or
more moieties and/or functional groups. A variety of moieties or
functional groups can be used in accordance with the present
invention. In some embodiments, polymers may be modified with
polyethylene glycol (PEG), with a carbohydrate, and/or with acyclic
polyacetals derived from polysaccharides (Papisov, 2001, ACS
Symposium Series, 786:301). Certain embodiments may be made using
the general teachings of U.S. Pat. No. 5,543,158 to Gref et al., or
WO publication WO2009/051837 by Von Andrian et al.
[0204] In some embodiments, polymers may be modified with a lipid
or fatty acid group. In some embodiments, a fatty acid group may be
one or more of butyric, caproic, caprylic, capric, lauric,
myristic, palmitic, stearic, arachidic, behenic, or lignoceric
acid. In some embodiments, a fatty acid group may be one or more of
palmitoleic, oleic, vaccenic, linoleic, alpha-linoleic,
gamma-linoleic, arachidonic, gadoleic, arachidonic,
eicosapentaenoic, docosahexaenoic, or erucic acid.
[0205] In some embodiments, polymers may be polyesters, including
copolymers comprising lactic acid and glycolic acid units, such as
poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide),
collectively referred to herein as "PLGA"; and homopolymers
comprising glycolic acid units, referred to herein as "PGA," and
lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid,
poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and
poly-D,L-lactide, collectively referred to herein as "PLA." In some
embodiments, exemplary polyesters include, for example,
polyhydroxyacids; PEG copolymers and copolymers of lactide and
glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG
copolymers, and derivatives thereof. In some embodiments,
polyesters include, for example, poly(caprolactone),
poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),
poly(serine ester), poly(4-hydroxy-L-proline ester),
poly[.alpha.-(4-aminobutyl)-L-glycolic acid], and derivatives
thereof.
[0206] In some embodiments, a polymer may be PLGA. PLGA is a
biocompatible and biodegradable co-polymer of lactic acid and
glycolic acid, and various forms of PLGA are characterized by the
ratio of lactic acid:glycolic acid. Lactic acid can be L-lactic
acid, D-lactic acid, or D,L-lactic acid. The degradation rate of
PLGA can be adjusted by altering the lactic acid:glycolic acid
ratio. In some embodiments, PLGA to be used in accordance with the
present invention is characterized by a lactic acid:glycolic acid
ratio of approximately 85:15, approximately 75:25, approximately
60:40, approximately 50:50, approximately 40:60, approximately
25:75, or approximately 15:85.
[0207] In some embodiments, polymers may be one or more acrylic
polymers. In certain embodiments, acrylic polymers include, for
example, acrylic acid and methacrylic acid copolymers, methyl
methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl
methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic
acid), poly(methacrylic acid), methacrylic acid alkylamide
copolymer, poly(methyl methacrylate), poly(methacrylic acid
anhydride), methyl methacrylate, polymethacrylate, poly(methyl
methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate
copolymer, glycidyl methacrylate copolymers, polycyanoacrylates,
and combinations comprising one or more of the foregoing polymers.
The acrylic polymer may comprise fully-polymerized copolymers of
acrylic and methacrylic acid esters with a low content of
quaternary ammonium groups.
[0208] In some embodiments, polymers can be cationic polymers. In
general, cationic polymers are able to condense and/or protect
negatively charged strands of nucleic acids. Amine-containing
polymers such as poly(lysine) (Zauner et al., 1998, Adv. Drug Del.
Rev., 30:97; and Kabanov et al., 1995, Bioconjugate Chem., 6:7),
poly(ethylene imine) (PEI; Boussif et al., 1995, Proc. Natl. Acad.
Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers
(Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA,
93:4897; Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler
et al., 1993, Bioconjugate Chem., 4:372) are positively-charged at
physiological pH, form ion pairs with nucleic acids. In
embodiments, the synthetic nanocarriers may not comprise (or may
exclude) cationic polymers.
[0209] In some embodiments, polymers can be degradable polyesters
bearing cationic side chains (Putnam et al., 1999, Macromolecules,
32:3658; Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon
et al., 1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am.
Chem. Soc., 121:5633; and Zhou et al., 1990, Macromolecules,
23:3399). Examples of these polyesters include
poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem.
Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,
Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam
et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am.
Chem. Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam
et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am.
Chem. Soc., 121:5633).
[0210] The properties of these and other polymers and methods for
preparing them are well known in the art (see, for example, U.S.
Pat. Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404;
6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600;
5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and U.S.
Pat. No. 4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123:9480;
Lim et al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Acc.
Chem. Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and
Uhrich et al., 1999, Chem. Rev., 99:3181). More generally, a
variety of methods for synthesizing certain suitable polymers are
described in Concise Encyclopedia of Polymer Science and Polymeric
Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980;
Principles of Polymerization by Odian, John Wiley & Sons,
Fourth Edition, 2004; Contemporary Polymer Chemistry by Allcock et
al., Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and
in U.S. Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and
6,818,732.
[0211] In some embodiments, polymers can be linear or branched
polymers. In some embodiments, polymers can be dendrimers. In some
embodiments, polymers can be substantially cross-linked to one
another. In some embodiments, polymers can be substantially free of
cross-links. In some embodiments, polymers can be used in
accordance with the present invention without undergoing a
cross-linking step. It is further to be understood that the
synthetic nanocarriers may comprise block copolymers, graft
copolymers, blends, mixtures, and/or adducts of any of the
foregoing and other polymers. Those skilled in the art will
recognize that the polymers listed herein represent an exemplary,
not comprehensive, list of polymers that can be of use in
accordance with the present invention.
[0212] In some embodiments, synthetic nanocarriers do not comprise
a polymeric component. In some embodiments, synthetic nanocarriers
may comprise metal particles, quantum dots, ceramic particles, etc.
In some embodiments, a non-polymeric synthetic nanocarrier is an
aggregate of non-polymeric components, such as an aggregate of
metal atoms (e.g., gold atoms).
[0213] Any immunosuppressant as provided herein can be, in some
embodiments, coupled to synthetic nanocarriers, antibodies or
antigen-binding fragments thereof (or other ligand that targets an
APC), erythrocyte-binding peptides, etc. Immunosuppressants
include, but are not limited to, statins; mTOR inhibitors, such as
rapamycin or a rapamycin analog; TGF-.beta. signaling agents;
TGF-.beta. receptor agonists; histone deacetylase (HDAC)
inhibitors; corticosteroids; inhibitors of mitochondrial function,
such as rotenone; P38 inhibitors; NF-.kappa..beta. inhibitors;
adenosine receptor agonists; prostaglandin E2 agonists;
phosphodiesterase inhibitors, such as phosphodiesterase 4
inhibitor; proteasome inhibitors; kinase inhibitors; G-protein
coupled receptor agonists; G-protein coupled receptor antagonists;
glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor
inhibitors; cytokine receptor activators; peroxisome
proliferator-activated receptor antagonists; peroxisome
proliferator-activated receptor agonists; histone deacetylase
inhibitors; calcineurin inhibitors; phosphatase inhibitors and
oxidized ATPs. Immunosuppressants also include IDO, vitamin D3,
cyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol,
azathiopurine, 6-mercaptopurine, aspirin, niflumic acid, estriol,
tripolide, interleukins (e.g., IL-1, IL-10), cyclosporine A, siRNAs
targeting cytokines or cytokine receptors and the like.
[0214] Examples of statins include atorvastatin (LIPITOR.RTM.,
TORVAST.RTM.), cerivastatin, fluvastatin (LESCOL.RTM., LESCOL.RTM.
XL), lovastatin (MEVACOR.RTM., ALTOCOR.RTM., ALTOPREV.RTM.),
mevastatin (COMPACTIN.RTM.), pitavastatin (LIVALO.RTM.,
PIAVA.RTM.), rosuvastatin (PRAVACHOL.RTM., SELEKTINE.RTM.,
LIPOSTAT.RTM.), rosuvastatin (CRESTOR.RTM.), and simvastatin
(ZOCOR.RTM., LIPEX.RTM.).
[0215] Examples of mTOR inhibitors include rapamycin and analogs
thereof (e.g., CCL-779, RAD001, AP23573, C20-methallylrapamycin
(C20-Marap), C16-(S)-butylsulfonamidorapamycin (C16-BSrap),
C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry
& Biology 2006, 13:99-107)), AZD8055, BEZ235 (NVP-BEZ235),
chrysophanic acid (chrysophanol), deforolimus (MK-8669), everolimus
(RAD0001), KU-0063794, PI-103, PP242, temsirolimus, and WYE-354
(available from Selleck, Houston, Tex., USA).
[0216] Examples of TGF-.beta. signaling agents include TGF-.beta.
ligands (e.g., activin A, GDF1, GDF11, bone morphogenic proteins,
nodal, TGF-.beta.s) and their receptors (e.g., ACVR1B, ACVR1C,
ACVR2A, ACVR2B, BMPR2, BMPR1A, BMPR1B, TGF.beta.RI, TGF.beta.RII),
R-SMADS/co-SMADS (e.g., SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD8),
and ligand inhibitors (e.g, follistatin, noggin, chordin, DAN,
lefty, LTBP1, THBS1, Decorin).
[0217] Examples of inhibitors of mitochondrial function include
atractyloside (dipotassium salt), bongkrekic acid (triammonium
salt), carbonyl cyanide m-chlorophenylhydrazone,
carboxyatractyloside (e.g., from Atractylis gummifera), CGP-37157,
(-)-Deguelin (e.g., from Mundulea sericea), F16, hexokinase II VDAC
binding domain peptide, oligomycin, rotenone, Ru360, SFK1, and
valinomycin (e.g., from Streptomyces fulvissimus) (EMD4Biosciences,
USA).
[0218] Examples of P38 inhibitors include SB-203580
(4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole)-
, SB-239063
(trans-1-(4hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxy-pyrimidin-4-y-
l) imidazole), SB-220025
(5-(2amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole))-
, and ARRY-797.
[0219] Examples of NF (e.g., NK-.kappa..beta.) inhibitors include
IFRD1, 2-(1,8-naphthyridin-2-yl)-Phenol, 5-aminosalicylic acid, BAY
11-7082, BAY 11-7085, CAPE (Caffeic Acid Phenethylester),
diethylmaleate, IKK-2 Inhibitor IV, IMD 0354, lactacystin, MG-132
[Z-Leu-Leu-Leu-CHO], NF.kappa.B Activation Inhibitor III,
NF-.kappa.B Activation Inhibitor II, JSH-23, parthenolide,
Phenylarsine Oxide (PAO), PPM-18, pyrrolidinedithiocarbamic acid
ammonium salt, QNZ, RO 106-9920, rocaglamide, rocaglamide AL,
rocaglamide C, rocaglamide I, rocaglamide J, rocaglaol, (R)-MG-132,
sodium salicylate, triptolide (PG490), and wedelolactone.
[0220] Examples of adenosine receptor agonists include CGS-21680
and ATL-146e.
[0221] Examples of prostaglandin E2 agonists include E-Prostanoid 2
and E-Prostanoid 4.
[0222] Examples of phosphodiesterase inhibitors (non-selective and
selective inhibitors) include caffeine, aminophylline, IBMX
(3-isobutyl-1-methylxanthine), paraxanthine, pentoxifylline,
theobromine, theophylline, methylated xanthines, vinpocetine, EHNA
(erythro-9-(2-hydroxy-3-nonyl)adenine), anagrelide, enoximone
(PERFAN.TM.), milrinone, levosimendon, mesembrine, ibudilast,
piclamilast, luteolin, drotaverine, roflumilast (DAXAS.TM.,
DALIRESP.TM.), sildenafil (REVATION.RTM., VIAGRA.RTM.), tadalafil
(ADCIRCA.RTM., CIALIS.RTM.), vardenafil (LEVITRA.RTM.,
STAXYN.RTM.), udenafil, avanafil, icariin, 4-methylpiperazine, and
pyrazolo pyrimidin-7-1.
[0223] Examples of proteasome inhibitors include bortezomib,
disulfiram, epigallocatechin-3-gallate, and salinosporamide A.
[0224] Examples of kinase inhibitors include bevacizumab, BIBW
2992, cetuximab (ERBITUX.RTM.), imatinib (GLEEVEC.RTM.),
trastuzumab (HERCEPTIN.RTM.), gefitinib (IRESSA.RTM.), ranibizumab
(LUCENTIS.RTM.), pegaptanib, sorafenib, dasatinib, sunitinib,
erlotinib, nilotinib, lapatinib, panitumumab, vandetanib, E7080,
pazopanib, and mubritinib.
[0225] Examples of glucocorticoids include hydrocortisone
(cortisol), cortisone acetate, prednisone, prednisolone,
methylprednisolone, dexamethasone, betamethasone, triamcinolone,
beclometasone, fludrocortisone acetate, deoxycorticosterone acetate
(DOCA), and aldosterone.
[0226] Examples of retinoids include retinol, retinal, tretinoin
(retinoic acid, RETIN-A.RTM.), isotretinoin (ACCUTANE.RTM.,
AMNESTEEM.RTM., CLARAVIS.RTM., SOTRET.RTM.), alitretinoin
(PANRETIN.RTM.), etretinate (TEGISON.TM.) and its metabolite
acitretin (SORIATANE.RTM.), tazarotene (TAZORAC.RTM., AVAGE.RTM.,
ZORAC.RTM.), bexarotene (TARGRETIN.RTM.), and adapalene
(DIFFERIN.RTM.).
[0227] Examples of cytokine inhibitors include IL1ra, IL1 receptor
antagonist, IGFBP, TNF-BF, uromodulin, Alpha-2-Macroglobulin,
Cyclosporin A, Pentamidine, and Pentoxifylline (PENTOPAK.RTM.,
PENTOXIL.RTM., TRENTAL.RTM.).
[0228] Examples of peroxisome proliferator-activated receptor
antagonists include GW9662, PPAR.gamma. antagonist III, G335, and
T0070907 (EMD4Biosciences, USA).
[0229] Examples of peroxisome proliferator-activated receptor
agonists include pioglitazone, ciglitazone, clofibrate, GW1929,
GW7647, L-165,041, LY 171883, PPAR.gamma. activator, Fmoc-Leu,
troglitazone, and WY-14643 (EMD4Biosciences, USA).
[0230] Examples of histone deacetylase inhibitors include
hydroxamic acids (or hydroxamates) such as trichostatin A, cyclic
tetrapeptides (such as trapoxin B) and depsipeptides, benzamides,
electrophilic ketones, aliphatic acid compounds such as
phenylbutyrate and valproic acid, hydroxamic acids such as
vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat
(LBH589), benzamides such as entinostat (MS-275), CI994, and
mocetinostat (MGCD0103), nicotinamide, derivatives of NAD,
dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes.
[0231] Examples of calcineurin inhibitors include cyclosporine,
pimecrolimus, voclosporin, and tacrolimus.
[0232] Examples of phosphatase inhibitors include BN82002
hydrochloride, CP-91149, calyculin A, cantharidic acid,
cantharidin, cypermethrin, ethyl-3,4-dephostatin, fostriecin sodium
salt, MAZ51, methyl-3,4-dephostatin, NSC 95397, norcantharidin,
okadaic acid ammonium salt from prorocentrum concavum, okadaic
acid, okadaic acid potassium salt, okadaic acid sodium salt,
phenylarsine oxide, various phosphatase inhibitor cocktails,
protein phosphatase 1C, protein phosphatase 2A inhibitor protein,
protein phosphatase 2A1, protein phosphatase 2A2, and sodium
orthovanadate.
[0233] Compositions according to the invention can comprise
pharmaceutically acceptable excipients, such as preservatives,
buffers, saline, or phosphate buffered saline. The compositions may
be made using conventional pharmaceutical manufacturing and
compounding techniques to arrive at useful dosage forms. In an
embodiment, compositions are suspended in sterile saline solution
for injection together with a preservative.
D. Methods of Using and Making the Compositions
[0234] Viral transfer vectors can be made with methods known to
those of ordinary skill in the art or as otherwise described
herein. For example, viral transfer vectors can be constructed
and/or purified using the methods set forth, for example, in U.S.
Pat. No. 4,797,368 and Laughlin et al., Gene, 23, 65-73 (1983).
[0235] As an example, replication-deficient adenoviral vectors can
be produced in complementing cell lines that provide gene functions
not present in the replication-deficient adenoviral vectors, but
required for viral propagation, at appropriate levels in order to
generate high titers of viral transfer vector stock. The
complementing cell line can complement for a deficiency in at least
one replication-essential gene function encoded by the early
regions, late regions, viral packaging regions, virus-associated
RNA regions, or combinations thereof, including all adenoviral
functions (e.g., to enable propagation of adenoviral amplicons).
Construction of complementing cell lines involve standard molecular
biology and cell culture techniques, such as those described by
Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d
edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989),
and Ausubel et al., Current Protocols in Molecular Biology, Greene
Publishing Associates and John Wiley & Sons, New York, N.Y.
