U.S. patent application number 17/351113 was filed with the patent office on 2021-12-09 for methods for the treatment of danon disease and other disorders of autophagy.
The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Eric D. Adler, Sherin Hashem, Bradley Nelson.
Application Number | 20210379201 17/351113 |
Document ID | / |
Family ID | 1000005782762 |
Filed Date | 2021-12-09 |
United States Patent
Application |
20210379201 |
Kind Code |
A1 |
Adler; Eric D. ; et
al. |
December 9, 2021 |
METHODS FOR THE TREATMENT OF DANON DISEASE AND OTHER DISORDERS OF
AUTOPHAGY
Abstract
This disclosure provides gene therapy vectors comprising a
polynucleotide encoding one or more isoforms of lysosome-associated
membrane protein 2 (LAMP-2), and methods of using such gene therapy
vectors for the treatment of Danon disease and other autophagy
disorders.
Inventors: |
Adler; Eric D.; (La Jolla,
CA) ; Nelson; Bradley; (La Jolla, CA) ;
Hashem; Sherin; (La Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA |
Oakland |
CA |
US |
|
|
Family ID: |
1000005782762 |
Appl. No.: |
17/351113 |
Filed: |
June 17, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16070233 |
Jul 13, 2018 |
11065347 |
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PCT/US2017/014164 |
Jan 19, 2017 |
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17351113 |
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62280269 |
Jan 19, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/86 20130101;
C12N 2750/14143 20130101; A61P 9/00 20180101; A61K 9/0019 20130101;
A61P 3/00 20180101; C07K 14/70596 20130101; A01K 2227/105 20130101;
C12N 15/8645 20130101; A01K 2267/0306 20130101; A01K 2217/075
20130101; A61K 48/005 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07K 14/705 20060101 C07K014/705; C12N 15/86 20060101
C12N015/86; C12N 15/864 20060101 C12N015/864; A61P 3/00 20060101
A61P003/00; A61P 9/00 20060101 A61P009/00; A61K 9/00 20060101
A61K009/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under
HL107755 awarded by the National Institutes of Health. The
government has certain rights in this invention.
Claims
1. A gene therapy vector comprising an expression cassette
comprising a polynucleotide encoding one or more isoforms of
lysosome-associated membrane protein 2 (LAMP-2).
2. The gene therapy vector of claim 1, wherein the vector is a
viral vector from a virus selected from the group consisting of
adenovirus, retrovirus, lentivirus, herpesvirus, and
adeno-associated virus (AAV).
3. The gene therapy vector of claim 2, wherein the vector is from
one or more of adeno-associated virus (AAV) serotypes 1-11, or any
subgroups thereof.
4. The gene therapy vector of claim 2, wherein the viral vector is
encapsulated in an anionic liposome.
5. The gene therapy vector of claim 1, wherein the vector is a
non-viral vector selected from the group consisting of naked DNA, a
cationic liposome complex, a cationic polymer complex, a cationic
liposome-polymer complex, and an exosome.
6. The gene therapy vector of claim 1, wherein the expression
cassette comprises operably linked in the 5' to 3' direction, a
first inverse terminal repeat, an enhancer/promoter region, the
polynucleotide encoding one or more isoforms of LAMP-2, a 3'
untranslated region including a polyadenylation signal, and a
second inverse terminal repeat.
7. The gene therapy vector of claim 6, wherein the promoter is
selected from the group consisting of cytomegalovirus (CMV)
promoter and CAG promoter.
8. The gene therapy vector of claim 1, wherein the polynucleotide
comprises DNA or cDNA.
9. The gene therapy vector of claim 1, wherein the polynucleotide
encoding one or more isoforms of LAMP-2 comprises one or more human
LAMP-2 isoforms.
10. The gene therapy vector of claim 1, wherein the polynucleotide
encoding one or more isoforms of LAMP-2 comprises one or more
LAMP-2 isoforms selected from the group consisting of LAMP-2A,
LAMP-2B, and LAMP-2C.
11. The gene therapy vector of claim 1, wherein the polynucleotide
encoding one or more isoforms of LAMP-2 has at least about 90%
sequence identity to one or more of SEQ ID NO:1, SEQ ID NO:2, and
SEQ ID NO:3.
12. The gene therapy vector of claim 11, wherein the polynucleotide
encoding one or more isoforms of LAMP-2 comprises one or more of
SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
13. A method of preventing, mitigating, ameliorating, reducing,
inhibiting, eliminating, and/or reversing one or more symptoms of
Danon disease or another autophagy disorder in a subject in need
thereof, comprising administering to the subject a gene therapy
vector of claim 1.
14. A method of preventing, mitigating, ameliorating, reducing,
inhibiting, eliminating, and/or reversing one or more symptoms of
Danon disease or another autophagy disorder in a subject in need
thereof, comprising administering to the subject an
adeno-associated virus (AAV) vector comprising an expression
cassette comprising a polynucleotide encoding one or more isoforms
of lysosome-associated membrane protein 2 (LAMP-2).
15. The method of claim 13, wherein the vector is administered via
a route selected from the group consisting of intravenous,
intra-arterial, intracardiac, intracoronary, intramyocardial,
intrarenal, intraurethral, epidural, and intramuscular.
16. The method of claim 13, wherein the vector is administered
multiple times.
17. The method of claim 13, wherein the autophagy disorder is
selected from the group consisting of end-stage heart failure,
myocardial infarction, drug toxicities, diabetes, end-stage renal
failure, and aging.
18. The method of claim 13, wherein the subject is exhibiting
symptoms of Danon disease or another autophagy disorder.
19. The method of claim 13, wherein the subject has been identified
as having reduced or non-detectable LAMP-2 expression.
20. The method of claim 13, wherein the subject has been identified
as having a mutated LAMP-2 gene.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 16/070,233, filed Jul. 13, 2018, now U.S. Pat.