(1994).
[0236] Complementing cell lines for producing adenoviral vectors
include, but are not limited to, 293 cells (described in, e.g.,
Graham et al., J. Gen. Virol., 36, 59-72 (1977)), PER.C6 cells
(described in, e.g., International Patent Application WO 97/00326,
and U.S. Pat. Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells
(described in, e.g., International Patent Application WO 95/34671
and Brough et al., J. Virol., 71, 9206-9213 (1997)). In some
instances, the complementing cell will not complement for all
required adenoviral gene functions. Helper viruses can be employed
to provide the gene functions in trans that are not encoded by the
cellular or adenoviral genomes to enable replication of the
adenoviral vector. Adenoviral vectors can be constructed,
propagated, and/or purified using the materials and methods set
forth, for example, in U.S. Pat. Nos. 5,965,358, 5,994,128,
6,033,908, 6,168,941, 6,329,200, 6,383,795, 6,440,728, 6,447,995,
and 6,475,757, U.S. Patent Application Publication No. 2002/0034735
A1, and International Patent Applications WO 98/53087, WO 98/56937,
WO 99/15686, WO 99/54441, WO 00/12765, WO 01/77304, and WO
02/29388, as well as the other references identified herein.
Non-group C adenoviral vectors, including adenoviral serotype 35
vectors, can be produced using the methods set forth in, for
example, U.S. Pat. Nos. 5,837,511 and 5,849,561, and International
Patent Applications WO 97/12986 and WO 98/53087.
[0237] AAV vectors may be produced using recombinant methods.
Typically, the methods involve culturing a host cell which contains
a nucleic acid sequence encoding an AAV capsid protein or fragment
thereof; a functional rep gene; a recombinant AAV vector composed
of AAV inverted terminal repeats (ITRs) and a transgene; and
sufficient helper functions to permit packaging of the recombinant
AAV vector into the AAV capsid proteins. In some embodiments, the
viral transfer vector may comprise inverted terminal repeats (ITR)
of AAV serotypes selected from the group consisting of: AAV1, AAV2,
AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11 and variants
thereof.
[0238] The components to be cultured in the host cell to package a
rAAV vector in an AAV capsid may be provided to the host cell in
trans. Alternatively, any one or more of the required components
(e.g., recombinant AAV vector, rep sequences, cap sequences, and/or
helper functions) may be provided by a stable host cell which has
been engineered to contain one or more of the required components
using methods known to those of skill in the art. Most suitably,
such a stable host cell can contain the required component(s) under
the control of an inducible promoter. However, the required
component(s) may be under the control of a constitutive promoter.
The recombinant AAV vector, rep sequences, cap sequences, and
helper functions required for producing the rAAV of the invention
may be delivered to the packaging host cell using any appropriate
genetic element. The selected genetic element may be delivered by
any suitable method, including those described herein. The methods
used to construct any embodiment of this invention are known to
those with skill in nucleic acid manipulation and include genetic
engineering, recombinant engineering, and synthetic techniques.
See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly,
methods of generating rAAV virions are well known and the selection
of a suitable method is not a limitation on the present invention.
See, e.g., K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S.
Pat. No. 5,478,745.
[0239] In some embodiments, recombinant AAV vectors may be produced
using the triple transfection method (e.g., as described in detail
in U.S. Pat. No. 6,001,650, the contents of which relating to the
triple transfection method are incorporated herein by reference).
Typically, the recombinant AAVs are produced by transfecting a host
cell with a recombinant AAV vector (comprising a transgene) to be
packaged into AAV particles, an AAV helper function vector, and an
accessory function vector. Generally, an AAV helper function vector
encodes AAV helper function sequences (rep and cap), which function
in trans for productive AAV replication and encapsidation.
Preferably, the AAV helper function vector supports efficient AAV
vector production without generating any detectable wild-type AAV
virions (i.e., AAV virions containing functional rep and cap
genes). The accessory function vector can encode nucleotide
sequences for non-AAV derived viral and/or cellular functions upon
which AAV is dependent for replication. The accessory functions
include those functions required for AAV replication, including,
without limitation, those moieties involved in activation of AAV
gene transcription, stage specific AAV mRNA splicing, AAV DNA
replication, synthesis of cap expression products, and AAV capsid
assembly. Viral-based accessory functions can be derived from any
of the known helper viruses such as adenovirus, herpesvirus (other
than herpes simplex virus type-1), and vaccinia virus.
[0240] Lentiviral vectors may be produced using any of a number of
methods known in the art. Examples of lentiviral vectors and/or
methods of their production can be found, for example, in U.S.
Publication Nos. 20150224209, 20150203870, 20140335607,
20140248306, 20090148936, and 20080254008, such lentiviral vectors
and methods of production are incorporated herein by reference. As
an example, when the lentiviral vector is integration-incompetent,
the lentiviral genome further comprises an origin of replication
(ori), whose sequence is dependent on the nature of cells where the
lentiviral genome has to be expressed. Said origin of replication
may be from eukaryotic origin, preferably of mammalian origin, most
preferably of human origin. Since the lentiviral genome does not
integrate into the cell host genome (because of the defective
integrase), the lentiviral genome can be lost in cells undergoing
frequent cell divisions; this is particularly the case in immune
cells, such as B or T cells. The presence of an origin of
replication can be beneficial in some instances. Vector particles
may be produced after transfection of appropriate cells, such as
293 T cells, by said plasmids, or by other processes. In the cells
used for the expression of the lentiviral particles, all or some of
the plasmids may be used to stably express their coding
polynucleotides, or to transiently or semi-stably express their
coding polynucleotides.
[0241] Methods for producing other viral vectors as provided herein
are known in the art and may be similar to the exemplified methods
above. Moreover, viral vectors are available commercially.
[0242] In embodiments, when preparing certain antigen-presenting
cell targeted immunosuppressants, methods for attaching components
to, for example, erythrocyte-binding peptides, antibodies or
antigen-binding fragments thereof (or other ligand that targets an
APC), or synthetic nanocarriers may be useful.
[0243] In certain embodiments, the attaching can be a covalent
linker. In embodiments, immunosuppressants according to the
invention can be covalently attached to the external surface via a
1,2,3-triazole linker formed by the 1,3-dipolar cycloaddition
reaction of azido groups with immunosuppressant containing an
alkyne group or by the 1,3-dipolar cycloaddition reaction of
alkynes with immunosuppressants containing an azido group. Such
cycloaddition reactions are preferably performed in the presence of
a Cu(I) catalyst along with a suitable Cu(I)-ligand and a reducing
agent to reduce Cu(II) compound to catalytic active Cu(I) compound.
This Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) can also be
referred as the click reaction.
[0244] Additionally, covalent coupling may comprise a covalent
linker that comprises an amide linker, a disulfide linker, a
thioether linker, a hydrazone linker, a hydrazide linker, an imine
or oxime linker, an urea or thiourea linker, an amidine linker, an
amine linker, and a sulfonamide linker.
[0245] An amide linker is formed via an amide bond between an amine
on one component such as an immunosuppressant with the carboxylic
acid group of a second component such as the nanocarrier. The amide
bond in the linker can be made using any of the conventional amide
bond forming reactions with suitably protected amino acids and
activated carboxylic acid such N-hydroxysuccinimide-activated
ester.
[0246] A disulfide linker is made via the formation of a disulfide
(S--S) bond between two sulfur atoms of the form, for instance, of
R1-S--S--R2. A disulfide bond can be formed by thiol exchange of a
component containing thiol/mercaptan group (--SH) with another
activated thiol group or a component containing thiol/mercaptan
groups with a component containing activated thiol group.
[0247] A triazole linker, specifically a 1,2,3-triazole of the
form
##STR00001##
wherein R1 and R2 may be any chemical entities, is made by the
1,3-dipolar cycloaddition reaction of an azide attached to a first
component with a terminal alkyne attached to a second component
such as the immunosuppressant. The 1,3-dipolar cycloaddition
reaction is performed with or without a catalyst, preferably with
Cu(I)-catalyst, which links the two components through a
1,2,3-triazole function. This chemistry is described in detail by
Sharpless et al., Angew. Chem. Int. Ed. 41(14), 2596, (2002) and
Meldal, et al, Chem. Rev., 2008, 108(8), 2952-3015 and is often
referred to as a "click" reaction or CuAAC.
[0248] A thioether linker is made by the formation of a
sulfur-carbon (thioether) bond in the form, for instance, of
R1-S--R2. Thioether can be made by either alkylation of a
thiol/mercaptan (--SH) group on one component with an alkylating
group such as halide or epoxide on a second component. Thioether
linkers can also be formed by Michael addition of a thiol/mercaptan
group on one component to an electron-deficient alkene group on a
second component containing a maleimide group or vinyl sulfone
group as the Michael acceptor. In another way, thioether linkers
can be prepared by the radical thiol-ene reaction of a
thiol/mercaptan group on one component with an alkene group on a
second component.
[0249] A hydrazone linker is made by the reaction of a hydrazide
group on one component with an aldehyde/ketone group on the second
component.
[0250] A hydrazide linker is formed by the reaction of a hydrazine
group on one component with a carboxylic acid group on the second
component. Such reaction is generally performed using chemistry
similar to the formation of amide bond where the carboxylic acid is
activated with an activating reagent.
[0251] An imine or oxime linker is formed by the reaction of an
amine or N-alkoxyamine (or aminooxy) group on one component with an
aldehyde or ketone group on the second component.
[0252] An urea or thiourea linker is prepared by the reaction of an
amine group on one component with an isocyanate or thioisocyanate
group on the second component.
[0253] An amidine linker is prepared by the reaction of an amine
group on one component with an imidoester group on the second
component.
[0254] An amine linker is made by the alkylation reaction of an
amine group on one component with an alkylating group such as
halide, epoxide, or sulfonate ester group on the second component.
Alternatively, an amine linker can also be made by reductive
amination of an amine group on one component with an aldehyde or
ketone group on the second component with a suitable reducing
reagent such as sodium cyanoborohydride or sodium
triacetoxyborohydride.
[0255] A sulfonamide linker is made by the reaction of an amine
group on one component with a sulfonyl halide (such as sulfonyl
chloride) group on the second component.
[0256] A sulfone linker is made by Michael addition of a
nucleophile to a vinyl sulfone. Either the vinyl sulfone or the
nucleophile may be on the surface of the nanocarrier or attached to
a component.
[0257] The component can also be conjugated via non-covalent
conjugation methods. For example, a negative charged
immunosuppressant can be conjugated to a positive charged component
through electrostatic adsorption. A component containing a metal
ligand can also be conjugated to a metal complex via a metal-ligand
complex.
[0258] In embodiments, the component can be attached to a polymer,
for example polylactic acid-block-polyethylene glycol, prior to the
assembly of a synthetic nanocarrier or the synthetic nanocarrier
can be formed with reactive or activatible groups on its surface.
In the latter case, the component may be prepared with a group
which is compatible with the attachment chemistry that is presented
by the synthetic nanocarriers' surface. In other embodiments, a
peptide component can be attached to VLPs or liposomes using a
suitable linker. A linker is a compound or reagent that capable of
coupling two molecules together. In an embodiment, the linker can
be a homobifuntional or heterobifunctional reagent as described in
Hermanson 2008. For example, an VLP or liposome synthetic
nanocarrier containing a carboxylic group on the surface can be
treated with a homobifunctional linker, adipic dihydrazide (ADH),
in the presence of EDC to form the corresponding synthetic
nanocarrier with the ADH linker. The resulting ADH linked synthetic
nanocarrier is then conjugated with a peptide component containing
an acid group via the other end of the ADH linker on nanocarrier to
produce the corresponding VLP or liposome peptide conjugate.
[0259] In embodiments, a polymer containing an azide or alkyne
group, terminal to the polymer chain is prepared. This polymer is
then used to prepare a synthetic nanocarrier in such a manner that
a plurality of the alkyne or azide groups are positioned on the
surface of that nanocarrier. Alternatively, the synthetic
nanocarrier can be prepared by another route, and subsequently
functionalized with alkyne or azide groups. The component is
prepared with the presence of either an alkyne (if the polymer
contains an azide) or an azide (if the polymer contains an alkyne)
group. The component is then allowed to react with the nanocarrier
via the 1,3-dipolar cycloaddition reaction with or without a
catalyst which covalently attaches the component to the particle
through the 1,4-disubstituted 1,2,3-triazole linker.
[0260] If the component is a small molecule it may be of advantage
to attach the component to a polymer prior to the assembly of
synthetic nanocarriers. In embodiments, it may also be an advantage
to prepare the synthetic nanocarriers with surface groups that are
used to attach the component to the synthetic nanocarrier through
the use of these surface groups rather than attaching the component
to a polymer and then using this polymer conjugate in the
construction of synthetic nanocarriers.
[0261] For detailed descriptions of available conjugation methods,
see Hermanson G T "Bioconjugate Techniques", 2nd Edition Published
by Academic Press, Inc., 2008. In addition to covalent attachment
the component can be attached by adsorption to a pre-formed
synthetic nanocarrier or it can be attached by encapsulation during
the formation of the synthetic nanocarrier.
[0262] Synthetic nanocarriers may be prepared using a wide variety
of methods known in the art. For example, synthetic nanocarriers
can be formed by methods such as nanoprecipitation, flow focusing
using fluidic channels, spray drying, single and double emulsion
solvent evaporation, solvent extraction, phase separation, milling,
microemulsion procedures, microfabrication, nanofabrication,
sacrificial layers, simple and complex coacervation, and other
methods well known to those of ordinary skill in the art.
Alternatively or additionally, aqueous and organic solvent
syntheses for monodisperse semiconductor, conductive, magnetic,
organic, and other nanomaterials have been described (Pellegrino et
al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci.,
30:545; and Trindade et al., 2001, Chem. Mat., 13:3843). Additional
methods have been described in the literature (see, e.g., Doubrow,
Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy,"
CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control.
Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275;
and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755; U.S.
Pat. Nos. 5,578,325 and 6,007,845; P. Paolicelli et al.,
"Surface-modified PLGA-based Nanoparticles that can Efficiently
Associate and Deliver Virus-like Particles" Nanomedicine.
5(6):843-853 (2010)).
[0263] Materials may be encapsulated into synthetic nanocarriers as
desirable using a variety of methods including but not limited to
C. Astete et al., "Synthesis and characterization of PLGA
nanoparticles" J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp.
247-289 (2006); K. Avgoustakis "Pegylated Poly(Lactide) and
Poly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties
and Possible Applications in Drug Delivery" Current Drug Delivery
1:321-333 (2004); C. Reis et al., "Nanoencapsulation I. Methods for
preparation of drug-loaded polymeric nanoparticles" Nanomedicine
2:8-21 (2006); P. Paolicelli et al., "Surface-modified PLGA-based
Nanoparticles that can Efficiently Associate and Deliver Virus-like
Particles" Nanomedicine. 5(6):843-853 (2010). Other methods
suitable for encapsulating materials into synthetic nanocarriers
may be used, including without limitation methods disclosed in U.S.
Pat. No. 6,632,671 to Unger issued Oct. 14, 2003.
[0264] In certain embodiments, synthetic nanocarriers are prepared
by a nanoprecipitation process or spray drying. Conditions used in
preparing synthetic nanocarriers may be altered to yield particles
of a desired size or property (e.g., hydrophobicity,
hydrophilicity, external morphology, "stickiness," shape, etc.).
The method of preparing the synthetic nanocarriers and the
conditions (e.g., solvent, temperature, concentration, air flow
rate, etc.) used may depend on the materials to be attached to the
synthetic nanocarriers and/or the composition of the polymer
matrix.
[0265] If synthetic nanocarriers prepared by any of the above
methods have a size range outside of the desired range, synthetic
nanocarriers can be sized, for example, using a sieve.
[0266] Elements of the synthetic nanocarriers may be attached to
the overall synthetic nanocarrier, e.g., by one or more covalent
bonds, or may be attached by means of one or more linkers.
Additional methods of functionalizing synthetic nanocarriers may be
adapted from Published US Patent Application 2006/0002852 to
Saltzman et al., Published US Patent Application 2009/0028910 to
DeSimone et al., or Published International Patent Application
WO/2008/127532 A1 to Murthy et al.
[0267] Alternatively or additionally, synthetic nanocarriers can be
attached to components directly or indirectly via non-covalent
interactions. In non-covalent embodiments, the non-covalent
attaching is mediated by non-covalent interactions including but
not limited to charge interactions, affinity interactions, metal
coordination, physical adsorption, host-guest interactions,
hydrophobic interactions, TT stacking interactions, hydrogen
bonding interactions, van der Waals interactions, magnetic
interactions, electrostatic interactions, dipole-dipole
interactions, and/or combinations thereof. Such attachments may be
arranged to be on an external surface or an internal surface of a
synthetic nanocarrier. In embodiments, encapsulation and/or
absorption is a form of attaching.