No. 11,065,347, which is a U.S. National Phase application of
International Patent Application PCT/US2017/014164 filed Jan. 19,
2017, which claims priority pursuant to 35 U.S.C. .sctn. 119(e) to
U.S. Provisional Patent Application Ser. No. 62/280,269, filed Jan.
19, 2016, both of which are hereby incorporated by reference in its
entirety.
BACKGROUND
[0003] Danon disease is a familial cardiomyopathy associated with
impaired autophagy due to mutations in the gene encoding
lysosome-associated membrane protein 2 (LAMP-2, also known as
CD107b). Emerging evidence has highlighted the importance of
autophagy in regulating cardiomyocyte bioenergetics, function, and
survival. However, the mechanisms responsible for cellular
dysfunction and death in cardiomyocytes with impaired autophagic
flux remain unclear. In a previous study, to investigate the
molecular mechanisms responsible for Danon disease, we created
human induced pluripotent stem cells (hiPSCs) from two patients
with different LAMP-2 mutations (Hashem, et al., Stem Cells. 2015
July; 33(7):2343-50). Danon hiPSC-derived cardiomyocytes
(hiPSC-CMs) exhibited impaired autophagic flux and key features of
heart failure such as increased cell size, increased expression of
natriuretic peptides, and abnormal calcium handling compared to
control hiPSC-CMs. Additionally, Danon hiPSC-CMs demonstrated
excessive amounts of mitochondrial oxidative stress and apoptosis.
Using the sulfhydryl antioxidant N-acetylcysteine to scavenge free
radicals resulted in a significant reduction in apoptotic cell
death in Danon hiPSC-CMs. We also used a lentiviral vector to
introduce the coding sequence of the LAMP-2B isoform under the
control of a doxycycline-inducible promoter in one of the Danon
hiPSC lines. Overexpression of LAMP-2B with the addition of
doxycycline also reduced oxidative stress levels and apoptotic cell
death in Danon hiPSC-CMs, confirming the importance of LAMP-2B in
disease pathophysiology. In summary, we modeled Danon disease using
hiPSC-CMs from patients with mutations in LAMP-2, allowing us to
gain mechanistic insight into the pathogenesis of this disease. We
demonstrated that LAMP-2 deficiency led to an impairment in
autophagic flux, which resulted in excessive oxidative stress, and
subsequent cardiomyocyte apoptosis. Scavenging excessive free
radicals with antioxidants and overexpression of LAMP-2B improved
the disease phenotype in vitro. The prior art does not disclose
effective treatment strategies for Danon disease or other diseases
associated with autophagy, thus the in vivo studies presented
herein were necessary to validate the approach.
SUMMARY
[0004] In one aspect, provided are gene therapy vectors, e.g., for
use in systemically or locally increasing the expression of one or
more isoforms of lysosome-associated membrane protein 2 (LAMP-2) in
a subject. The gene therapy vectors find use in preventing,
mitigating, ameliorating, reducing, inhibiting, and/or treating one
or more symptoms of Danon disease or another disorder of
insufficient autophagic flux. In varying embodiments, the gene
therapy vectors comprise an expression cassette comprising a
polynucleotide encoding one or more isoforms of lysosome-associated
membrane protein 2 (LAMP-2). In varying embodiments, the vector is
a viral vector. In varying embodiments, the viral vector is from a
virus selected from the group consisting of adenovirus, retrovirus,
lentivirus, herpesvirus and adeno-associated virus (AAV). In
varying embodiments, the vector is from one or more of
adeno-associated virus (AAV) serotypes 1-11, or any subgroups
thereof. In varying embodiments, the viral vector is encapsulated
in an anionic liposome. In varying embodiments, the vector is a
non-viral vector. In varying embodiments, the non-viral vector is
selected from the group consisting of naked DNA, a cationic
liposome complex, a cationic polymer complex, a cationic
liposome-polymer complex, and an exosome. In varying embodiments,
the expression cassette comprises operably linked in the 5' to 3'
direction (from the perspective of the mRNA to be transcribed), a
first inverse terminal repeat, an enhancer, a promoter, the
polynucleotide encoding one or more isoforms of LAMP-2, a 3'
untranslated region, polyadenylation (polyA) signal, and a second
inverse terminal repeat. In varying embodiments, the promoter is
selected from the group consisting of cytomegalovirus (CMV)
promoter and chicken-beta actin (CAG) promoter. In varying
embodiments, the polynucleotide comprises DNA or cDNA. In varying
embodiments, the polynucleotide encoding one or more isoforms of
LAMP-2 comprises one or more human LAMP-2 isoforms. In varying
embodiments, the polynucleotide encoding one or more isoforms of
LAMP-2 comprises one or more LAMP-2 isoforms selected from the
group consisting of LAMP-2A, LAMP-2B, and LAMP-2C. In varying
embodiments, the polynucleotide encoding one or more isoforms of
LAMP-2 has at least about 90% sequence identity, e.g., at least
about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence
identity, to one or more of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID
NO:3. In varying embodiments, the polynucleotide encoding one or
more isoforms of LAMP-2 comprises one or more of SEQ ID NO:1, SEQ
ID NO:2 and SEQ ID NO:3.
[0005] In a further aspect, provided are methods of preventing,
mitigating, ameliorating, reducing, inhibiting, eliminating and/or
reversing one or more symptoms of Danon disease or another
autophagy disorder in a subject in need thereof, comprising
administering to the subject a gene therapy vector as described
above and herein (see for example FIG. 2). In a further aspect,
provided are methods of preventing, mitigating, ameliorating,
reducing, inhibiting, eliminating and/or reversing one or more
symptoms of Danon disease or another autophagy disorder in a
subject in need thereof, comprising administering to the subject an
adeno-associated virus (AAV) vector comprising an expression
cassette comprising a polynucleotide encoding one or more isoforms
of lysosome-associated membrane protein 2 (LAMP-2). In varying
embodiments, the vector is administered via a route selected from
the group consisting of intravenous, intraarterial, intracardiac,
intracoronary, intramyocardial, intrarenal, intraurethral,
epidural, intracranial, subcutaneous, and intramuscular. In varying
embodiments, the vector is delivered or administered via a physical
or mechanical method selected from the group consisting of
microinjection, jet injection, particle bombardment, hydrodynamic
infusion, electroporation, sonoporation, laser irradiation,
magnetofection. In varying embodiments, the vector is administered
multiple times with or without immunosuppression or plasmapheresis
of the patient. In varying embodiments, the autophagy disorder is
selected from the group consisting of end-stage heart failure,
myocardial infarction, drug toxicities, diabetes, end-stage renal
failure, and aging. In varying embodiments, the subject is human.