[0268] Compositions provided herein may comprise inorganic or
organic buffers (e.g., sodium or potassium salts of phosphate,
carbonate, acetate, or citrate) and pH adjustment agents (e.g.,
hydrochloric acid, sodium or potassium hydroxide, salts of citrate
or acetate, amino acids and their salts) antioxidants (e.g.,
ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate
20, polysorbate 80, polyoxyethylene9-10 nonyl phenol, sodium
desoxycholate), solution and/or cryo/lyo stabilizers (e.g.,
sucrose, lactose, mannitol, trehalose), osmotic adjustment agents
(e.g., salts or sugars), antibacterial agents (e.g., benzoic acid,
phenol, gentamicin), antifoaming agents (e.g.,
polydimethylsilozone), preservatives (e.g., thimerosal,
2-phenoxyethanol, EDTA), polymeric stabilizers and
viscosity-adjustment agents (e.g., polyvinylpyrrolidone, poloxamer
488, carboxymethylcellulose) and co-solvents (e.g., glycerol,
polyethylene glycol, ethanol).
[0269] Compositions according to the invention may comprise
pharmaceutically acceptable excipients. The compositions may be
made using conventional pharmaceutical manufacturing and
compounding techniques to arrive at useful dosage forms. Techniques
suitable for use in practicing the present invention may be found
in Handbook of Industrial Mixing: Science and Practice, Edited by
Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004
John Wiley & Sons, Inc.; and Pharmaceutics: The Science of
Dosage Form Design, 2nd Ed. Edited by M. E. Auten, 2001, Churchill
Livingstone. In an embodiment, compositions are suspended in
sterile saline solution for injection with a preservative.
[0270] It is to be understood that the compositions of the
invention can be made in any suitable manner, and the invention is
in no way limited to compositions that can be produced using the
methods described herein. Selection of an appropriate method of
manufacture may require attention to the properties of the
particular moieties being associated.
[0271] In some embodiments, compositions are manufactured under
sterile conditions or are terminally sterilized. This can ensure
that resulting compositions are sterile and non-infectious, thus
improving safety when compared to non-sterile compositions. This
provides a valuable safety measure, especially when subjects
receiving the compositions have immune defects, are suffering from
infection, and/or are susceptible to infection.
[0272] Administration according to the present invention may be by
a variety of routes, including but not limited to subcutaneous,
intravenous, intramuscular and intraperitoneal routes. The
compositions referred to herein may be manufactured and prepared
for administration, in some embodiments concomitant administration,
using conventional methods.
[0273] The compositions of the invention can be administered in
effective amounts, such as the effective amounts described
elsewhere herein. In some embodiments, the antigen-presenting cell
targeted immunosuppressants and/or viral transfer vectors are
present in dosage forms in an amount effective to attenuate an
anti-viral transfer vector immune response or allow for
readministration of a viral transfer vector to a subject. In some
embodiments, the antigen-presenting cell targeted
immunosuppressants and/or viral transfer vectors are present in
dosage forms in an amount effective to escalate transgene
expression in a subject. In preferable embodiments, the
antigen-presenting cell targeted immunosuppressants and/or viral
transfer vectors are present in dosage forms in an amount effective
to reduce immune responses to the viral transfer vector, such as
when concomitantly administered to a subject. Dosage forms may be
administered at a variety of frequencies. In some embodiments,
repeated administration of antigen-presenting cell targeted
immunosuppressant with a viral transfer vector is undertaken.
[0274] Aspects of the invention relate to determining a protocol
for the methods of administration as provided herein. A protocol
can be determined by varying at least the frequency, dosage amount
of the viral transfer vector and antigen-presenting cell targeted
immunosuppressant and subsequently assessing a desired or undesired
immune response. A preferred protocol for practice of the invention
reduces an immune response against the viral transfer vector,
attenuates an anti-viral transfer vector response and/or escalates
transgene expression. The protocol comprises at least the frequency
of the administration and doses of the viral transfer vector and
antigen-presenting cell targeted immunosuppressant.
Examples
Example 1: Polymeric Nanocarrier Containing Polymer-Rapamycin
Conjugate (Prophetic)
[0275] Preparation of PLGA-Rapamycin Conjugate:
[0276] PLGA polymer with acid end group (7525 DLG1A, acid number
0.46 mmol/g, Lakeshore Biomaterials; 5 g, 2.3 mmol, 1.0 eq) is
dissolved in 30 mL of dichloromethane (DCM).
N,N-Dicyclohexylcarbodimide (1.2 eq, 2.8 mmol, 0.57 g) is added
followed by rapamycin (1.0 eq, 2.3 mmol, 2.1 g) and
4-dimethylaminopyridine (DMAP) (2.0 eq, 4.6 mmol, 0.56 g). The
mixture is stirred at rt for 2 days. The mixture is then filtered
to remove insoluble dicyclohexylurea. The filtrate is concentrated
to ca. 10 mL in volume and added to 100 mL of isopropyl alcohol
(IPA) to precipitate out the PLGA-rapamycin conjugate. The IPA
layer is removed and the polymer is then washed with 50 mL of IPA
and 50 mL of methyl t-butyl ether (MTBE). The polymer is then dried
under vacuum at 35 C for 2 days to give PLGA-rapamycin as a white
solid (ca. 6.5 g).
[0277] Nanocarrier containing PLGA-rapamycin is prepared as
follows:
[0278] Solutions for nanocarrier formation are prepared as
follows:
[0279] Solution 1: PLGA-rapamycin @ 100 mg/mL in methylene
chloride. The solution is prepared by dissolving PLGA-rapamycin in
pure methylene chloride. Solution 2: PLA-PEG @ 100 mg/mL in
methylene chloride. The solution is prepared by dissolving PLA-PEG
in pure methylene chloride. Solution 3: Polyvinyl alcohol @ 50
mg/mL in 100 mM pH 8 phosphate buffer.
[0280] A primary water-in-oil emulsion is prepared first. W1/01 is
prepared by combining solution 1 (0.75 mL), and solution 2 (0.25
mL) in a small pressure tube and sonicating at 50% amplitude for 40
seconds using a Branson Digital Sonifier 250. A secondary emulsion
(W1/O1/W2) is then prepared by combining solution 3 (3.0 mL) with
the primary W1/01 emulsion, vortexing for 10 s, and sonicating at
30% amplitude for 60 seconds using the Branson Digital Sonifier
250. The W1/O1/W2 emulsion is added to a beaker containing 70 mM pH
8 phosphate buffer solution (30 mL) and stirred at room temperature
for 2 hours to allow the methylene chloride to evaporate and for
the nanocarriers to form. A portion of the nanocarriers is washed
by transferring the nanocarrier suspension to a centrifuge tube and
centrifuging at 75,600.times.g and 4.degree. C. for 35 min,
removing the supernatant, and re-suspending the pellet in phosphate
buffered saline. The washing procedure is repeated, and the pellet
is re-suspended in phosphate buffered saline for a final
nanocarrier dispersion of about 10 mg/mL.
Example 2: Preparation of Gold Nanocarriers (AuNCs) Containing
Rapamycin (Prophetic)
[0281] Preparation of HS-PEG-Rapamycin:
[0282] A solution of PEG acid disulfide (1.0 eq), rapamycin
(2.0-2.5 eq), DCC (2.5 eq) and DMAP (3.0 eq) in dry DMF is stirred
at rt overnight. The insoluble dicyclohexylurea is removed by
filtration and the filtrate is added to isopropyl alcohol (IPA) to
precipitate out the PEG-disulfide-di-rapamycin ester and washed
with IPA and dried. The polymer is then treated with
tris(2-carboxyethyl)phosphine hydrochloride in DMF to reduce the
PEG disulfide to thiol PEG rapamycin ester (HS-PEG-rapamycin). The
resulting polymer is recovered by precipitation from IPA and dried
as previously described and analyzed by H NMR and GPC.
[0283] Formation of Gold NCs (AuNCs):
[0284] An aq. solution of 500 mL of 1 mM HAuCl4 is heated to reflux
for 10 min with vigorous stirring in a 1 L round-bottom flask
equipped with a condenser. A solution of 50 mL of 40 mM of
trisodium citrate is then rapidly added to the stirring solution.
The resulting deep wine red solution is kept at reflux for 25-30
min and the heat is withdrawn and the solution is cooled to room
temperature. The solution is then filtered through a 0.8 .mu.m
membrane filter to give the AuNCs solution. The AuNCs are
characterized using visible spectroscopy and transmission electron
microscopy. The AuNCs are ca. 20 nm diameter capped by citrate with
peak absorption at 520 nm.
[0285] AuNCs Conjugate with HS-PEG-Rapamycin:
[0286] A solution of 150 .mu.l of HS-PEG-rapamycin (10 .mu.M in 10
mM pH 9.0 carbonate buffer) is added to 1 mL of 20 nm diameter
citrate-capped gold nanocarriers (1.16 nM) to produce a molar ratio
of thiol to gold of 2500:1. The mixture is stirred at room
temperature under argon for 1 hour to allow complete exchange of
thiol with citrate on the gold nanocarriers. The AuNCs with
PEG-rapamycin on the surface is then purified by centrifuge at
12,000 g for 30 minutes. The supernatant is decanted and the pellet
containing AuNC-S-PEG-rapamycin is then pellet washed with
1.times.PBS buffer. The purified Gold-PEG-rapamycin nanocarriers
are then resuspend in suitable buffer for further analysis and
bioassays.
Example 3: Mesoporous Silica Nanoparticles with Attached Ibuprofen
(Prophetic)
[0287] Mesoporous SiO2 nanoparticle cores are created through a
sol-gel process. Hexadecyltrimethyl-ammonium bromide (CTAB) (0.5 g)
is dissolved in deionized water (500 mL), and then 2 M aqueous NaOH
solution (3.5 mL) is added to the CTAB solution. The solution is
stirred for 30 min, and then Tetraethoxysilane (TEOS) (2.5 mL) is
added to the solution. The resulting gel is stirred for 3 h at a
temperature of 80.degree. C. The white precipitate which forms is
captured by filtration, followed by washing with deionized water
and drying at room temperature. The remaining surfactant is then
extracted from the particles by suspension in an ethanolic solution
of HCl overnight. The particles are washed with ethanol,
centrifuged, and redispersed under ultrasonication. This wash
procedure is repeated two additional times.
[0288] The SiO2 nanoparticles are then functionalized with amino
groups using (3-aminopropyl)-triethoxysilane (APTMS). To do this,
the particles are suspended in ethanol (30 mL), and APTMS (50
.mu.L) is added to the suspension. The suspension is allowed to
stand at room temperature for 2 h and then is boiled for 4 h,
keeping the volume constant by periodically adding ethanol.
Remaining reactants are removed by five cycles of washing by
centrifugation and redispersing in pure ethanol.
[0289] In a separate reaction, 1-4 nm diameter gold seeds are
created. All water used in this reaction is first deionized and
then distilled from glass. Water (45.5 mL) is added to a 100 mL
round-bottom flask. While stirring, 0.2 M aqueous NaOH (1.5 mL) is
added, followed by a 1% aqueous solution of
tetrakis(hydroxymethyl)phosphonium chloride (THPC) (1.0 mL). Two
minutes after the addition of THPC solution, a 10 mg/mL aqueous
solution of chloroauric acid (2 mL), which has been aged at least
15 min, is added. The gold seeds are purified through dialysis
against water.
[0290] To form the core-shell nanocarriers, the
amino-functionalized SiO2 nanoparticles formed above are first
mixed with the gold seeds for 2 h at room temperature. The
gold-decorated SiO2 particles are collected through centrifugation
and mixed with an aqueous solution of chloroauric acid and
potassium bicarbonate to form the gold shell. The particles are
then washed by centrifugation and redispersed in water. Ibuprofen
is loaded by suspending the particles in a solution of sodium
ibuprofen (1 mg/L) for 72 h. Free ibuprofen is then washed from the
particles by centrifugation and redispersing in water.
Example 4: Liposomes Containing Cyclosporine A (Prophetic)
[0291] The liposomes are formed using thin film hydration.
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 .mu.mol),
cholesterol (32 .mu.mol), and cyclosporin A (6.4 .mu.mol) are
dissolved in pure chloroform (3 mL). This lipid solution is added
to a 50 mL round-bottom flask, and the solvent is evaporated on a
rotary evaporator at a temperature of 60.degree. C. The flask is
then flushed with nitrogen gas to remove remaining solvent.
Phosphate buffered saline (2 mL) and five glass beads are added to
the flask, and the lipid film is hydrated by shaking at 60.degree.
C. for 1 h to form a suspension. The suspension is transferred to a
small pressure tube and sonicated at 60.degree. C. for four cycles
of 30 s pulses with a 30 s delay between each pulse. The suspension
is then left undisturbed at room temperature for 2 h to allow for
complete hydration. The liposomes are washed by centrifugation
followed by resuspension in fresh phosphate buffered saline.
Example 5: Synthetic Nanocarriers Comprising Rapamycin
Materials
[0292] Rapamycin was purchased from TSZ CHEM (185 Wilson Street,
Framingham, Mass. 01702; Product Catalogue # R1017). PLGA with 76%
lactide and 24% glycolide content and an inherent viscosity of 0.69
dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin
Drive, Birmingham, Ala. 35211. Product Code 7525 DLG 7A.) PLA-PEG
block co-polymer with a PEG block of approximately 5,000 Da and PLA
block of approximately 40,000 Da was purchased from SurModics
Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211;
Product Code 100 DL mPEG 5000 5CE). Polyvinyl alcohol (85-89%
hydrolyzed) was purchased from EMD Chemicals (Product Number
1.41350.1001).
Method
[0293] Solutions were prepared as follows:
[0294] Solution 1: PLGA at 75 mg/mL and PLA-PEG at 25 mg/mL in
methylene chloride. The solution was prepared by dissolving PLGA
and PLA-PEG in pure methylene chloride.
[0295] Solution 2: Rapamycin at 100 mg/mL in methylene chloride.
The solution was prepared by dissolving rapamycin in pure methylene
chloride.
[0296] Solution 3: Polyvinyl alcohol at 50 mg/mL in 100 mM pH 8
phosphate buffer.
[0297] An oil-in-water emulsion was used to prepare the
nanocarriers. The O/W emulsion was prepared by combining solution 1
(1 mL), solution 2 (0.1 mL), and solution 3 (3 mL) in a small
pressure tube and sonicating at 30% amplitude for 60 seconds using
a Branson Digital Sonifier 250. The O/W emulsion was added to a
beaker containing 70 mM pH 8 phosphate buffer solution (30 mL) and
stirred at room temperature for 2 hours to allow the methylene
chloride to evaporate and for the nanocarriers to form. A portion
of the nanocarriers was washed by transferring the nanocarrier
suspension to a centrifuge tube and centrifuging at 75,000.times.g
and 4.degree. C. for 35 min, removing the supernatant, and
re-suspending the pellet in phosphate buffered saline. The washing
procedure was repeated, and the pellet was re-suspended in
phosphate buffered saline for a final nanocarrier dispersion of
about 10 mg/mL.
[0298] Nanocarrier size was determined by dynamic light scattering.
The amount rapamycin in the nanocarrier was determined by HPLC
analysis. The total dry-nanocarrier mass per mL of suspension was
determined by a gravimetric method.
TABLE-US-00001 Effective Diameter Rapamycin Content (nm) (% w/w)
227 6.4
Example 6: Synthetic Nanocarriers Comprising GSK1059615
Materials
[0299] GSK1059615 was purchased from MedChem Express (11 Deer Park
Drive, Suite 102D Monmouth Junction, N.J. 08852), product code
HY-12036. PLGA with a lactide:glycolide ratio of 1:1 and an
inherent viscosity of 0.24 dL/g was purchased from Lakeshore
Biomaterials (756 Tom Martin Drive, Birmingham, Ala. 35211),
product code 5050 DLG 2.5A. PLA-PEG-OMe block co-polymer with a
methyl ether terminated PEG block of approximately 5,000 Da and an
overall inherent viscosity of 0.26 DL/g was purchased from
Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, Ala.
35211; Product Code 100 DL mPEG 5000 5K-E). Cellgro phosphate
buffered saline 1.times.pH 7.4 (PBS 1.times.) was purchased from
Corning (9345 Discovery Blvd. Manassas, Va. 20109), product code
21-040-CV.
Method
[0300] Solutions were prepared as follows:
[0301] Solution 1: PLGA (125 mg), and PLA-PEG-OMe (125 mg), were
dissolved in 10 mL of acetone. Solution 2: GSK1059615 was prepared
at 10 mg in 1 mL of N-methyl-2-pyrrolidinone (NMP).