In varying embodiments, the subject is exhibiting symptoms of Danon
disease or another autophagy disorder. In varying embodiments, the
subject has been identified as having reduced or non-detectable
LAMP-2 expression. In varying embodiments, the subject has been
identified as having a mutated LAMP-2 gene.
Definitions
[0006] The term "Danon disease" refers to an X-linked dominant
skeletal and cardiac muscle disorder with multisystem clinical
manifestations. Danon disease mutations lead to an absence of
lysosome-associated membrane protein 2 (LAMP-2) protein expression.
Major clinical features include skeletal and cardiac myopathy,
cardiac conduction abnormalities, cognitive difficulties, and
retinal disease. Men are typically affected earlier and more
severely than women.
[0007] The terms "lysosome-associated membrane protein 2" and
"LAMP-2" interchangeably refer to nucleic acids and polypeptide
polymorphic variants, alleles, mutants, and interspecies homologs
that: (1) have an amino acid sequence that has greater than about
90% amino acid sequence identity, for example, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity,
preferably over a region of at least about 25, 50, 100, 200, 300,
400, or more amino acids, or over the full-length, to an amino acid
sequence encoded by a LAMP-2 nucleic acid (see, e.g., GenBank
Accession Nos. NM_002294.2 (isoform A). NM_013995.2 (isoform B),
NM_001122606.1 (isoform C)) or to an amino acid sequence of a
LAMP-2 polypeptide (see e.g. GenBank Accession Nos. NP_002285.1
(isoform A), NP_054701.1 (isoform B), NP_001116078.1 (isoform C));
(2) bind to antibodies, e.g., polyclonal antibodies, raised against
an immunogen comprising an amino acid sequence of a LAMP-2
polypeptide (e.g., LAMP-2 polypeptides described herein); or an
amino acid sequence encoded by a LAMP-2 nucleic acid (e.g., LAMP-2
polynucleotides described herein), and conservatively modified
variants thereof; (3) specifically hybridize under stringent
hybridization conditions to an anti-sense strand corresponding to a
nucleic acid sequence encoding a LAMP-2 protein, and conservatively
modified variants thereof; (4) have a nucleic acid sequence that
has greater than about 90%, preferably greater than about 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher nucleotide sequence
identity, preferably over a region of at least about 25, 50, 100,
200, 500, 1000, 2000 or more nucleotides, or over the full-length,
to a LAMP-2 nucleic acid (e.g., LAMP-2 polynucleotides, as
described herein, and LAMP-2 polynucleotides that encode LAMP-2
polypeptides, as described herein).
[0008] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0009] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .alpha.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0010] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0011] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence.
[0012] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0013] The following eight groups each contain amino acids that are
conservative substitutions for one another: [0014] 1) Alanine (A),
Glycine (G); [0015] 2) Aspartic acid (D), Glutamic acid (E); [0016]
3) Asparagine (N), Glutamine (Q); [0017] 4) Arginine I, Lysine (K);
[0018] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
[0019] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); and
[0020] 7) Serine (S), Threonine (T)
[0021] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When the term is applied to
double-stranded molecules it is used to denote overall length and
will be understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the two strands
of a double-stranded polynucleotide may differ slightly in length
and that the ends thereof may be staggered as a result of enzymatic
cleavage; thus all nucleotides within a double-stranded
polynucleotide molecule may not be paired.
[0022] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., share at least about 80% identity, for example, at
least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
identity over a specified region to a reference sequence, e.g.,
LAMP-2 polynucleotide or polypeptide sequence as described herein,
when compared and aligned for maximum correspondence over a
comparison window, or designated region as measured using one of
the following sequence comparison algorithms or by manual alignment
and visual inspection. Such sequences are then said to be
"substantially identical." This definition also refers to the
compliment of a test sequence. Preferably, the identity exists over
a region that is at least about 25 amino acids or nucleotides in
length, for example, over a region that is 50, 100, 200, 300, 400
amino acids or nucleotides in length, or over the full-length of a
reference sequence.
[0023] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters. For sequence comparison of nucleic acids and
proteins to LAMP-2 nucleic acids and proteins, the BLAST and BLAST
2.0 algorithms and the default parameters are used.
[0024] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Ausubel et al., eds.,
Current Protocols in Molecular Biology (1995 supplement)). Examples
of algorithms that are suitable for determining percent sequence
identity and sequence similarity are the BLAST and BLAST 2.0
algorithms, which are described in Altschul et al., J. Mol. Biol.
215:403-410 (1990) and Altschul et al., Nucleic Acids Res.
25:3389-3402 (1977), respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information (on the worldwide web at
ncbi.nlm.nih.gov/).
[0025] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as described below. Thus, a polypeptide is
typically substantially identical to a second polypeptide, for
example, where the two peptides differ only by conservative
substitutions. Another indication that two nucleic acid sequences
are substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below. Yet another indication that two nucleic acid
sequences are substantially identical is that the same primers can
be used to amplify the sequence.
[0026] As used herein, "administering" refers to local and systemic
administration, e.g., including enteral, parenteral, pulmonary, and
topical/transdermal administration. Routes of administration for
compounds (e.g., polynucleotide encoding one or more LAMP-2
isoforms) that find use in the methods described herein include,
e.g., oral (per os (P.O.)) administration, nasal or inhalation
administration, administration as a suppository, topical contact,
transdermal delivery (e.g., via a transdermal patch), intrathecal
(IT) administration, intravenous ("iv") administration,
intraperitoneal ("ip") administration, intramuscular ("im")
administration, intralesional administration, or subcutaneous
("sc") administration, or the implantation of a slow-release device
e.g., a mini-osmotic pump, a depot formulation, etc., to a subject.