[0302] Nanocarriers were prepared by combining Solution 1 (4 mL)
and Solution 2 (0.25 mL) in a small glass pressure tube and adding
the mixture drop wise to a 250 mL round bottom flask containing 20
mL of ultra-pure water under stirring. The flask was mounted onto a
rotary evaporation device, and the acetone was removed under
reduced pressure. A portion of the nanocarriers was washed by
transferring the nanocarrier suspension to centrifuge tubes and
centrifuging at 75,600 rcf and 4.degree. C. for 50 minutes,
removing the supernatant, and re-suspending the pellet in PBS
1.times.. The washing procedure was repeated, and the pellet was
re-suspended in PBS 1.times. to achieve a nanocarrier suspension
having a nominal concentration of 10 mg/mL on a polymer basis. The
washed nanocarrier solution was then filtered using 1.2 .mu.m PES
membrane syringe filters from Pall, part number 4656. An identical
nanocarrier solution was prepared as above, and pooled with the
first after the filtration step. The homogenous suspension was
stored frozen at -20.degree. C.
[0303] Nanocarrier size was determined by dynamic light scattering.
The amount of GSK1059615 in the nanocarrier was determined by UV
absorption at 351 nm. The total dry-nanocarrier mass per mL of
suspension was determined by a gravimetric method.
TABLE-US-00002 Effective Diameter GSK1059615 Content (nm) (% w/w)
143 1.02
Example 7: Erythrocyte-Binding Therapeutic with a Viral Transfer
Vector Antigen (Prophetic)
[0304] An erythrocyte-binding therapeutic is prepared based on the
teachings of U.S. Publication No. 20120039989 and used as an
antigen-presenting cell targeted immunosuppressant. The
erythrocyte-binding therapeutic may comprise any one of ERY1,
ERY19, ERY59, ERY64, ERY123, ERY141 and ERY162 and any one of the
viral transfer vector antigens described herein, such as a viral
vector antigen, e.g., a capsid protein (or peptide antigen derived
therefrom), or a protein (or peptide antigen derived therefrom),
such as a therapeutic protein (or peptide antigen derived
therefrom) that is encoded by a transgene as described herein.
Example 8: Particles Containing an Inhibitor of the NF-kB Pathway
(Prophetic)
[0305] An antigen-presenting cell targeted immunosuppressant is
prepared according to the teachings of U.S. Publication No.
20100151000. The particle may be a liposome or polymeric particle
and comprises any one of the immunosuppressants provided herein or
any one of the inhibitors of the NF-kB pathway provided in U.S.
Publication No. 20100151000, which inhibitors are incorporated
herein by reference in their entirety. In addition, the liposome or
polymeric particle may further comprise any one of the viral
transfer vector antigens described herein, such as a viral vector
antigen, e.g., a capsid protein (or peptide antigen derived
therefrom), or a protein (or peptide antigen derived therefom),
such as a therapeutic protein (or peptide antigen derived
therefrom), that is encoded by a transgene as described herein.
Example 9: Adenoviral Transfer Vector with a Gene Therapy Transgene
(Prophetic)
[0306] An adenoviral transfer vector is generated according to the
methods provided in U.S. Patent Publication 2004/0005293. Such a
vector may comprise any one of the transgenes as provided herein.
For example, an Ad-AAT-hFVIII vector that expresses the human
B-domain deleted FVIII cDNA from the human alfa1-antitrypsin
promoter (AAT) is prepared. An HPRT stuffer fragment is employed to
optimize vector size and to avoid vector rearrangements (Parks R J,
Graham F L. A helper-dependent system for adenovirus vector
production helps define a lower limit for efficient DNA packaging.
J Virol. 1997, 71:3293-3298). The Cre66 packaging cell line is
used.
Example 10: Concomitant Administration of a Viral Transfer Vector
with Synthetic Nanocarriers Coupled to Immunosuppressant
(Prophetic)
[0307] The viral transfer vector of any one of the Examples, such
as Example 9, is administered concomitantly, such as on the same
day, as any one of the antigen-presenting cell targeted
immunosuppressants provided herein, such as in Examples 1-8 or 12,
to subjects recruited for a clinical trial. One or more immune
responses against the viral transfer vector is evaluated. The
level(s) of the one or more immune responses against the viral
transfer vector can be evaluated by comparison with the level(s) of
the one or more immune responses in the subjects, or another group
of subjects, administered the viral transfer vector in the absence
of the antigen-presenting cell targeted immunosuppressant, such as
when administered the viral transfer vector alone. In embodiments,
repeated concomitant administration is evaluated in a similar
manner.
[0308] In an application of the information established during such
trials, the viral transfer vector and antigen-presenting cell
targeted immunosuppressant can be administered concomitantly to
subjects in need of viral transfer vectors when such subjects are
expected to have an undesired immune response against the viral
transfer vector when not administered concomitantly with the
antigen-presenting cell targeted immunosuppressant. In a further
embodiment, a protocol using the information established during the
trials can be prepared to guide the concomitant dosing of the viral
transfer vector and synthetic nanocarriers of subjects in need of
treatment with a viral transfer vector and have or are expected to
have an undesired immune response against the viral transfer vector
without the benefit of the antigen-presenting cell targeted
immunosuppressant. The protocol so prepared can then be used to
treat subjects, particularly human subjects.
Example 11: Administration of a Viral Transfer Vector with a Gene
Therapy Transgene with Synthetic Nanocarriers Coupled to
Immunosuppressant
[0309] Two successive intravenous (i.v.) inoculations of
adeno-associated virus expressing recombinant green fluorescent
protein (AAV-GFP) led to higher GFP expression in liver cells in
vivo if nanocarrier-encapsulated immunosuppressant (NCs) was
co-injected at boost stage.
Experimental Methods
[0310] Male C57BL/6 mice were used (5 mice/group). Animals were
injected with 200 .mu.L of AAV-GFP or AAV-GFP+synthetic
nanocarriers comprising rapamycin (NCs) mixture once or twice over
a 21d interval at different iterations (see Table 1 below). At d33
after the first injection (=d12 after the second injection for
those groups that were injected twice) animals were sacrificed,
their livers treated with collagenase 4 (Worthington, Lakewood,
N.J.), meshed and total cell suspensions analyzed by FACS for GFP
expression. Briefly, tissue was initially perfused with collagenase
(100 U), incubated at 37.degree. C. (30 min), collagenase
supernatant removed, and quenched with 2% FBS. Tissue samples were
then cut into .about.2 mm squares, digested (collagenase, 400 U)
with repeated agitation, filtered (nylon mesh), spun down (1,500
rpm), and pellets re-suspended in ice-cold 2% FBS.
[0311] At day 14 after the first injections all animals were bled
and their serum analyzed for antibodies against AAV with ELISA as
follows. 96-well plates were coated with 50 .mu.L of AAV at
2.times.10.sup.9 vg/mL in carbonate buffer for 92 hours, and then
blocked for 2 hours with 300 .mu.L of casein. Samples were added at
a 1:40 dilution in 50 .mu.L of casein, and incubated for 2 hours at
RT. Rabbit Anti-mouse IgG (Jackson ImmunoResearch, West Grove, Pa.,
315-035-008) was used as a secondary antibody (0.5 .mu.g/mL, 1
hour) and then TMB substrate was added (10 min) followed by the
stop solution. Plates were then read at wavelength of 450 nm with a
subtraction of background at 570 nm. Mouse monoclonal anti-AAV8
antibody (Fitzgerald, Acton, Mass., 10R-2136) served as a positive
control.
Amounts of AAV-GFP:
[0312] 1.times.10.sup.10 viral genomes (VG) at d0 prime,
5.times.10.sup.10 VG at d21 boost.
Amounts of Nanocarrier-Encapsulated Immunosuppressant (Rapamycin or
Rapa) Used:
[0313] 50 .mu.g of nanocarrier-entrapped Rapa at either prime (gr.
2, 3 and 5) or boost (gr. 3 and 4).
TABLE-US-00003 TABLE 1 Experimental Groups Immunization, NCs Gr. #
i.v. (i.v., day 0) Boost, d. 21 1 AAV-GFP None AAV-GFP; 5 .times.
10.sup.10 VG (1 .times. 10.sup.10 VG) 2 Same 50 .mu.g of Rapa Same
3 Same Same AAV-GFP, 5 .times. 10.sup.10 VG + NCs 4 Same None
AAV-GFP, 5 .times. 10.sup.10 VG + NCs 5 Same 50 .mu.g of Rapa None
6 Same None None
Results
[0314] Statistically higher levels of GFP expression in the liver
of AAV-injected mice were seen if NCs was utilized at the boost
stage after prime with AAV-GFP only compared to both prime and
boost with AAV-GFP only (FIG. 1). There was also a trend towards
higher GFP expression if AAV-GFP was co-injected with NCs at both
prime and boost, but due to a single outlier it did not manifest a
clear statistical superiority to prime-boost with AAV-GFP only.
Utilization of NCs only at prime injection did not result in any
elevation of GFP expression (FIG. 1, gr. 1 vs. gr. 2 and gr. 5 vs.
gr. 6). Collectively, it appeared that co-administration of AAV-GFP
and NCs at boost drives the higher GFP expression in animals, which
received two injections of recombinant AAV according to the current
regimen. This was pronounced if all the animals boosted with
AAV-GFP only (whether or not treated with NCs at prime) are plotted
against all the animals boosted with AAV-GFP+NCs (FIG. 2) with 9/10
animals boosted in presence of NCs exhibiting higher GFP expression
than all (10/10) animals boosted without NCs (average expression
increase in the former being >50%). Similarly, if only
highly-GFP positive liver cells were considered, utilization of NCs
during boost resulted in statistically higher numbers than boost
with AAV-GFP without NCs, whether or not NCs was utilized at the
prime stage (FIG. 3). It was also apparent that AAV-GFP boost
without NCs led to decreased GFP expression even compared to a
single prime immunization.
[0315] Separately, mice were bled at d14 and their serum tested for
the presence of antibodies to AAV. At this point, all mice had been
injected with AAV-GFP once with or without co-administration of NCs
(resulting in two groups of 15 mice each). As seen in FIG. 4, all
15/15 mice which received a single AAV-GFP injection without NCs
had exhibited antibody reactivity against AAV, resulting in top ODs
higher than normal serum control (OD=0.227), while no mouse which
received AAV co-administered with NCs exhibited a detectable level
of antibodies to AAV. If only a single AAV immunization was
employed, levels of anti-AAV antibodies stayed below the baseline
at d21 in mice to which NCs was co-administered with AAV, while
being elevated in mice that received AAV without NCs (FIG. 5). At
33 days after a single injection, levels of these in untreated mice
were still moderately growing, while in NCs-treated group 4 out of
5 mice had no detectable antibodies to AAV (FIG. 5).
[0316] If mice were boosted with AAV-GFP at day 21, then antibody
levels in untreated mice continued to grow significantly while
being blunted in those mice that received NCs only at boost (FIGS.
6 and 7). Interestingly, two mice in this latter group while being
positive at d14 (no treatment at prime) had their levels of
antibodies to AAV fall below background by d33 (FIG. 7). At the
same time, 8/10 mice treated with NCs at prime had no detectable
antibodies at d33 even after d21 boost (FIGS. 6 and 7). Application
of NCs at AAV boost may have had a minor effect in blocking
generation of antibodies to AAV although at this point it was not
statistically significant from no NCs treatment at boost (FIGS. 6
and 7). Thus, NCs treatment at prime appears to be important for
blocking the development of antibodies to AAV with its
administration at later time-points also being beneficial.
[0317] The results demonstrate the benefit of administering
synthetic nanocarriers coupled to an immunosuppressant in
conjunction with a viral transfer vector for reducing antibody
responses against the viral transfer vector. Such benefits were
seen with concomitant administration of synthetic nanocarriers
coupled to an immunosuppressant in conjunction with a viral
transfer vector encoding a protein for expression. Accordingly,
protocols for reducing anti-viral transfer vector antibody
responses are hereinabove exemplified.
Example 12: Synthetic Nanocarriers Comprising Rapamycin
Materials
[0318] PLA with an inherent viscosity of 0.41 dL/g was purchased
from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, Ala.
35211), product code 100 DL 4A.
[0319] PLA-PEG-OMe block co-polymer with a methyl ether terminated
PEG block of approximately 5,000 Da and an overall inherent
viscosity of 0.50 DL/g was purchased from Lakeshore Biomaterials
(756 Tom Martin Drive, Birmingham, Ala. 35211), product code 100 DL
mPEG 5000 5CE.
[0320] Rapamycin was purchased from Concord Biotech Limited
(1482-1486 Trasad Road, Dholka 382225, Ahmedabad India), product
code SIROLIMUS.
[0321] Sorbitan monopalmitate was purchased from Sigma-Aldrich
(3050 Spruce St., St. Louis, Mo. 63103), product code 388920.
[0322] EMPROVE.RTM. Polyvinyl Alcohol (PVA) 4-88, USP (85-89%
hydrolyzed, viscosity of 3.4-4.6 mPas) was purchased from EMD
Chemicals Inc. (480 South Democrat Road Gibbstown, N.J. 08027),
product code 1.41350.
[0323] Dulbecco's phosphate buffered saline 1.times. (DPBS) was
purchased from Lonza (Muenchensteinerstrasse 38, CH-4002 Basel,
Switzerland), product code 17-512Q.
Method
[0324] Solutions were prepared as follows:
[0325] Solution 1:
[0326] A polymer, rapamycin, and sorbitan monopalmitate mixture was
prepared by dissolving PLA at 37.5 mg/mL, PLA-PEG-Ome at 12.5
mg/mL, rapamycin at 8 mg/mL, and sorbitan monopalmitate at 2.5 in
dichloromethane.
[0327] Solution 2:
[0328] Polyvinyl alcohol was prepared at 50 mg/mL in 100 mM pH 8
phosphate buffer.
[0329] An O/W emulsion was prepared by combining Solution 1 (1.0
mL) and Solution 2 (3 mL) in a small glass pressure tube, vortex
mixed for 10 seconds. The formulation was then homogenized by
sonication at 30% amplitude for 1 minute. The emulsion was then
added to an open beaker containing DPBS (30 mL). A second O/W
emulsion was prepared using the same materials and method as above
and then added to the same beaker containing the first emulsion and
DPBS. The combined emulsion was then stirred at room temperature
for 2 hours to allow the dichloromethane to evaporate and for the
nanocarriers to form. A portion of the nanocarriers was washed by
transferring the nanocarrier suspension to a centrifuge tube and
centrifuging at 75,600.times.g and 4.degree. C. for 50 minutes,
removing the supernatant, and re-suspending the pellet in DPBS
containing 0.25% w/v PVA. The wash procedure was repeated and then
the pellet was re-suspended in DPBS containing 0.25% w/v PVA to
achieve a nanocarrier suspension having a nominal concentration of
10 mg/mL on a polymer basis. The nanocarrier suspension was then
filtered using a 0.22 .mu.m PES membrane syringe filter (Millipore
part number SLGP033RB). The filtered nanocarrier suspension was
then stored at -20.degree. C.
[0330] Nanocarrier size was determined by dynamic light scattering.
The amount of rapamycin in the nanocarrier was determined by HPLC
analysis. The total dry-nanocarrier mass per mL of suspension was
determined by a gravimetric method.
TABLE-US-00004 Effective Diameter Rapamycin Content Nanocarrier
Conc (nm) (% w/w) (mg/mL) 150 11.5 11.1
Example 13. Single Administration of a Viral Transfer Vector with a
Gene Therapy Transgene Induces Anti-Vector Antibody Responses that
can be Inhibited by Concomitant Administration with Synthetic
Nanocarriers Coupled to Immunosuppressant
[0331] A single intravenous (i.v.) administration of
adeno-associated virus encoding a recombinant green fluorescent
protein (AAV-GFP) (Virovek, Hayward, Calif.) under a CMV promoter
led to an anti-AAV antibody response that was inhibited by
concomitant treatment with nanocarrier-encapsulated
immunosuppressant (produced according to Example 12).
Experimental Methods
[0332] Male C57BL/6 mice were used (5-15 mice/group). Animals were
injected i.v. with 200 .mu.L of AAV8-GFP or an admixture of
AAV8-GFP+NCs, a PLGA nanocarrier containing rapamycin (see Table 2
below). On day 14 after treatment, all animals were bled and their
sera analyzed for antibodies against AAV8 by ELISA. Briefly,
96-well plates were coated with 50 .mu.L of AAV8 at
2.times.10.sup.9 vector genomes (vg)/mL in carbonate buffer for 92
hours, and then blocked for 2 hours with 300 .mu.L of casein.
Samples were added at a 1:40 dilution in 50 .mu.L of casein, and
incubated for 2 hours at room temperature (RT). Horse radish
peroxidase-conjugated rabbit anti-mouse IgG (Jackson
ImmunoResearch, West Grove, Pa., 315-035-008) was used as a
secondary antibody (0.5 .mu.g/mL, 1 hour) and then TMB substrate
was added (10 min) followed by the stop solution. Plates were then
read at a wavelength of 450 nm with a subtraction of background at
570 nm. Mouse monoclonal anti-AAV8 antibody (Fitzgerald, Acton,
Mass., 10R-2136) served as a positive control.
Amounts of AAV-GFP:
[0333] 1.times.10.sup.10 viral genomes (vg) at day 0 (prime) and
5.times.10.sup.10 vg at day 21 (boost).
Amounts of Nanocarrier-Encapsulated Rapamycin (Rapa) Used:
[0334] 50 .mu.g of nanocarrier-entrapped rapamycin.
TABLE-US-00005 TABLE 2 Experimental Groups Gr. # Immunization, i.v.
day 0 NCs (i.v. day 0) 1 AAV-GFP (1 .times. 10.sup.10 VG) None 2
AAV-GFP (1 .times. 10.sup.10 VG) 50 .mu.g of Rapa
[0335] Mice were bled at d14 and their sera tested for the presence
of antibodies to AAV8. At this point, all mice had been injected
with AAV8-GFP once with or without co-administration of the
nanocarriers (15 mice each). As seen in FIG. 8, all mice which
received a single AAV-GFP injection without nanocarriers had
exhibited antibody reactivity against AAV8, resulting in antibody
levels higher than the normal serum control (OD=0.227), while mice
which received AAV8 co-administered with NCs exhibited little or no
detectable levels of antibodies to AAV8. Levels of anti-AAV8
antibodies stayed at or below the baseline at d21 in the NCs
treated group (n=5), while being elevated in mice that received
AAV8-GFP without NCs (FIG. 9). At 33 days after a single injection
of AAV8-GFP, anti-AAV8 antibody levels in untreated mice continued
to increase moderately, while in the NCs-treated group 4 out of 5
mice had no detectable antibodies to AAV (FIG. 9).
[0336] The results demonstrate the benefit of administering
synthetic nanocarriers coupled to an immunosuppressant in
conjunction with a viral transfer vector for reducing antibody
responses against the viral transfer vector. Such benefits were
seen with concomitant administration of synthetic nanocarriers
coupled to an immunosuppressant in conjunction with an viral
transfer vector comprising a transgene encoding a protein for
expression. Accordingly, protocols for reducing anti-viral transfer
vector antibody responses are herein exemplified.
Example 14: Concomitant Administration of a Viral Transfer Vector
with a Gene Therapy Transgene with Synthetic Nanocarriers Coupled
to Immunosuppressant Inhibits the Anti-AAV Antibody Response
Experimental Methods
[0337] Male C57BL/6 mice were used (5 mice/group). Animals were
injected with 200 .mu.L of AAV8-GFP (Virovek, Hayward, Calif.) or
an admixture of AAV8-GFP+NCs (as produced in Example 12) on day 0
and/or day 21 as indicated in Table 3. Sera were collected on day 0
and 33 and analyzed for anti-AAV8 antibody levels by ELISA as
described above.
Amounts of AAV-GFP:
[0338] 1.times.10.sup.10 viral genomes (vg) at d0 prime,
5.times.10.sup.10 vg at d21 boost.
Amounts of Nanocarrier-Encapsulated Immunosuppressant (Rapamycin or
Rapa) Used:
[0339] 50 .mu.g of nanocarrier-entrapped Rapa at either prime (gr.
2 and 4) or boost (gr. 3 and 4).
TABLE-US-00006 TABLE 3 Experimental Groups Day 0 Day 21 Viral
transfer Viral transfer Gr. # vector (i.v.) NCs (i.v.) vector
(i.v.) NCs (i.v.) 1 AAV-GFP None AAV-GFP None (1 .times. 10.sup.10
vg) (5 .times. 10.sup.10 vg) 2 AAV-GFP None AAV-GFP 50 .mu.g of
Rapa (1 .times. 10.sup.10 vg) (5 .times. 10.sup.10 vg) 3 AAV-GFP 50
.mu.g of Rapa AAV-GFP None (1 .times. 10.sup.10 vg) (5 .times.
10.sup.10 vg) 4 AAV-GFP 50 .mu.g of Rapa AAV-GFP 50 .mu.g of Rapa
(1 .times. 10.sup.10 vg) (5 .times. 10.sup.10 vg)
Results
[0340] Mice injected with AAV8-GFP on day 0 in the absence of NCs
showed a robust anti-AAV8 antibody response that increased
significantly after the second injection of AAV8-GFP on day 21
(FIG. 10). However if NCs was concomitantly administered with the
AAV8-GFP on day 21, the antibody response on average was
significantly blunted. Interestingly, two mice in this latter group
which were antibody positive on d14 had no detectable levels of
antibodies to AAV8 on d33 (FIG. 10). However anti-AAV8 antibody
titers increased in 2 other mice. In contrast, NCs concomitantly
administered at the time of the first AAV8-GFP injection (day 0)
completely inhibited the anti-AAV8 antibody response at day 14. The
anti-AAV8 antibody was also inhibited in 4 of 5 mice at day 33
after a second administration of AAV8-GFP alone on day 21.
Concomitant administration of NCs at both day 0 and 21 showed a
similar trend. Thus, NCs treatment at the time of the first
administration of AAV is important for blocking the development of
antibodies to AAV. Additional administration of NCs upon repeat
dosing of AAV may be potentially beneficial.
[0341] The results demonstrate the benefit of administering
synthetic nanocarriers coupled to an immunosuppressant in
conjunction with a viral transfer vector for reducing antibody
responses against the viral transfer vector. Such benefits were
seen with concomitant administration of synthetic nanocarriers
coupled to an immunosuppressant in conjunction with a viral
transfer vector encoding a protein for expression. Accordingly,
protocols for reducing anti-viral transfer vector antibody
responses are herein exemplified.
Example 15: Therapeutic Administration of Synthetic Nanocarriers
Coupled to Immunosuppressant Enhances the Maintenance of Transgene
Expression Upon Repeat Dosing of a Viral Transfer Vector
[0342] Two successive intravenous (i.v.) inoculations of
adeno-associated virus encoding recombinant green fluorescent
protein (AAV8-GFP) (Virovek, Hayward, Calif.) led to higher GFP
expression in liver cells in vivo if nanocarrier-encapsulated
immunosuppressant (NCs) (as produced in Example 12) was co-injected
at the time of a repeat administration of a viral transfer vector
encoding a protein for expression.
Experimental Methods
[0343] Male C57BL/6 mice were used (5 mice/group). Animals were
injected with 200 .mu.L of AAV8-GFP in the absence of NCs on day 0.
One group of animals received no further treatment, while other
groups received a second dose of AAV8-GFP on day 21 with or without
concomitant administration of NCs carrying 50 .mu.g rapamycin (see
Table 4 below). At d33 after the first injection (12 days after the
second injection for those groups that were injected twice) animals
were sacrificed, their livers treated with collagenase 4
(Worthington, Lakewood, N.J.), meshed and total cell suspensions
were analyzed by flow cytometry for GFP expression. Briefly, tissue
was initially perfused with collagenase (100 U) and incubated at
37.degree. C. for 30 min. The collagenase supernatant was removed,
and quenched with 2% FBS. Tissue samples were then cut into
.about.2 mm squares, digested (collagenase, 400 U) with repeated
agitation, filtered (nylon mesh), spun down (1,500 rpm), and
pellets re-suspended in ice-cold 2% FBS.
Amounts of AAV-GFP:
[0344] 1.times.10.sup.10 viral genomes (vg) at d0 and
5.times.10.sup.10 vg at d21 (groups 2 and 3 only).
Amounts of Nanocarrier-Encapsulated Immunosuppressant (Rapamycin or
Rapa) Used:
[0345] 50 .mu.g of nanocarrier-entrapped Rapa at day 21 (gr.
3).
TABLE-US-00007 TABLE 4 Experimental Groups Day 0 Day 21 Viral
transfer Viral transfer Gr. # vector (i.v.) vector (i.v.) NCs
(i.v.) 1 AAV-GFP None None (1 .times. 10.sup.10 vg) 2 AAV-GFP
AAV-GFP None (1 .times. 10.sup.10 vg) (5 .times. 10.sup.10 vg) 3
AAV-GFP AAV-GFP 50 .mu.g of Rapa (1 .times. 10.sup.10 vg) (5
.times. 10.sup.10 vg)
Results
[0346] Statistically higher levels of GFP expression in the liver
of AAV8-GFP treated mice were seen if NCs was concomitantly
administered with the second injection of AAV8-GFP on d21 compared
to animals that received a second injection of AAV8-GFP in the
absence of NCs (FIG. 11). The level of GFP expression observed in
mice that received a second injection of AAV8-GFP plus NCs was
similar to that observed in mice that received only a single dose
of AAV8-GFP on day 0. These results indicate that the
co-administration of NCs at the time of the second dose of AAV8-GFP
was important to maintain expression of GFP, perhaps by inhibiting
cytolytic T cells which could eliminate transduced liver cells
expressing GFP.
[0347] The results demonstrate the benefit of administering
synthetic nanocarriers coupled to an immunosuppressant in
conjunction with a viral transfer vector for maintaining expression
of the vector transgene. Such benefits were seen with concomitant
administration of synthetic nanocarriers coupled to an
immunosuppressant in conjunction with a viral transfer vector
comprising a transgene encoding a protein for expression.
Example 16: Concomitant Administration of a Synthetic Nanocarriers
Coupled to Immunosuppressant Enhances Transgene Expression
Experimental Methods
[0348] Male C57BL/6 mice were used (5 mice/group). Animals were
injected with 200 .mu.L of AAV-red fluorescence protein (RFP)
(Virovek, Hayward, Calif.) on day 0 (groups 1-5) and/or day 21
(groups 1-4, 6) (see Table 5 below). NCs carrying 50 .mu.g
rapamycin was concomitantly administered on day 0 (groups 2, 4)
and/or day 21 (groups 3, 4). At d33 after the first injection (12
days after the second injection for those groups that were injected
twice) animals were sacrificed, their livers treated with
collagenase 4 (Worthington, Lakewood, N.J.), meshed and total cell
suspensions were analyzed by flow cytometry for RFP expression.
Briefly, tissue was initially perfused with collagenase (100 U) and
incubated at 37.degree. C. for 30 min. The collagenase supernatant
was removed and quenched with 2% FBS. Tissue samples were then cut
into .about.2 mm squares, digested (collagenase, 400 U) with
repeated agitation, filtered (nylon mesh), spun down (1,500 rpm),
and pellets re-suspended in ice-cold 2% FBS.
TABLE-US-00008 TABLE 5 Experimental Groups Day 0 Day 21 Viral
transfer Viral transfer Gr. # vector (i.v.) NCs (i.v.) vector
(i.v.) NCs (i.v.) 1 AAV-RFP None AAV-RFP None 2 AAV-RFP 50 .mu.g of
Rapa AAV-RFP None 3 AAV-RFP None AAV-RFP 50 .mu.g of Rapa 4 AAV-RFP
50 .mu.g of Rapa AAV-RFP 50 .mu.g of Rapa 5 AAV-RFP None None None
6 None None AAV-RFP None
Results
[0349] Animals administered one or two injections of AAV8-RFP in
the absence of NCs showed similar low levels of RFP expression at
day 33 (FIG. 12). Mice treated with NCs concomitantly at the time
of the first injection of AAV8-RFP showed a trend towards increased
expression of RFP that was not statistically significant. In
contrast, mice that were treated concomitantly with NCs at the time
of the second injection of AAV8-RFP (day 21) showed a statistically
significant increase in RFP expression. Mice that were treated with
NCs at both day 0 and day 21 also showed a significant increase in
RFP expression compared to control animals that received AAV8-RFP
on days 0 and 21 in the absence of NCs.
[0350] The results demonstrate the benefit of administering
synthetic nanocarriers coupled to an immunosuppressant in
conjunction with a viral transfer vector for enhancing expression
of the vector transgene. Such benefits were seen with concomitant
administration of synthetic nanocarriers coupled to an
immunosuppressant in conjunction with a viral transfer vector
comprising a transgene encoding a protein for expression.
Example 17: Administration of a Viral Transfer Vector with a Gene
Therapy Transgene with Synthetic Nanocarriers Coupled to
Immunosuppressant Inhibits CD8+ T Cell Activation
[0351] Two successive intravenous (i.v.) inoculations of
adeno-associated virus expressing recombinant green fluorescent
protein (AAV-GFP) (Virovek, Hayward, Calif.) led to lower cytolytic
T cell (CTL) activity against AAV capsid protein and GFP in vivo if
nanocarrier-encapsulated immunosuppressant (NCs) was co-injected
with both AAV8-GFP injections.
Experimental Methods
[0352] Male C57BL/6 mice were used (3 or 6 mice/group). Animals
were injected with 200 .mu.L of AAV-GFP or an admixture of
AAV-GFP+NCs on day 0 and day 17 or 21 (see Table 6 below). Groups
injected at days 0 and 21 (n=3 mice per group) were assayed for
antigen-specific CTL activity at 28 days after the first injection
(7 days after the second injection). Briefly, splenocytes from
syngeneic naive mice were labeled with either 0.5 .mu.M, or 5 .mu.M
CFSE, resulting in CFSE.sup.low and CFSE.sup.high cell populations,
respectively. CFSE.sup.high cells were incubated with 1 .mu.g/mL
dominant MHC class I-binding peptide from AAV capsid (sequence
NSLANPGIA (SEQ ID NO: 1), amino acids 517-525) and dominant MHC
class I peptide from GFP (HYLSTQSAL (SEQ ID NO: 2), aa 200-208) at
37.degree. C. for 1 h, while CFSElow cells were incubated in medium
alone. The control CFSElow cells and peptide-pulsed CFSEhigh target
cells were mixed in a 1:1 ratio (2.0.times.107 cells total) and
injected i.v. Eighteen hours after the injection of labeled cells,
spleens were harvested, processed and analyzed by flow cytometry.
Specific cytotoxicity was calculated based on a control ratio of
recovery (RR) in naive mice: (percentage of CFSElow
cells)/(percentage of CFSEhigh cells). Percent specific lysis
(%)=100.times.[1-(RR of cells from naive mice/RR of cells from
immunized mice) or 100.times.[1-(RRnaive/RRimm)].
[0353] Groups injected at days 0 and 17 (n=6 mice per group) were
assayed for antigen-specific IFN-.gamma. production on d25 after
the first injection (7 days after the second injection). Briefly,
splenocytes were isolated, plated in wells with pre-absorbed
anti-IFN-.gamma. antibody and re-stimulated with AAV capsid or GFP
peptides (1 .mu.g/mL) for 7 days in vitro. ELISpots were developed
by biotinylated anti-IFN-.gamma. antibody and streptavidin-HRP, and
spots were counted. Nonspecific background was subtracted.
Amounts of AAV-GFP:
[0354] 1.times.10.sup.10 viral genomes (vg) at d0 prime,
5.times.10.sup.10 vg at d21 boost.
Amounts of Nanocarrier-Encapsulated Immunosuppressant (Rapamycin or
Rapa) Used:
[0355] 50 .mu.g of nanocarrier-entrapped Rapa at both prime and
boost (gr. 2).
TABLE-US-00009 TABLE 6 Experimental Groups First Injection Second
Injection i.v. (Day 0) i.v. (day 17 or 21) Gr. # AAV8-GFP NCs
Challenge NCs 1 1 .times. 10.sup.10 vg None 5 .times. 10.sup.10 vg
None 2 1 .times. 10.sup.10 vg 50 .mu.g rapamycin 5 .times.
10.sup.10 vg 50 .mu.g rapamycin
Results
[0356] Animals concomitantly treated with NCs showed lower levels
of in vivo CTL activity against targets cells pulsed with a
combination of AAV capsid and GFP dominant MHC class I peptides
(FIG. 13). Similarly, mice concomitantly treated with NCs showed a
significant reduction in antigen-specific IFN-.gamma.-producing
cells compared to the non-NCs-treated group (FIGS. 14 and 15). In
particular, 4/6 mice demonstrated a recall response to the AAV
capsid protein at 250,000 cells per well density, while no (0/6)
mice responded to this peptide in the NCs-treated group (FIG. 14,
p<0.05). Moreover, 3/6 mice in the AAV8-GFP-immunized group
showed a response to an immunodominant GFP peptide, while no mice
(0/6) responded to this peptide in the NCs-treated group (FIG. 15,
p=0.01).
[0357] Collectively, it appeared that co-administration of AAV-GFP
and NCs at prime and boost results in suppression of cytotoxic T
cell responses against viral capsid and transgenic proteins.