Administration can be by any route including parenteral and
transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal).
Parenteral administration includes, e.g., intravenous,
intramuscular, intraarterial, intrarenal, intraurethral,
intracardiac, intracoronary, intramyocardial, intradermal,
epidural, subcutaneous, intraperitoneal, intraventricular,
ionophoretic and intracranial. Other modes of delivery include, but
are not limited to, the use of liposomal formulations, intravenous
infusion, transdermal patches, etc.
[0027] The terms "systemic administration" and "systemically
administered" refer to a method of administering a compound or
composition to a mammal so that the compound or composition is
delivered to sites in the body, including the targeted site of
pharmaceutical action, via the circulatory system. Systemic
administration includes, but is not limited to, oral, intranasal,
rectal and parenteral (e.g., other than through the alimentary
tract, such as intramuscular, intravenous, intra-arterial,
transdermal and subcutaneous) administration.
[0028] The term "co-administering" or "concurrent administration",
when used, for example with respect to the compounds (e.g., LAMP-2
polynucleotides) and/or analogs thereof and another active agent,
refers to administration of the compound and/or analogs and the
active agent such that both can simultaneously achieve a
physiological effect. The two agents, however, need not be
administered together. In certain embodiments, administration of
one agent can precede administration of the other. Simultaneous
physiological effect need not necessarily require presence of both
agents in the circulation at the same time. However, in certain
embodiments, co-administering typically results in both agents
being simultaneously present in the body (e.g., in the plasma) at a
significant fraction (e.g., 20% or greater, e.g., 30% or 40% or
greater, e.g., 50% or 60% or greater, e.g., 70% or 80% or 90% or
greater) of their maximum serum concentration for any given
dose.
[0029] The term "effective amount" or "pharmaceutically effective
amount" refer to the amount and/or dosage, and/or dosage regime of
one or more compounds (e.g., gene therapy vectors) necessary to
bring about the desired result e.g., increased expression of one or
more LAMP-2 isoforms in an amount sufficient to reduce the ultimate
severity of a disease characterized by impaired or deficient
autophagy (e.g., Danon disease).
[0030] The phrase "cause to be administered" refers to the actions
taken by a medical professional (e.g., a physician), or a person
controlling medical care of a subject, that control and/or permit
the administration of the agent(s)/compound(s) at issue to the
subject. Causing to be administered can involve diagnosis and/or
determination of an appropriate therapeutic or prophylactic
regimen, and/or prescribing particular agent(s)/compounds for a
subject. Such prescribing can include, for example, drafting a
prescription form, annotating a medical record, and the like.
[0031] The phrase "in conjunction with" when used in reference to
the use of the active agent(s) described herein (e.g., one or more
isoforms of a LAMP-2 polynucleotide) in conjunction with one or
more other drugs described herein (e.g., an acetylcholinesterase
inhibitor) the active agent(s) and the other drug(s) are
administered so that there is at least some chronological overlap
in their physiological activity on the organism. When they are not
administered in conjunction with each other, there is no
chronological overlap in physiological activity on the organism. In
certain preferred embodiments, the "other drug(s)" are not
administered at all (e.g., not co-administered) to the
organism.
[0032] As used herein, the terms "treating" and "treatment" refer
to delaying the onset of, retarding or reversing the progress of,
reducing the severity of, or alleviating or preventing either the
disease or condition to which the term applies, or one or more
symptoms of such disease or condition.
[0033] The term "mitigating" refers to reduction or elimination of
one or more symptoms of that pathology or disease, and/or a
reduction in the rate or delay of onset or severity of one or more
symptoms of that pathology or disease, and/or the prevention of
that pathology or disease. In certain embodiments, the reduction or
elimination of one or more symptoms of pathology or disease can
include, e.g., measurable and sustained increase in the expression
levels of one or more isoforms of LAMP-2.
[0034] As used herein, the phrase "consisting essentially of"
refers to the genera or species of active pharmaceutical agents
recited in a method or composition, and further can include other
agents that, on their own do not have substantial activity for the
recited indication or purpose.
[0035] The terms "subject," "individual," and "patient"
interchangeably refer to a mammal, preferably a human or a
non-human primate, but also domesticated mammals (e.g., canine or
feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster,
guinea pig) and agricultural mammals (e.g., equine, bovine,
porcine, ovine). In various embodiments, the subject can be a human
(e.g., adult male, adult female, adolescent male, adolescent
female, male child, female child) under the care of a physician or
other healthworker in a hospital, psychiatric care facility, as an
outpatient, or other clinical context. In certain embodiments, the
subject may not be under the care or prescription of a physician or
other healthworker.
[0036] The terms "gene transfer" or "gene delivery" refer to
methods or systems for reliably inserting foreign DNA into host
cells. Such methods can result in transient expression of
non-integrated transferred DNA, extrachromosomal replication and
expression of transferred replicons (e.g. episomes), or integration
of transferred genetic material into the genomic DNA of host
cells.
[0037] By an "AAV vector" is meant a vector derived from an
adeno-associated virus serotype, including without limitation,
AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, etc. AAV vectors can have one
or more of the AAV wild-type genes deleted in whole or part, e.g.,
the rep and/or cap genes, but retain functional flanking inverted
terminal repeat (ITR) sequences. Functional ITR sequences are
necessary for the rescue, replication and packaging of the AAV
virion. Thus, an AAV vector is defined herein to include at least
those sequences required in cis for replication and packaging
(e.g., functional ITRs) of the virus. The ITRs need not be the
wild-type nucleotide sequences, and may be altered, e.g. by the
insertion, deletion or substitution of nucleotides, as long as the
sequences provide for functional rescue, replication and packaging.