Example 18: Administration of Viral Transfer Vector with a Gene
Therapy Transgene with Synthetic Nanocarriers Comprising
Immunosuppressant
Experimental Methods
[0358] Male C57BL/6 mice were used (5 mice/group). Animals were
injected i.v. with 10.sup.10 vg of rAAV2/8-luciferase
(rAAV2/8-Luc)) (produced in a manner similar to the methods
provided herein such as in Example 21 or 22) or
rAAV2/8-Luc+synthetic nanocarriers containing 100 .mu.g rapamycin
(NCs) on Day 0 (see Table 7 below). On day 14, all animals received
an i.v. injection of 10.sup.10 vg of rAAV2/8 encoding human factor
IX (hFIX) (rAAV2/8-hFIX)) (produced in a manner similar to the
methods provided herein such as in Example 21 or 22). Sera were
collected at various time points and assayed for anti-AAV antibody
levels and hFIX protein levels by ELISA.
[0359] FIG. 16 illustrates the protocol and timing of
administration of synthetic nanocarriers comprising an
immunosuppressant. Synthetic PLGA nanocarriers containing 100 .mu.g
rapamycin (NCs) or control empty nanoparticles (Empty NP) were
administered i.v. concomitantly with rAAV2/8-Luc vector (10.sup.10
vg) on Day 0 (N=5/group). All groups received an injection (i.v.)
of rAAV2/8 vector encoding human coagulation factor IX (hFIX) on
Day 14. The data show that a single administration of synthetic
nanocarriers comprising immunosuppressant concomitantly
administered with AAV8-Luc can prevent or delay the onset of
anti-AAV8 antibodies (FIG. 17). Importantly, the concomitant
administration of NCs with rAAV2/8-luciferase inhibited anti-AAV8
antibody formation sufficiently to enable efficient expression of
hFIX from the rAAV2/8-hFIX administered on day 14. In contrast,
animals treated with empty NP developed anti-AAV8 antibodies which
prevented efficient expression of hFIX from the rAAV2/8-hFIX vector
administered on day 14. These data indicate that concomitant
administration of synthetic nanocarriers comprising
immunosuppressant at the time of the first application of AAV
enables efficacious repeat dosing of the same serotype of AAV.
TABLE-US-00010 TABLE 7 Treatment Groups Groups Nanocarriers
rAAV2/8-Luc rAAV2/8-FIX N 1 NCs, D0 D0 D14 5 2 Empty NP, D0 D0 D14
5
Example 19: Multiple Administrations of Viral Transfer Vector with
a Gene Therapy Transgene with Synthetic Nanocarriers Comprising
Immunosuppressant
[0360] The experimental design is shown in FIG. 18. Male C57BL/6
mice were used (5 mice/group). Synthetic nanocarriers containing
rapamycin (NCs) (100 .mu.g rapamycin) were administered i.v.
concomitantly with rAAV2/8-Luc vector) (produced in a manner
similar to the methods provided herein such as in Example 21 or 22)
(1.times.10.sup.11 vg) on day 0 (N=5/group) (Table 8). Mice were
then challenged with rAAV2/8-hFIX) (produced in a manner similar to
the methods provided herein such as in Example 21 or 22)
concomitantly administered with synthetic nanocarriers containing
rapamycin (100 .mu.g rapamycin) on day 21. The control group
received empty NP instead of NCs on days 0 and 21.
TABLE-US-00011 TABLE 8 Treatment Groups rAAV2/8-Luc rAAV2/8-FIX
Groups Nanoparticles (10.sup.11 vg) (10.sup.11 vg) N 1 NCs, d 0, 21
d0 d21 5 2 Empty NP, d0, 21 d0 d21 5
[0361] The results showed that concomitant administration of
synthetic nanocarrier injections with both the first (rAAV2/8-Luc)
and second (rAAV2/8-hFIX) injections of viral transfer vector
inhibited the anti-AAV8 antibody response (FIG. 19, left panel) and
reduced the titer of neutralizing antibodies to AAV8 (Table 9). The
inhibition of the anti-AAV8 antibodies enabled higher levels of
AAV2/8-hFIX vector copy numbers (FIG. 19, middle panel), which in
turn provided for robust expression of the hFIX transgene (FIG. 19,
right panel). Note that in the control group treated with empty
nanoparticles, several animals had low levels of antibodies at day
20. Two of these animals had an intermediate level of vector copy
numbers and some expression of hFIX in response to administration
of rAAV2/8-hFIX on day 21. However three of the control animals
showed very low vector copy numbers and no detectable levels of FIX
expression.
[0362] Accordingly, it was demonstrated that multiple
administrations of synthetic nanocarriers comprising
immunosuppressant can completely prevent the induction of
antigen-specific anti-AAV8 antibodies, allowing for high levels of
transgene expression upon a second injection of AAV.
TABLE-US-00012 TABLE 9 Neutralizing anti-AAV antibody titers AAV8
Neutralizing Antibody Titer NCs Empty NP Animal # Day 20 Day 41 Day
20 Day 41 1 1:3.16 1:31.6 1:31.6 1:1000 2 <1:1 1:31.6 1:31.6
1:316 3 <1:1 1:31.6 1:31.6 1:316 4 <1:1 1:31.6 1:31.6 1:1000
5 <1:1 1:31.6 1:31.6 1:1000
Example 20: Antigen Specificity
[0363] The experimental design is shown in FIG. 20. Synthetic
nanocarriers comprising immunosuppressant (100 .mu.g rapamycin) or
control empty nanoparticles were administered i.v. concurrently
with rAAV2/8-Luc vector) (produced in a manner similar to the
methods provided herein such as in Example 21 or 22)
(1.times.10.sup.11 vg/mouse) on Day 0. On day 21 mice received
either an i.v. injection of rAAV5-hFIX) (produced in a manner
similar to the methods provided herein such as in Example 21 or 22)
(1.times.10.sup.11 vg/mouse) or an i.m. injection of human Factor
IX (hFIX) protein emulsified in complete Freund's adjuvant (CFA)
(Table 10).
TABLE-US-00013 TABLE 10 Treatment Groups FIX rAAV2/8-Luc rAAV5-hFIX
protein Groups Nanoparticles (10.sup.11 vg) (10.sup.11 vg) CFA N 1
NCs, d0 d0 d21 -- 5 2 Empty NP, d0 d0 d21 -- 5 3 NCs, d0 d0 -- d21
5 4 Empty NP, d0 d0 -- d21 5
[0364] The results showed that concomitant i.v. administration of
synthetic nanocarrier carrying rapamycin with an rAAV2/8 vector
(AAV2/8-Luc) on day 0 did not have a profound impact on the
antibody response to an AAV5 vector (AAV5-hFIX) administered on day
21 (FIG. 21, left panel). The animals treated with the NCs
containing rapamycin had a short delay in the anti-AAV5 antibody
response compared to mice treated with empty NP, perhaps because of
the presence of B cells in the empty NP-treated mice that were
primed against AAV8 and crossreactive to AAV5. However the
anti-AAV5 antibody response of the NCs-treated mice rapidly
parallels the anti-AAV5 antibody response of the empty NP-treated
group.
[0365] In contrast, animals that received AAV2/8-FIX on day 21
showed little or no anti-AAV8 antibodies. These data indicate that
the effect of the NCs treatment on inhibiting anti-AAV antibody
responses were specific to AAV serotype with which it was
co-administered (i.e., AAV8) and does not render the mice
chronically immunosuppressed. Similarly, mice concomitantly treated
with NCs and rAAV2/8-Luc on day 0 showed a robust response to
immunization with recombinant hFIX protein in complete Freund's
adjuvant (CFA) on day 21 (FIG. 21, right panel). The anti-hFIX
antibody response was indistinguishable from that of mice that were
treated with empty NP instead of NCs on day 0. Accordingly, it was
demonstrated that concomitant administration of synthetic
nanocarriers comprising immunosuppressant with AAV does not result
in chronic immunosuppression.
Example 21: AAV5 Transfer Vector with a Gene Therapy Transgene
(Prophetic)
[0366] ART-I02 is produced as described previously (Matsushita T,
et al. Gene Ther. 1998; 5: 938-945). The plasmid encodes any one of
the transgenes provided herein under the control of the NF-.kappa.B
promoter and a human growth hormone polyadenylation signal. The
gene of interest may also be under the control of the
cytomegalovirus (CMV) promoter. The transgene cassette is flanked
by AAV-2 inverted terminal repeats and is packaged in capsid from
AAV5 as described in Gao G P, et al. Proc Natl Acad Sci USA. 2002;
99: 11854-11859. The vector is purified by combined chromatography
and cesium chloride density gradient centrifugation, resulting in
empty capsid-free fractions. Vector titers can be determined by
qPCR using specific primers and probe. Similarly, as an example, a
rAAV5 vector can be produced coding for Firefly Luciferase.
Example 22: AAV2/8 and AAV2/5 Transfer Vector with a Gene Therapy
Transgene (Prophetic)
[0367] An scAAV backbone plasmid is constructed by ligating
MscIBsaI and BsaITsp45I fragments from AAV2-HCR-hAAT-FIX to the
simian virus 40 late polyA (SV40 LpA). The resulting plasmid
contains the modified AAV2 backbone with an intact 5' terminal
resolution site (trs) and a deleted 3' trs. The LP1
enhancer/promoter can be constructed using standard polymerase
chain reaction (PCR) methods with amplification of consecutive
segments of the human apolipoprotein hepatic control region (HCR),
the human alphalantitrypsin (hAAT) gene promoter including the 5'
untranslated region and cloned upstream of a modified SV40 small t
antigen intron (SV40 intron) modified at positions 4582 (g to c),
4580 (g to c), 4578 (a to c), and 4561 (a to t) into the modified
AAV2 backbone. The wild-type hFIX cDNA, or other cDNA of interest,
without the 3' untranslated region (UTR) regions is PCR amplified
from AAV-HCR-hAAT-hFIX and inserted downstream of the modified SV40
intron to make scAAV-LP1-hFIX. A codon-optimized hFIX is generated
using codons most frequently found in highly expressed eukaryotic
genes, synthesized as oligonucleotides, and subsequently assembled
by ligation, PCR amplified, and sequenced prior to cloning into the
AAVLP1 backbone to create sc-AAV-LP1-hFIXco. ss and scAAV vectors
are made by the adenovirus-free transient transfection method.
[0368] AAV5-pseudotyped vector particles are generated using a
chimeric AAV2 Rep-5Cap packaging plasmid called pLT-RCO3, which is
based on XX2 and pAAV5-2. Additionally, AAV8-pseudotyped vectors
are made using the packaging plasmid pAAV8-2. AAV2/5 and 2/8
vectors are purified by the ion exchange chromatography method.
Vector genome (vg) titers can be determined by quantitative
slotblot using supercoiled plasmid DNA as standards. Such a viral
vector can comprise any one of the transgenes as provided
herein.
Example 23: AAV8 Transfer Vector with a Gene Therapy Transgene
(Prophetic)
[0369] A mouse genomic Alb segment (90474003-90476720 in NCBI
reference sequence:NC_0000710.6) can be PCR-amplified and inserted
between AAV2 inverted terminal repeats into BsrGI and SpeI
restriction sites in a modified pTRUF backbone. An optimized P2A
coding sequence preceded by a linker coding sequence
(glycine-serine-glycine) and followed by anNheI restriction site
can be into the Bpu10I restriction site. A codon-optimized F9
coding sequence can be inserted into the NheI site to get pAB269
that can serve in the construction of the rAAV8 vector. To
construct the inverse control, an internal segment from the BsiWI
restriction site to the NheI restriction site can be amplified
using appropriate PCR primers. Final rAAV production plasmids can
be generated using an EndoFree Plasmid Megaprep Kit (Qiagen).
[0370] rAAV8 vectors can be produced as described in Grimm, et al.,
J. Virol. 80, 426-439 (2006) using a Ca.sub.3(PO.sub.4).sub.2
transfection protocol followed by CsCl gradient purification.
Vectors can be titred by quantitative dot blot.
[0371] As described in Barzel, et al., 364, Nature, Vol. 517, 2015,
amelioration of the bleeding diathesis in haemophilia B mice was
demonstrated using such vectors as described above. In particular,
the vectors achieved integration into the albumin alleles in
hepatocytes. F9 was produced from on-target integration, and
ribosomal skipping was highly efficient. Stable F9 plasma levels
were obtained, and treated F9-deficient mice had normal coagulation
times.
Example 24: AAV9 Transfer Vector with a Gene Therapy Transgene
(Prophetic)
[0372] Adenoviral constructs using a "first-generation"
E1/E3-deleted replication-deficient adenovirus can be produced as
described in Kypson, et al. J Thorac Cardiovasc Surg. 1998 and
Akhter, et al. Proc Natl Acad Sci USA. 1997; 94:12100-12105. The
b.sub.2AR construct (Adeno-b.sub.2AR) and a transgene can be driven
by an appropriate promoter. Large-scale preparations of
adenoviruses can be purified from infected Epstein-Barr nuclear
antigen-transfected 293 cells.
[0373] As described in Shah et al., Circulation. 2000; 101:408-414,
rabbits that underwent percutaneous subselective catheterization of
either the left or right coronary artery and infusion of adenoviral
vectors such as those produced as above containing a marker
transgene expressed the transgene in a chamber-specific manner. In
addition, it was concluded that percutaneous adenovirus-mediated
intracoronary delivery of a therapeutic transgene is feasible, and
that acute global left ventricular function can be enhanced.
Example 25: Lentiviral Transfer Vector with a Gene Therapy
Transgene (Prophetic)
[0374] The following can be prepared: a lentiviral expression
plasmid containing a packaging sequence and a transgene inserted
between the lentiviral LTRs to allow target cell integration; a
packaging plasmid, encoding the pol, gag, rev and tat viral genes
and containing the rev-response element; and a pseudotyping
plasmid, encoding a protein, of a virus envelope gene. HEK 293T
cells can be transfected by the foregoing. After transfection of
HEK 293T cells, the lentiviral vectors can be obtained from the
cell supernatant which contains recombinant lentiviral vectors.
Example 26: HIV Lentiviral Transfer Vector with a Gene Therapy
Transgene (Prophetic)
[0375] An HIV lentiviral transfer vector is prepared according the
methods of U.S. Publication No. 20150056696. An hPEDF CDS fragment
is PCR amplified from cDNA of the human Retinal pigment epithelium
cell strain ARPE-19 (American Type Culture Collection, ATCC) as a
template and using appropriate primers. An alternative fragment can
be similarly obtained for any one of the proteins described herein.
The hPEDF fragment is obtained by gel recovery and ligated into the
pLenti6.3/V5-TOPO.RTM. vector (Invitrogen) by TA cloning procedure
following the manufacturer's instruction. The sequence of the
ligated hPEDF fragment can be verified by sequencing.
Example 27: SIV Lentiviral Transfer Vector with a Gene Therapy
Transgene (Prophetic)
[0376] An SIV lentiviral transfer vector is prepared according the
methods of U.S. Publication No. 20150056696. A SIV gene transfer
vector, a packaging vector, a rev expression vector, and a VSV-G
expression vector are obtained, and an hPEDF fragment is introduced
into the gene transfer vector. An alternative fragment can be
similarly obtained for any one of the proteins described herein for
introduction into the gene transfer vector.
[0377] The cell line 293T cells derived from human fetal kidney
cells are seeded at a cell density of approximate 1.times.10.sup.7
cells per plastic Petri dish having the diameter of 15 cm (cell
density of 70-80% next day) and cultured in 20 ml of D-MEM culture
medium (Gibco BRL) supplemented with 10% fetal bovine serum for 24
hrs. After 24 h of cultivation, the culture medium is replaced with
10 ml of OPTI-MEM culture medium (Gibco BRL).
[0378] For one petri dish, 10 .mu.g of the gene transfer vector, 5
.mu.g of the packaging vector, 2 .mu.g of the rev expression vector
and 2 .mu.g of VSV-G expression vector are dissolved in 1.5 ml of
OPTI-MEM medium, and 40 .mu.l of PLUS Reagent reagent (Invitro Co.)
is added. The resulting mixture is stirred and left at room
temperature for 15 min. A dilute solution is obtained by diluting
60 .mu.l of LIPOFECT AMINE Reagent with 1.5 ml of OPTI-MEM medium;
the resulting mixture is stirred and left at room temperature for
15 min. The resulting DNA-complex is dropped onto the cells in the
above-described Petri dish. The Petri dish is shaken carefully to
achieve uniform mixing, and then incubated. 13 ml of D-MEM medium
comprising 20% of fetal bovine serum is added. Supernatant is
recovered.
Example 28: HSV Transfer Vector with a Gene Therapy Transgene
(Prophetic)
[0379] An HSV transfer vector is prepared according the methods of
U.S. Publication No. 20090186003. HSV-1 (F) strain is a low passage
clinical isolate used as the prototype HSV-1 strain. M002, which
expresses murine interleukin 12 (mIL-12) under the transcriptional
control of the murine early-growth response-1 promoter (Egr-1), is
constructed. Alternatively, similar constructs may be prepared
encoding any one of the proteins described herein under the control
of an appropriate promoter. The plasmids containing the p40 and p35
subunits of mIL-12 in pBluescript-SK+(Stratagene) are obtained. The
p40 subunit is removed by digestion with HindIII (5' end) and BamHI
(3' end) and the p35 subunit is removed by digestion with Ncol (5'
end) and EcoRI (3' end). The internal ribosome entry site, or IRES,
sequence is amplified from vector pCITE-4a+(Novagen, Madison, Wis.)
using polymerase chain reaction (PCR) and appropriate primers.