AAV expression vectors are constructed using known techniques to at
least provide as operatively linked components in the direction of
transcription, control elements including a transcriptional
initiation region, the DNA of interest (i.e. the LAMP-2 gene) and a
transcriptional termination region.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIGS. 1A-O illustrate schematics of Adeno-Associated Virus
LAMP-2 Gene Delivery Constructs. The indicated lysosome-associated
membrane protein (LAMP) LAMP-2 coding region generally should be
understood to include the upstream ribosome binding sequence and
start codon, but in some embodiments the native element can be
substituted with a heterologous one. The upstream ribosome binding
site is not used in combinations with coding regions downstream of
an IRES or self-cleaving peptide. The start codon is not
necessarily present in coding regions downstream of a self-cleaving
peptide. FIGS. 1A-1F show vector genomes containing one LAMP-2
isoform. FIGS. 1G-1K show vector genomes containing two LAMP-2
isoforms. FIGS. 1L-1O show vector genomes containing all three
LAMP-2 isoforms. The following abbreviations are used in the
figure: ITR: Inverse terminal repeat; LAMP-2: Lysosomal associated
membrane protein type 2; UTR: Untranslated region; Poly A:
Polyadenylation signal; CAG: Promoter region comprising CMV
enhancer and CBA promoter sequences; CMV: Cytomegalovirus; CBA:
Chicken beta-actin; WPRE: Woodchuck hepatitis virus
posttranscriptional regulatory element; RBG: Rabbit beta-globin
polyadenylation signal; EF-1: Human elongation factor-1; IRES:
Internal ribosome entry site; P2A: 2A peptide.
[0039] FIG. 1A shows a schematic of a construct containing protein
coding information for one LAMP-2 isoform--A, B, or C--with generic
5' and 3' control regions.
[0040] FIG. 1B shows a schematic of the construct of some
embodiments and used in the Examples below consisting of the 5' and
3' inverse terminal repeat elements, a CAG promoter region
containing the CMV enhancer and CBA promoter sequences, the CBA
intron, the coding sequence for one of the LAMP-2 isoforms
including the upstream ribosomal binding sequence and start codon,
the WPRE sequence as the 3' UTR, and the rabbit beta-globin
polyadenylation signal.
[0041] FIG. 1C shows a schematic of a construct containing the
native human LAMP-2 promoter region. In some embodiments, this
construct is used to express the transgene in response to cellular
signals that would typically result in LAMP-2 expression in normal
cells.
[0042] FIG. 1D shows a schematic of a construct containing the
human elongation factor-1 alpha promoter. In some embodiments, this
construct is used to express the transgene constitutively under the
control of a human promoter region. Other constitutively active
human promoters could also be used in place of EF-1 alpha.
[0043] FIG. 1E shows a schematic of a construct containing a
cardiac-specific promoter such as, but not limited to, the cardiac
troponin T2 promoter. In some embodiments, this construct is used
to express the transgene only in cardiac tissue (and, for example,
avoid hepatic expression).
[0044] FIG. 1F shows a schematic of a construct containing a
muscle-specific promoter such as, but not limited to, the
creatinine muscle kinase promoter. In some embodiments, this
construct is used to express the transgene in cardiac and skeletal
muscle (and avoid extra-muscular expression).
[0045] FIG. 1G shows a schematic of a construct containing the
coding sequences for two LAMP-2 isoforms (using any potential
combination) under the control of different promoter regions. In
some embodiments, this construct (and those of FIGS. 1H to 1K) is
used to express two different LAMP-2 isoforms using the same viral
genome.
[0046] FIG. 1H shows a schematic of a construct containing the
sequences for two LAMP-2 isoforms (using any potential combination)
under the control of different promoter regions. One isoform is
coded in the (+) direction on the single-stranded AAV genome and
the other isoform is coded in (-) direction. In some embodiments,
this construct is used to express two different LAMP-2 isoforms on
separate DNA strands.
[0047] FIG. 1I shows a schematic of a construct containing the
coding sequences for two LAMP-2 isoforms (using any potential
combination) under the control of a promoter region and an internal
ribosome entry site, respectively, and followed by 3' UTR and Poly
A signals. In some embodiments, this construct is used to express
two different LAMP-2 isoforms using the same viral genome.
[0048] FIG. 1J shows a schematic of a construct containing the
coding sequences for two LAMP-2 isoforms (using any potential
combination) separated by a P2A cleavage site. In some embodiments,
this construct is used to express an mRNA encoding two different
LAMP-2 isoforms in a single polypeptide, which would spontaneously
cleave into the individual LAMP-2 protein isoforms following
translation via the 2A peptide self-cleavage, using the same viral
genome.
[0049] FIG. 1K shows a schematic of a construct containing the
coding sequence for LAMP-2 exons 1-8 followed by an intronic region
(containing all necessary splice signals), the exon 9 coding
sequence for one of the LAMP-2 isoforms with the 3' UTR and Poly A
signal, a second intronic region (containing all necessary splice
signals), and the exon 9 coding sequence for a different LAMP-2
isoform (in any potential combination) with the 3' UTR and Poly A
signal. In some embodiments, this construct is used to express
mRNAs for two different LAMP-2 isoforms via alternative splicing,
which is the mechanism that generates the different LAMP-2 mRNAs in
normal cells, using the same viral genome.
[0050] FIG. 1L shows a schematic of a construct containing the
coding sequences for all three LAMP-2 isoforms, with 3' UTR and
Poly A signals, under the control of different promoter regions. In
some embodiments, this construct is used to express all three of
the different LAMP-2 isoforms using the same viral genome and would
allow for restoration of all potential LAMP-2 functions.
[0051] FIG. 1M shows a schematic of a construct containing the
coding sequences for all three LAMP-2 isoforms, in any order, under
the control of a promoter region and two different internal
ribosome entry sites, followed by 3' UTR and Poly A signals. In
some embodiments, this construct is used to express all three
LAMP-2 isoforms using the same viral genome.