Plasmid pBS-IL12 is constructed by three-way ligation of the murine
p40, murine p35 and IRES sequences into HindIII and EcoRI sites of
pBS-SK+ such that the IRES sequence separates the p40 and p35
coding sequences.
[0380] A HSV shuttle plasmid pRB4878 can be prepared as previously
described (Andreansky et al. (1998) Gene Ther. 5, 121-130). Plasmid
4878-IL12 is constructed as follows: pBS-mIL-12 is digested with
XhoI and SpeI to remove a 2.2 kb fragment containing the entire
IL-12 subunit coding regions, including the IRES, ends filled in
using the Klenow fragment, and ligated into a blunted KpnI site
located between the Egr-1 promoter and hepatitis B virus polyA
sequences within pRB4878. M001 (tk-) and M002 (tk repaired at
native locus) are constructed via homologous recombination as
described previously (Andreansky et al. (1998) Gene Ther. 5,
121-130). Two tk-repaired viruses M002.29 and M002.211, are
confirmed by Southern blot hybridization of restriction
enzyme-digested viral DNAs which are electrophoretically separated
on a 1% agarose, 1.times.TPE gel and transferred to a Zeta-Probe
membrane (Bio-Rad). The blot is hybridized with the appropriate DNA
probe labeled with alkaline phosphatase using the Gene Images
AlkPhos Direct DNA labeling system (Amersham-Pharmacia Biotech,
Piscataway, N.J.). IL-12 production is demonstrated by
enzyme-linked immunosorbent assay (ELISA).
Example 29: Viral Transfer Vector with a CRISPR/Cas-9 Transgene
(Prophetic)
[0381] Any one of the viral vectors described herein, such as in
the above Examples, may be used to produce a viral transfer vector
with a gene editing transgene. Alternatively, and as an example,
the following provides a method for producing a viral transfer
vector with a gene editing transgene that encodes Cas9, such as
Cas9 wild-type (Type II).
[0382] HEK293T cells can be cultured in DMEM medium (Life
Technologies, Darmstadt, Germany) containing 10% fetal bovine serum
(Sigma, Steinheim, Germany), 100 U/mL penicillin and 100 .mu.g/mL
streptomycin (Life Technologies). Huh7 and Hep56D cell media can
additionally contain 1% non-essential amino acids (Life
Technologies). Jurkat cells can be grown in RPMI 1640 medium (GE
Healthcare, Pasching, Austria) containing 10% fetal bovine serum
(Sigma), 100 U/mL penicillin, 100 .mu.g/mL streptomycin, and 2 mM
L-glutamine (all Life Technologies). All cell lines can be cultured
at 37.degree. C. and 5% CO2. For large-scale AAV vector production,
HEK293T cells can be seeded in ten 15 cm2 dishes (4.times.106 cells
per dish). Two days later, they can be triple-transfected with (i)
the AAV vector plasmid (encoding gRNA and/or Cas9), (ii) an AAV
helper plasmid carrying AAV rep and cap genes, and (iii) an
adenoviral plasmid providing helper functions for AAV
production.
[0383] The AAV cap gene can be either derived from the synthetic
isolate AAV-DJ (Grimm, et al., J. Virol. 2008, 82, 5887-5911) or
from a new variant AAVrh10A2. Briefly, AAVrh10A2 can be created
through insertion of a seven amino acid long peptide into an
exposed region of the capsid of AAV serotype rh10. Further details
on AAV production plasmids and protocols can be performed as
reported in Borner, et al., Nucleic Acids Res. 2013, 41, e199 and
Grimm, Methods 2002, 28, 146-157. To generate small-scale AAV
stocks, 2.times.105 HEK293T cells per well can be seeded in 6-well
plates and the next day triple-transfected with the aforementioned
plasmids. Three days later, the cells can be scraped off into the
media, collected via 10 min centrifugation at 1500 rpm, resuspended
in 300 .mu.L 1.times.PBS (Life Technologies) and subjected to three
freeze-thaw cycles in liquid nitrogen and at 37.degree. C. A 10 min
centrifugation can be performed at 13,200 rpm to remove cell
debris, and supernatants containing viral transfer vector particles
can be used directly in transduction experiments or frozen at
-20.degree. C.
[0384] For small-scale transfections and subsequent T7 assays,
2.8.times.104 HEK293T or 1.2.times.104 Huh7 cells can be seeded per
well in a 96-well plate and the next day transfected using
lipofectamine 2000 (Life Technologies) following the manufacturer's
recommendations for this format (200 ng DNA and 0.5 .mu.L
lipofectamine 2000, each in 25 .mu.L serum-free medium). The 200 ng
DNA can consist of an all-in-one Cas9/gRNA vector, or, in the case
of separate Cas9 and gRNA constructs, of 100 ng of each. To obtain
lysates for Western blotting, HEK293T cells can be transfected in
24-well plates (one well per lysate), using lipofectamine 2000
according to the manufacturer's recommendations for this format. In
transduction experiments, cells can be grown in 96-well plates and
transduced with either 10 pt non-purified AAV or with purified
vector 1 day after seeding. Following a three (transfections) to
five (transductions) day incubation, the cells can be lysed with
DirectPCR Lysis Reagent Cell (PeqLab, Erlangen, Germany)
supplemented with 0.2 .mu.g/mL proteinase K (Roche, Mannheim,
Germany) following the manufacturer's protocol.
[0385] As described in Senis, et al. Biotechnol. J. 2014, 9,
1402-1412, plasmids and vectors such as those above can achieve
delivery of the CRISPR components--Cas9 and chimeric g(uide) RNA.
In addition, it was demonstrated that Cas9 expression could be
directed to or away from hepatocytes, using a liver-specific
promoter or a hepatic miRNA binding site, respectively. Further
evidence was provided that such vectors can be used for gene
engineering in vivo. This was accomplished in the exemplified liver
of adult mice.
Example 30: Viral Transfer Vector with a Cas9 Variant Transgene
(Prophetic)
[0386] The methodology in Example 30 can also be used to produce a
viral transfer vector with a gene editing transgene, such as a
transgene encoding a Cas9 variant, such as any one of the Cas9
variants described herein. Alternatively, any one of the other
viral vectors described herein, may be used instead to produce such
a viral transfer vector. Any one of the Cas9 variants provided can
be encoded by any one of the gene editing transgenes provided
herein.
[0387] To make Cas9 variants, the human codon-optimized
streptococcus pyogenes Cas9 nuclease with NLS and 3.times.FLAG tag
(Addgene plasmid 43861) can be used as the wild-type Cas9
expression plasmid. PCR products of wild-type Cas9 expression
plasmid as template with Cas9_Exp primers can be assembled with
Gibson Assembly Cloning Kit (New England Biolabs) to construct Cas9
and Fokl-dCas9 variants. Expression plasmids encoding a single gRNA
construct (gRNA G1 through G13) can also be cloned.
Example 31: Viral Transfer Vector with a Zinc Finger Nuclease
Transgene (Prophetic)
[0388] Any one of the viral vectors described herein, such as in
the above Examples, may be used to produce a viral transfer vector
with a gene editing transgene that encodes a zinc finger nuclease.
Alternatively, and as an example, the following provides a method
for producing such a viral transfer vector with a gene editing
transgene.
[0389] TRBC- and TRAC-ZFNs can be designed and assembled as
described in Urnov, et al. Nature 435, 646-651 (2005). The
recognition helices used can be as provided Provasi, et al., Nature
Medicine, Vol. 18, No. 5, May 2012. Lentiviral vectors encoding
TRBC- and TRAC-ZFNs can be generated from the HIV-derived
self-inactivating transfer construct pCCLsin.cPPT.SFFV.eGFP.Wpre,
which can be packaged by an integrase-defective third generation
packaging construct carrying the D64V mutation in the HIV integrase
and pseudotyped by the VSV envelope. The Ad5/F35 adenoviral vectors
can be generated on an E1-E3-deleted backbone. The ZFNs targeting
either the TRBC or TRAC gene can be linked using a 2A peptide
sequence and cloned into the pAdEasy-1/F35 vector under the control
of an appropriate promoter, and the Ad5/F35 virus for each
construct can be generated using TREx 293T cells. Lentiviral
vectors encoding both WT1-specific TCR chains and single a21 or
(321 WT1-specific TCR chains from the bidirectional
self-inactivating transfer vector pCCLsin.cPPT..DELTA.
LNGFR.minCMV.hPGK.eGFP.Wpre and from pCCLsin.cPPT.hPGK.eGFP.Wpre
can be generated and packaged by an integrase-competent
third-generation construct and pseudotyped by the VSV envelope.
[0390] Using vectors such as those above, and as described in
Provasi, et al., Nature Medicine, Vol. 18, No. 5, May 2012, it has
been shown that ZFNs promoted the disruption of endogenous TCR
.beta.- and .alpha.-chain genes. Lymphocytes treated with ZFNs
lacked surface expression of CD3-TCR and expanded with the addition
of interleukin-7 (IL-7) and IL-15. Further, after lentiviral
transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, the
TCR-edited cells expressed new TCR at high levels (also as
described in Provasi, et al., Nature Medicine, Vol. 18, No. 5, May
2012).
Example 32: Viral Transfer Vector with a Zinc Finger Nuclease
Transgene (Prophetic)
[0391] Zinc finger nucleases (ZFNs) targeting the hF9mut locus and
F9-targeting vectors can be prepared as described in Li, et al.
Nature. 2011; 475(7355):217-221. Such vectors have been shown to be
successfully used for in vivo gene targeting in a neonatal mouse
model of hemophilia B (HB). Systemic codelivery of the AAV vectors,
encoding the ZFN pair targeting the human F9 gene and a
gene-targeting vector with arms of homology flanking a corrective
cDNA cassette resulted in the correction of a defective hF9 gene
engineered into the mouse genome in the livers of such mice.
Further, stable levels of human factor IX expression sufficient to
normalize clotting times was achieved.
Example 33: Viral Transfer Vector with a Meganuclease Transgene
(Prophetic)
[0392] Any one of the viral vectors described herein, such as in
the above Examples, may be used to produce a viral transfer vector
with a gene editing transgene that encodes a meganuclease.
Alternatively, and as an example, the following describes a general
methodology for producing such a viral transfer vector with a gene
editing transgene. The meganuclease may be any one of the
meganucleases provided in U.S. Publication Nos. 20110033935 and
20130224863.
[0393] In some embodiments, particular viral genes are inactivated
to prevent reproduction of the virus. Preferably, in some
embodiments, a virus is altered so that it is capable only of
delivery and maintenance within a target cell, but does not retain
the ability to replicate within the target cell or tissue. One or
more DNA sequences encoding a meganuclease can be introduced to the
altered viral genome, so as to produce a viral genome that acts
like a vector. In some embodiments, the viral vector is a
retroviral vector such as, but not limited to, the MFG or pLJ
vectors. An MFG vector is a simplified Moloney murine leukemia
virus vector (MoMLV) in which the DNA sequences encoding the pol
and env proteins are deleted to render it replication defective. A
pll retroviral vector is also a form of the MoMLV (see, e.g.,
Korman et al. (1987), Proc. Nat'l Acad. Sci., 84:2150-2154). In
other embodiments, a recombinant adenovirus or adeno-associated
virus can be used to produce a viral vector.
Example 34: Viral Transfer Vector with an Exon Skipping Transgene
(Prophetic)
[0394] Any one of the viral vectors described herein, such as in
the above Examples, may be used to produce a viral transfer vector
with an exon skipping transgene. Alternatively, and as an example,
the following provides a method for producing a viral transfer
vector with a specific exon skipping transgene.
[0395] A three-plasmid transfection protocol can be used with
pAAV(U7smOPT-SD23/BP22) and pAAV(U7smOPT-scr) plasmids for
generation of single-strand AAV1-U7ex235 and AAV1-U7scr; and
scAAV-U7ex23 plasmid for self-complementary scAAV9-U7ex239.
pAAV(U7smOPT-scr) plasmid can contain the non specific sequence
GGTGTATTGCATGATATGT (SEQ ID NO: 3) that does not match to any
murine cDNAs.
[0396] Use of a viral transfer vector produced according to the
above, as described in Le Hir et al., Molecular Therapy vol. 21 no.
8, 1551-1558 August 2013 showed that such a vector can be used to
restore dystrophin. However, the restoration decreased
significantly between 3 and 12 months, which was correlated with
viral genome loss. Accordingly, the compositions and methods
provided herein can help maintain the effect of such a
treatment.
Example 35: Viral Transfer Vector with an Exon Skipping Transgene
(Prophetic)
[0397] Clone U1#23 can be obtained by inverse PCR on the human U1
snRNA gene, with oligos mU1anti5
(5'-CGAAATTTCAGGTAAGCCGAGGTTATGAGATCTTGGGCCTCTGC-3' (SEQ ID NO: 4))
and mU1anti3
(5'-GAACTTTGCAGAGCCTCAAAATTAAATAGGGCAGGGGAGATACCATGATC-3' (SEQ ID
NO: 5)). The antisense-containing insert can be amplified from
corresponding plasmid with oligos U1cas-up-NheI
(5'-CTAGCTAGCGGTAAGGACCAGCTTCTTTG-3' (SEQ ID NO: 6)) and
U1cas-down-NheI (5'-CTAGCTAGCGGTTAGCGTACAGTCTAC-3' (SEQ ID NO: 7)).
The resulting fragment can be NheI-digested and cloned in the
forward orientation of the pAAV2.1-CMV-EGFP plasmid.
[0398] AAV-U1#23 vector can be produced by triple transfection of
293 cells, purified by CsCl2 ultracentrifugation and titered by
using both real-time PCR-based and dot-blot assays. The number of
green-forming units can be assessed by serial dilution on 293
cells. AAV vector can be produced by the AAV TIGEM Vector Core.
[0399] Six-week-old mdx mice can be administered with
3-4.times.10.sup.12 genome copies of AAV vector via tail vein. Six
and 12 weeks after virus administration, animals can be killed, and
muscles from different districts can be harvested. EGFP analysis
and dissections can be performed under a fluorescent
stereomicroscope (Leica MZ16FA).
[0400] Use of a viral transfer vector produced according to the
above, as described in Denti et al., 3758-3763, PNAS, Mar. 7, 2006,
vol. 103, no. 10, resulted in persistent exon skipping in mdx mice
by tail vein injection. Systemic delivery of the vector resulted in
effective body-wide colonization, significant recovery of
functional properties in vivo, and lower creatine kinase serum
levels. The results suggest that there was a decrease in muscle
wasting.
Example 36: Viral Transfer Vector with an Exon Skipping Transgene
(Prophetic)
[0401] Different U7snRNA constructs specific to certain exons can
be engineered, such as from U7smOPT-SD23/BP22 (modified murine
U7snRNA gene). Antisense sequences targeting certain exons can be
replaced by antisense sequences targeting exons of dystrophin mRNA
that induce exon skipping as antisense oligonucleotides. Sequences
can be inserted into U7snRNA constructs. Resulting U7snRNA
fragments can then be introduced either in a lentiviral vector
construct for further lentiviral production or into an AAV vector
construct for AAV production.
[0402] Lentiviral vectors can be based on pRRLcPPT-hPGK-eGFP-WPRE
constructs where the hPGK-GFP cassette is removed and replaced with
the U7snRNA construct. Lentiviral vectors can be generated by
transfection into 293T cells of a packaging construct,
pCMV.DELTA.R8.74, a plasmid producing the vesicular stomatosis
virus-G envelope (pMD.G) and the vector itself as previously
described. Viral titers (infectious particles) can be determined by
transduction of NIH3T3 cells with serial dilutions of the vector
preparation in a 12-well plate. Seventy-two hours later, genomic
DNA from transduced cells can be extracted using a genomic DNA
purification kit (Qiagen, Crawley, UK). The infectious particles
titer (infectious particle/ml) can be determined by quantitative
real-time PCR as described elsewhere.
[0403] For subsequent AAV vector production, different U7snRNA
fragments can be introduced at the XbaI site of the pSMD2 AAV2
vector. AAV2/1 pseudotyped vectors can be prepared by
cotransfection in 293 cells of pAAV2-U7snRNA, pXX6 encoding
adenovirus helper functions and pAAV 1pITRCO2 that contains the
AAV2 rep and AAV1 cap genes. Vector particles can be purified on
Iodixanol gradients from cell lysates obtained 48 hours after
transfection and titers can be measured by quantitative real time
PCR.