[0052] FIG. 1N shows a schematic of a construct containing the
coding sequences for all three LAMP-2 isoforms, in any potential
order, separated by a P2A cleavage sites. In some embodiments, this
construct is used to express an mRNA encoding the three different
LAMP-2 isoforms in a single polypeptide, which would spontaneously
cleave into the individual LAMP-2 protein isoforms following
translation via 2A peptide self-cleavage, using the same viral
genome.
[0053] FIG. 1O shows a schematic of a construct containing the
coding sequence for LAMP-2 exons 1-8 followed by an intronic region
(containing all necessary splice signals), the exon 9 coding
sequence for one of the LAMP-2 isoforms with the 3' UTR and Poly A
signal, a second intronic region (containing all necessary splice
signals), and the exon 9 coding sequence for a second LAMP-2
isoform with the 3' UTR and Poly A signal, a third intronic region
(containing all necessary splice signals), and the exon 9 coding
sequence for a third LAMP-2 isoform with 3' UTR and Poly A signal.
In some embodiments, this construct is used to express mRNAs for
all three LAMP-2 isoforms via alternative splicing, which is the
mechanism that generates the different LAMP-2 mRNAs in normal
cells, using the same viral genome. The exon 9 coding sequences
could be placed in any potential order and combination.
[0054] FIG. 2 illustrates a flow chart of a method of therapeutic
treatment (e.g., of Danon disease or another disease caused at
least in part by an autophagy deficiency) by promoting the
expression of one or more isoforms of LAMP-2.
[0055] FIG. 3A-F depict LAMP-2 isoform coding and protein
sequences: FIG. 3A: LAMP-2A coding sequence; FIG. 3B: LAMP-2A
protein sequence; FIG. 3C: LAMP-2B coding sequence; FIG. 3D:
LAMP-2B protein sequence; FIG. 3E: LAMP-2C coding sequence; and
FIG. 3F: LAMP-2C protein sequence.
[0056] FIG. 4A-C presents a comparison of the last 90 amino acids
of the human and mouse protein sequences of LAMP-2A (FIG. 4A),
LAMP-2B (FIG. 4B), and LAMP-2C (FIG. 4C) showing sequence
identities.
[0057] FIG. 5A-B illustrates the dose dependent increase in LAMP-2B
(FIG. 5A) and LAMP-2A (FIG. 5B) mRNA expression following
administration of AAV9 vectors carrying those genes.
[0058] FIG. 6 illustrates the dose dependent increase in LAMP-2B
and LAMP-2A protein expression following administration of AAV9
vectors carrying those genes.
[0059] FIG. 7A-C shows fluorescent micrographs of heart sections
stained with DAPI (blue) and fluorescently-tagged anti-LAMP-2
antibody (white). FIG. 7A shows a heart section from a Lamp-2 knock
out (KO) mouse treated with AAV9.LAMP-2B. FIG. 7B shows a heart
section from a Lamp-2 KO mouse treated with AAV9.EGFP. FIG. 7C
shows a heart section from an untreated wild type (WT) mouse.
[0060] FIG. 8A-B shows fluorescent micrographs of heart sections
stained with DAPI (blue) and fluorescently-tagged anti-LAMP-2
antibody (white; some examples highlighted with arrows). FIG. 7A
shows a heart section from a Lamp-2 KO mouse treated with
AAV9.LAMP-2B. FIG. 7B shows a heart section from a Lamp-2 KO mouse
treated with AAV9.EGFP.
[0061] FIG. 9A-H illustrates the CAG-RFP-EGFP-LC3B autophagy
reporter system. FIG. 9A presents a schematic representation of the
operation of the system. FIG. 9B-G' are fluorescence microscopy
images; the X' (X prime) images being enlargements of the insets
outlined on the images labeled with the same letter without the
prime. With respect to the FIG. 9B-G' grid; the first column are
images of red fluorescence; the second column are images of green
fluorescence; the third column are merged images of the first and
second columns, yellow representing red+green fluorescence; the top
two rows are images of heart sections from WT mice expressing the
CAG-RFP-EGFP-LC3B autophagy reporter construct; the bottom two rows
are images of heart sections from Lamp-2 KO mice expressing the
CAG-RFP-EGFP-LC3B autophagy reporter construct. Immature
autophagosomes fluoresce both green and red (or yellow in the
merged images). Mature autolysosomes fluoresce only red. Yellow
arrows highlight autophagic vacuoles fluorescing in both colors
while white arrows highlight autophagic vacuoles fluorescing only
in red.
[0062] FIG. 9H illustrates the number of autophagic vacuoles that
are autophagosomes or autolysosomes in WT and Lamp-2 KO mice.
[0063] FIG. 10A-E illustrates the effect of -2 gene therapy using
the CAG-RFP-EGFP-LC3B autophagy reporter system. FIG. 10A is an
image of untreated WT mouse heart section. FIG. 10B is an image of
a heart section showing transduced cells from a Lamp-2 KO mouse
treated with the control vector AAV9.EGFP. FIG. 10C is an image of
a heart section from a Lamp-2 KO mouse treated with the gene
therapy vector AAV9.LAMP-2A. FIG. 10D is an image of a heart
section from a Lamp-2KO mouse treated with the gene therapy vector
AAV9.LAMP-2B. In the images of FIGS. 10A, 10C and 10D red arrows
are used to highlight some of the autolysosomes present. FIG. 10E
illustrates the proportion of total autophagic vacuoles represented
by autophagosomes and autolysosomes for the four conditions.
[0064] FIG. 11A-C' presents electron micrographs of heart tissue
from WT (FIG. 11A,A') and Lamp-2 KO (FIG. 11B, B') mice, and a
Lamp-2 KO mouse treated with AAV9.LAMP-2B (FIG. 11C, C'). White
arrows highlight some of the autophagic vacuoles. Black arrows
highlight some of the damaged mitochondria.
DETAILED DESCRIPTION
1. Introduction
[0065] Danon disease is caused by mutations resulting in reduced or
absent expression of the lysosome-associated membrane protein 2
(LAMP-2, also known as CD107b) gene. The present methods are based
in part, on the introduction of one or more polynucleotides
encoding one or more LAMP-2 isoforms, e.g., packaged in an
adeno-associated virus (AAV) vector, to deliver the LAMP-2
gene/individual isoforms to Danon patients. After delivery of the
one or more polynucleotides encoding one or more LAMP-2 isoforms,
the LAMP-2 transgene is then expressed by the patient's own cells.
Restoration of LAMP-2 gene expression in Danon patients can result
in amelioration of the disease phenotype, serving as a therapy for
this disease. Delivery to a subject of one or more polynucleotides
encoding one or more LAMP-2 isoforms also can be used for the
treatment of other disorders of autophagy including, but not
limited to, end-stage heart failure, myocardial infarction drug
toxicities, diabetes, end-stage renal failure, and aging.
Abnormalities in autophagy, particularly decreased autophagic flux,
have been implicated in these disorders as well as many others.
Expressing higher levels of the LAMP-2 gene increases autophagic
flux and therefore serves as a treatment for these disorders.
[0066] No current art exists for treating Danon disease by gene
therapy methods. Danon disease is not a traditional lysosomal
storage disorder, which is generally defined as a deficiency in a
lysosomal protein (e.g. a transporter or enzyme) that is required
for processing a specific cellular substrate, which results in a
toxic accumulation of that specific substrate within lysosomes.
Danon disease is understood to be a disorder of autophagy or an
autophagic vacuolar myopathy, which affects the degradation of all
cellular components processed by the autophagic pathway and is not
caused by the accumulation of specific substrate. Additionally, no
existing art explicitly describes the delivery of the LAMP-2
gene/individual isoforms for treatment of Danon disease or other
disorders of autophagy. Therefore, the present methods provide a
unique method for treating Danon disease and improves upon existing
AAV technologies by explicitly including delivery of the LAMP-2
gene as a means for improving disorders of autophagy.
2. Patients Subject to Treatment
[0067] Subjects/patients amenable to treatment using the methods
described herein include individuals at risk of a disease or
disorder characterized by insufficient autophagic flux (e.g., Danon
disease as well as other known disorders of autophagy including,
but not limited to, systolic and diastolic heart failure,
myocardial infarction, drug toxicities (for example, anthracyclines
chloroquine and its derivatives), diabetes, end-stage renal
disease, and aging) but not showing symptoms, as well as subjects
presently showing symptoms. Such subject may have been identified
as having a mutated LAMP-2 gene or as having reduced or
non-detectable levels of LAMP-2 expression.
[0068] In some embodiments, the subject is exhibiting symptoms of a
disease or disorder characterized by insufficient autophagic flux
(e.g., Danon disease as well as other known disorders of autophagy
including, but not limited to, systolic and diastolic heart
failure, myocardial infarction, drug toxicities, diabetes,
end-stage renal disease, and aging). The symptoms may be actively
manifesting, or may be suppressed or controlled (e.g., by
medication) or in remission. The subject may or may not have been
diagnosed with the disorder, e.g., by a qualified physician.
[0069] The subject can be any mammal at any stage of development at
the time of delivery, e.g., embryonic, fetal, infantile, juvenile
or adult. In varying embodiments, the subject is a child, a
juvenile or an adult. In varying embodiments, the subject is a
mammal, for example, a human or a domesticated mammal (e.g., a
canine or a feline).
3. Delivery Vectors of LAMP-2 Polynucleotide
[0070] Generally, the gene therapy vectors described herein
comprise an expression cassette comprising a polynucleotide
encoding one or more isoforms of lysosome-associated membrane
protein 2 (LAMP-2), that allows for the expression of LAMP-2 to
partially or wholly rectify deficient LAMP-2 protein expression
levels and autophagic flux in a subject in need thereof (e.g., a
subject having Danon disease or another disorder characterized by
deficient autophagic flux at least in part due to deficient LAMP-2
expression). The gene therapy vectors can be viral or non-viral
vectors. Illustrative non-viral vectors include, e.g., naked DNA,
cationic liposome complexes, cationic polymer complexes, cationic
liposome-polymer complexes, and exosomes.
[0071] In some embodiments the vector carries a single isoform,
LAMP-2A, LAMP-2B, or LAMP-2C; see for example FIG. 1A-F showing a
schematic of an AAV vector carrying a gene for a single LAMP-2
isoform. In other embodiments the vector carries genes for two
LAMP-2 isoforms; see for example FIG. 1G-K showing a schematic of
AAV vectors carrying a gene for a two LAMP-2 isoforms, LAMP-2B on
one DNA strand and LAMP-2A on the other DNA strand. Still other
embodiments carry all three isoforms (See FIG. 1L-O). In addition
to the depicted genome structures three isoform vectors could also
be constructed by making hybrids of the various embodiments shown.
For example, there could be two promoters, one driving expression
of a single isoform and the other driving expression of a two
isoform cassette making use of an IRES, a self-cleaving peptide, or
alternative splicing. In various embodiments comprising multiple
isoforms, the isoforms occur in any order, either mirroring the
natural order of B, A, C, or altering it. By carrying multiple
isoforms in a single vector one can ensure co-expression of the
isoforms in the transfected cells and use fewer total vector
particles in a dose.
[0072] Examples of viral vector include, but are not limited, to
adenoviral, retroviral, lentiviral, herpesvirus and
adeno-associated virus (AAV) vectors. Gene delivery viral vectors
useful in the practice of the present invention can be constructed
utilizing methodologies well known in the art of molecular biology.
Typically, viral vectors carrying transgenes are assembled from
polynucleotides encoding the transgene, suitable regulatory
elements and elements necessary for production of viral proteins
which mediate cell transduction,
[0073] Such recombinant viruses may be produced by techniques known
in the art, e.g., by transfecting packaging cells or by transient
transfection with helper plasmids or viruses. Typical examples of
virus packaging cells include but are not limited to PA317 cells,
PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for
producing such replication-defective recombinant viruses may be
found for instance in WO95/14785, WO96/22378, U.S. Pat. Nos.