[0404] As described in Goyenvalle, et al. The American Society of
Gene & Cell Therapy, Vol. 20 No. 6, 1212-1221 June 2012, viral
transfer vectors such as those produced and encoding U7
small-nuclear RNAs with the above methods can induce efficient exon
skipping both in vitro and in vivo.
Example 37: Viral Transfer Vector with an Exon Skipping Transgene
(Prophetic)
[0405] An HSV transfer vector can prepared according the methods of
U.S. Publication No. 20090186003 and Example 28 above except that
the methodology can be altered so that the transgene is instead an
exon skipping transgene. The exon skipping transgene may be any one
of such transgenes as described herein or otherwise known in the
art.
Example 38: Viral Transfer Vector with an Exon Skipping Transgene
(Prophetic)
[0406] An HIV lentiviral transfer vector is prepared according the
methods of U.S. Publication No. 20150056696 and Example 26 above
except that the methodology can be altered so that the transgene is
instead an exon skipping transgene. The exon skipping transgene may
be any one of such transgenes as described herein or otherwise
known in the art.
Example 39: Viral Transfer Vector with a Gene Expression Modulating
Transgene (Prophetic)
[0407] Any one of the viral vectors described herein, such as in
the above Examples, may be used to produce a viral transfer vector
with a gene expression modulating transgene. Alternatively, and as
an example, the following provides a method for producing a viral
transfer vector with a specific gene expression modulating
transgene.
[0408] A viral transfer vector is produced according to the methods
described in Brown et al., Nat Med. 2006 May; 12(5):585-91.
Briefly, a plasmid is constructed using reverse transcription of
RNA, quantitative PCR analysis to quantify the concentration of
mRNA, and GAPDH expression for normalization. VSV-pseudotyped
third-generation lentiviral vectors (LVs) are produced by transient
four-plasmid cotransfection into 293T cells and purified by
ultracentrifugation. Vector particles can be measured by HIV-1 gag
p24 antigen immunocapture.
[0409] As described in Brown et al., Nat Med. 2006 May;
12(5):585-91, such lentiviral vectors encoding target sequences of
endogenous miRNAs were shown to result in the production of miRNAs
that could segregate gene expression in different tissues. Evidence
of miRNA regulation was provided and demonstrates that such vectors
may be used in therapeutic applications.
Example 40: Viral Transfer Vector with a Gene Expression Modulating
Transgene (Prophetic)
[0410] To produce an AAV2/1 serotype vector encoding an miRNA-based
hairpin against a gene (e.g., huntingtin gene; AAV2/1-miRNA-Htt),
the cDNA for the specified gene (e.g., human HTT), can be cloned
into a shuttle plasmid containing the AAV2 inverted terminal
repeats (ITRs) and a 1.6-kb cytomegalovirus enhancer/chicken
b-actin (CBA) promoter. Control vectors can also be developed and
contain either an empty vector backbone (e.g., AAV2/1-Null) or
express a reporter such as enhanced green fluorescent protein under
the control of the same promoter (AAV2/1-eGFP). Viral transfer
vectors can be generated by triple-plasmid cotransfection of a cell
line, such as human 293 cells, and the recombinant virions can then
be column-purified as previously described in Stanek et al., Human
Gene Therapy. 2014; 25:461-474. The resulting titer of
AAV2/1-miRNA-Htt can then be determined using quantitative PCR.
[0411] Data generated using such viral transfer vectors, as
described in Stanek et al., Human Gene Therapy. 2014; 25:461-474,
demonstrated that AAV-mediated RNAi can be effective at transducing
cells in the striatum and can partially reduce the levels of both
wild-type and mutant Htt in this region.
Example 41: Viral Transfer Vector with a Gene Expression Modulating
Transgene (Prophetic)
[0412] The CD81 gene can be amplified by reverse transcription.
cDNA can be PCR amplified with appropriate primers. The forward
primer can contain a BamHI (Biolabs, Allschwill, Switzerland)
restriction site followed by a 5' CD81 cDNA-specific sequence; the
reverse primer can contain a 3' CD81 cDNA-specific sequence, a 6
His-tag, a stop codon and an Xho I (Biolabs) restriction site. The
PCR product can be digested and cloned into similar sites in
pTK431. The pTK431 is a self-inactivating HIV-1 vector which
contains the entire tet-off-inducible system, the central
polypurine tract (cPPT) and the woodchuck hepatitis virus
post-transcriptional regulatory element. Plasmids can be CsCl.sub.2
purified.
[0413] Targets can be designed according to the CD81 mRNA sequence
based on Hannon's design criterion
(katandincshl.org:9331/RNAi/html/rnai.html). Using the pSilencer
1.0-U6 (Ambion) as a template and a U6 promoter-specific forward
primer containing a restriction site, each shRNA target can be
added to the mouse U6 promoter by PCR. The PCR product can be
digested, cloned into similar sites in pTK431 and sequenced to
verify the integrity of each construct.
[0414] The vector plasmids, together with a packaging construct
plasmid p.DELTA.NRF and the envelope plasmid pMDG-VSVG, can be
cotransfected into HEK293T cells to produce viral particles. The
viral titres can be determined by p24 antigen measurements (KPL,
Lausanne, Switzerland).
[0415] As shown in Bahi, et al. J. Neurochem. (2005) 92, 1243-1255,
lentiviruses expressing short hairpin RNA (shRNA) targeted against
CD81 (Lenti-CD81-shRNAs) resulted in gene silencing after infection
of HEK293T cells in vitro. In addition, in vivo delivery of
Lenti-CD81-shRNA resulted in silencing of endogenous CD81.
Example 42: Viral Transfer Vector with a Gene Expression Modulating
Transgene (Prophetic)
[0416] Any one of the viral vectors described herein, such as in
the above Examples, may be used to produce a viral transfer vector
with a gene expression modulating transgene that encodes a RNAi
agent. An example of a RNAi agent that can be encoded by a gene
expression modulating transgene as provided herein is described
below.
[0417] An expression construct can include a promoter driving the
expression of three or more individual shRNA species. The synthesis
of small nuclear RNAs and transfer RNAs can be directed by RNA
polymerase III (pol III) under the control of pol III-specific
promoters. Because of the relatively high abundance of transcripts
directed by these regulatory elements, pol III promoters, including
those derived from the U6 and H1 genes, can be used to drive the
expression of 1-x RNAi (see, e.g., Domitrovich and Kunkel. Nucl.
Acids Res. 31(9): 2344-52 (2003); Boden, et al. Nucl. Acids Res.
31(17): 5033-38 (2003a); and Kawasaki, et al. Nucleic Acids Res.
31(2): 700-7 (2003)). RNAi expression constructs using the U6
promoter can comprise three RNAi agents targeting three different
regions of the HCV genome. Further examples of RNAi agents that may
be encoded by a gene expression modulating transgene include any
one of the RNAi agents described herein.
Example 43: Viral Transfer Vector with a Gene Expression Modulating
Transgene (Prophetic)
[0418] Any one of the viral vectors described herein, such as in
the above Examples, may be used to produce a viral transfer vector
with a gene expression modulating transgene that encodes a Serpinal
RNAi agent, such as one of such agents described in U.S. Patent
Publication No. 20140350071. A viral transfer vector with such a
transgene can be produced following similar methodology as provided
herein or otherwise known in the art.
[0419] For example, adeno-associated virus (AAV) vectors may be
used (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);
U.S. Pat. No. 5,436,146). The iRNA can be expressed as two
separate, complementary single-stranded RNA molecules from a
recombinant AAV vector having, for example, either the U6 or H1 RNA
promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV
vectors for expressing the dsRNA featured in the invention, methods
for constructing the recombinant AV vector, and methods for
delivering the vectors into target cells are described in Samulski
R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996),
J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63:
3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International
Patent Application No. WO 94/13788; and International Patent
Application No. WO 93/24641, the entire disclosures of such
information are herein incorporated by reference.
Example 44: Establishing an Anti-Viral Transfer Vector Attenuated
Response in a Subject (Prophetic)
[0420] Any one of the viral transfer vectors provided herein, such
as any one of the Examples, is administered concomitantly, such as
simultaneously i.v., i.m., s.c. or i.p., with any one of the
antigen-presenting cell targeted immunosuppressants as provided
herein, such as any one of the Examples, that is also administered
i.v., i.m., s.c. or i.p., respectively. The administration occurs
according to a protocol, including at least the frequency and dose
of the viral transfer vector and antigen-presenting cell targeted
immunosuppressant, that establishes an anti-viral transfer vector
attenuated response in the subject. The subject may be any one of
the subjects described herein, such as one that does not have a
pre-existing immunity to the viral transfer vector or one in which
repeated administration of the viral transfer vector is
desired.
[0421] In some embodiments, when the anti-viral transfer vector
attenuated response is a T cell response against the viral transfer
vector, the viral transfer vector is administered to the subject
without an antigen-presenting cell targeted immunosuppressant prior
to the concomitant administration of the antigen-presenting cell
targeted immunosuppressant and viral transfer vector. In such
embodiments, one or more repeat doses of the viral transfer vector
is administered to the subject subsequent to both the concomitant
administration and the administration of the viral transfer vector
prior thereto.
[0422] In some embodiments, when the anti-viral transfer vector
attenuated response is a B cell response against the viral transfer
vector, the subject is not administered the viral transfer vector
prior to the concomitant administration of the viral transfer
vector and antigen-presenting cell targeted immunosuppressant. In
such embodiments, one or more repeat doses of the viral transfer
vector is administered to the subject and each repeat dose is
concomitantly administered with the antigen-presenting cell
targeted immunosuppressant.
[0423] In other embodiments, when the anti-viral transfer vector
attenuated response is an anti-viral transfer vector antibody
response, the subject is not administered the viral transfer vector
prior to the concomitant administration of the viral transfer
vector and antigen-presenting cell targeted immunosuppressant. In
such embodiments, one or more repeat doses of the viral transfer
vector is administered to the subject and each repeat dose is
concomitantly administered with the antigen-presenting cell
targeted immunosuppressant.
[0424] The method for determining the level of antibodies may be
with the use of an ELISA assay. Assays for antigen-specific B cell
or T cell recall responses include, but are not limited to,
ELISpot, intracellular cytokine staining, cell proliferation, and
cytokine production assays.
[0425] In any one of the embodiments, the anti-viral transfer
vector attenuated response is evaluated after the concomitant
administration of the viral transfer vector and the
antigen-presenting cell targeted immunosuppressant.
[0426] In any one of the embodiments, a protocol for establishing
the anti-viral transfer vector attenuated response may be
determined. In such an embodiment, the protocol is determined in
another subject, such as a test subject. The protocol so determined
can be used to treat other subjects in need of treatment with the
viral transfer vector.
Example 45: Determining a Level of Pre-existing Immunity in a
Subject Prior to Administration of a Viral Transfer Vector
(Prophetic)
[0427] A sample, such as a blood sample, may be obtained from a
subject that is in need of treatment with a viral transfer vector
as provided herein, such as the viral transfer vector of any one of
the viral transfer vectors provided herein, such as in any one of
the Examples.
[0428] With the sample from the subject, the level of antibodies,
such as neutralizing antibodies or antigen recall responses of
immune cells, such as T cells or B cells, can be determined. The
method for determining the level of antibodies may be with the use
of an ELISA assay. Assays for antigen-specific B cell or T cell
recall responses include, but are not limited to, ELISpot,
intracellular cytokine staining, cell proliferation, and cytokine
production assays. The recall response can be assessed by
contacting the sample with the viral transfer vector or an antigen
thereof. Alternatively, the recall response can also be assessed by
taking the sample from the subject after administration of the
viral transfer vector or an antigen thereof to the subject and then
determining the level of antibodies or a B cell or T cell recall
response that was generated.
[0429] In some embodiments, where the subject does not have a
pre-existing immunity to the viral transfer vector, determined by
the measurement of a level of anti-viral transfer vector antibodies
in the subject (or a B cell response), the subject is administered,
i.v., i.m., s.c. or i.p., any one of the viral transfer vectors
provided herein, such as in any one of the Examples, concomitantly,
such as simultaneously, with any one of the antigen-presenting cell
targeted immunosuppressants provided herein, such as in any one of
the Examples. The antigen-presenting cell targeted
immunosuppressant is administered by the same route.
[0430] In other embodiments, where the subject does not have a
pre-existing immunity to the viral transfer vector, determined by
the level of a T cell response against the viral transfer vector in
the subject, the antigen-presenting cell targeted immunosuppressant
and viral transfer vector are concomitantly, such as
simultaneously, administered, i.v., i.m., s.c. or i.p., to the
subject after the subject is administered a dose of the viral
transfer vector without concomitant administration of the
antigen-presenting cell targeted immunosuppressant.
[0431] In any one of the embodiments, one or more repeat doses of
the viral transfer vector is/are administered to the subject. These
repeat doses may be concomitantly administered with the
antigen-presenting cell targeted immunosuppressant.
Example 46: Escalating Transgene Expression of a Viral Transfer
Vector in a Subject (Prophetic)
[0432] Any one of the viral transfer vectors provided herein, such
as in any one of the Examples, is administered concomitantly, such
as simultaneously, i.v., i.m., s.c. or i.p., with any one of the
antigen-presenting cell targeted immunosuppressants as provided
herein, such as in any one of the Examples, according to a
frequency and dosing that escalates transgene expression (the
transgene being delivered by the viral transfer vector). This can
be determined by measuring transgene protein concentrations in
various tissues or systems of interest in the subject. Whether or
not transgene expression is escalated can be determined according
to a method, such as that described in the Examples above. The
administration occurs according to the frequency and dose of the
viral transfer vector and antigen-presenting cell targeted
immunosuppressant, that escalates transgene expression in the
subject. The subject may be any one of the subjects described
herein, such as one that does not have a pre-existing immunity to
the viral transfer vector or one in which repeated administration
of the viral transfer vector is desired.
[0433] In any one of the embodiments, the frequency and dose that
achieves escalating transgene expression is determined in another
subject, such as a test subject. This can also be determined by
measuring transgene protein concentrations in various tissues or
systems of interest in the other subject, such as with a method as
described above. If the frequency and dose achieves escalated
transgene expression, as determined by the measured transgene
protein concentrations, in the other subject, the concomitant, such
as simultaneous, administration of the viral transfer vector and
antigen-presenting cell targeted immunosuppressant according to the
frequency and dose can be used to treat other subjects in need of
treatment with the viral transfer vector.
Example 47: Repeated, Concomitant Administration with Lower Doses
(Prophetic)
[0434] As provided herein, a subject can be evaluated for the level
of a pre-exisiting immunity to any one of the viral transfer
vectors provided herein, such as any one of the viral transfer
vectors any one of the Examples. Alternatively, a clinician may
evaluate a subject and determine whether or not, if administered
the viral transfer vector, the subject is expected to develop an
anti-viral transfer vector immune response if the viral transfer
vector is repeatedly administered to the subject. This
determination may be made based on the likelihood that the viral
transfer vector will produce such a result and may be based on such
a result in other subects, such as test subjects, information about
the virus that was used to generate the viral transfer vector,
information about the subject, etc. Generally, if the expectation
is that an anti-viral transfer vector immune response is the likely
result, the clinician selects a certain dose of the viral transfer
vector as a result of the expectation. However, in light of the
inventor's findings, a clinician may now select and use lower doses
of the viral transfer vector than would have been selected for the
subject. Benefits of lower doses can include reduced toxicity
associated with dosing of the viral transfer vector, and reduction
of other off-target effects.
[0435] Accordingly, any one of the subjects provided herein can be
treated with repeated, concomitant, such as simultaneous,
administration of any one of the viral transfer vectors provided
herein and any one of the antigen-presenting cell targeted
immunosuppressants provided herein where the doses of the viral
transfer vector are selected to be less than the dose of the viral
transfer vector that would have been selected for the subject if
the subject were expected to develop anti-viral transfer vector
immune responses due to repeated dosing of the viral transfer
vector. Each dose of the viral transfer vector of the repeated,
concomitant administration may be less than what would have
otherwise been selected.
Sequence CWU 1
1
719PRTArtificial SequenceSynthetic Polypeptide 1Asn Ser Leu Ala Asn
Pro Gly Ile Ala 1 5 29PRTArtificial SequenceSynthetic Polypeptide
2His Tyr Leu Ser Thr Gln Ser Ala Leu 1 5 319DNAArtificial
SequenceSynthetic Polynucleotide 3ggtgtattgc atgatatgt
19444DNAArtificial SequenceSynthetic Polynucleotide 4cgaaatttca
ggtaagccga ggttatgaga tcttgggcct ctgc 44550DNAArtificial
SequenceSynthetic Polynucleotide 5gaactttgca gagcctcaaa attaaatagg
gcaggggaga taccatgatc 50629DNAArtificial SequenceSynthetic
Polynucleotide 6ctagctagcg gtaaggacca gcttctttg 29727DNAArtificial
SequenceSynthetic Polynucleotide 7ctagctagcg gttagcgtac agtctac
27
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