5,882,877, 6,013,516, 4,861,719, 5,278,056 and WO94/19478, the
complete contents of each of which is hereby incorporated by
reference.
[0074] In some embodiments, the gene viral vector is an adenoviral
vector or an adeno-associated viral (AAV) vector. In varying
embodiments, the AAV vector is selected from AAV serotypes AAV1,
AAV2, AAV3, AAV4, AAS, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,
AAVrh10, and subgroups and mixtures thereof including
self-complementary AAV (scAAV) genomes, or any other serotypes of
AAV that can infect humans, monkeys or other species. In one
embodiment, the AAV vector is an AAVrh10. In another embodiment the
AAV vector is AAV9. In still another embodiment the AAV vector is
AAV8. Recombinant AAV (rAAV) vectors are frequently utilized for
the delivery of therapeutic genes and have been studied in human
clinical trials. rAAV vectors can be designed to deliver specified
transgenes to a patient's cells for expression. After infection and
introduction of the viral genome by the rAAV vector, the viral
genes exist mostly as extrachromosomal structures that do not
integrate into the host's genome, but are expressed by the host
cell's translational machinery. For successful host cell infection
and gene expression, rAAV vectors require several components (see
FIG. 1): inverse terminal repeat elements (ITRs), promoter and/or
enhancer region, transgene, and 3' untranslated region, and
polyadenylation signal. After viral infection of host cells, the
promoter region initiates signals for translation of the virally
delivered transgene by the host cell's translational machinery.
[0075] Generally, the control elements are selected to be
functional in a mammalian cell. The resulting construct which
contains the operatively linked components is bounded (5' and 3)
with functional AAV ITR sequences. By "adeno-associated virus
inverted terminal repeats" or "AAV ITRs" is meant the
art-recognized regions found at each end of the AAV genome which
function together in cis as origins of DNA replication and as
packaging signals for the virus. AAV ITRs, together with the AAV
rep coding region, provide for the efficient excision and rescue
from, and integration of a nucleotide sequence interposed between
two flanking ITRs into a mammalian cell genome. The nucleotide
sequences of AAV ITR regions are known. See, e.g., Kotin, 1994;
Berns, K I "Parvoviridae and their Replication" in Fundamental
Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds., the
complete contents of which is hereby incorporated by reference) for
the AAV2 sequence. As used herein, an "AAV ITR" does not
necessarily comprise the wild-type nucleotide sequence, but may be
altered, e.g., by the insertion, deletion or substitution of
nucleotides.
[0076] Additionally, the AAV ITR may be derived from any of several
AAV serotypes, including without limitation, AAV1, AAV2, AAV3,
AAV4, AAS, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10.
Furthermore, 5' and 3' ITRs which flank a selected nucleotide
sequence in an AAV vector need not necessarily be identical or
derived from the same AAV serotype or isolate, so long as they
function as intended, i.e., to allow for excision and rescue of the
sequence of interest from a host cell genome or vector, and to
allow integration of the heterologous sequence into the recipient
cell genome when AAV Rep gene products are present in the cell.
Additionally, AAV ITRs may be derived from any of several AAV
serotypes, including without limitation, AAV1, AAV2, AAV3, AAV4,
AAS, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10. Furthermore, 5'
and 3' ITRs which flank a selected nucleotide sequence in an AAV
expression vector need not necessarily be identical or derived from
the same AAV serotype or isolate, so long as they function as
intended, i.e., to allow for excision and rescue of the sequence of
interest from a host cell genome or vector, and to allow
integration of the DNA molecule into the recipient cell genome when
AAV Rep gene products are present in the cell.
[0077] In varying embodiments, vectors derived from AAV serotypes
having tropism for, and high transduction efficiencies in, cells of
the mammalian myocardium are employed, particularly cardiomyocytes
and cardiomyocyte progenitors. A review and comparison of
transduction efficiencies of different serotypes is provided in
Cearley C N et al., Molecular Therapy 16(10); 1710-1718, 2008, the
complete contents of which is hereby incorporated by reference. In
other non-limiting examples, preferred vectors include vectors
derived from any serotypes like AAV1, AAV2, AAV3, AAV4, AAS, AAV6,
AAV7, AAV8, AAV9, AAV10, AAV11 or AAVrh10, which have also been
shown to transduce cells of cardiomyocytes.
[0078] In varying embodiments, the selected nucleotide sequence is
operably linked to control elements that direct the transcription
or expression thereof in the subject in vivo. Such control elements
can comprise control sequences normally associated with the
selected gene.
[0079] Alternatively, heterologous control sequences can be
employed. Useful heterologous control sequences generally include
those derived from sequences encoding mammalian or viral genes.
Examples include, but are not limited to, the phophoglycerate
kinase (PKG) promoter, CAG (CMV enhancer with chicken beta-actin
promoter including sequence through the 1st intron and the splice
acceptor of the rabbit beta-globin gene) promoter, MCK (muscle
creatine kinase) promoter, the SV40 early promoter, mouse mammary
tumor virus LTR promoter; adenovirus major late promoter (Ad MLP);
a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV)
promoter such as the CMV immediate early promoter region (CMVIE),
rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid
promoters, and the like. The promoters can be of human origin or
from other species, including from mice. In addition, sequences
derived from non-viral genes, such as the marine metallothionein
gene, will also find use herein. Such promoter sequences are
commercially available from, e.g. Stratagene (San Diego, Calif.).
Examples of heterologous promoters include but are not limited to
the CMV promoter. Examples of inducible promoters include but are
not limited to DNA responsive elements for ecdysone, tetracycline,
and hypoxia andaufin. When multiple isoforms are encoded in a
single vector they are preferably, but not necessarily, operably
linked to different control sequences.
[0080] Similarly, control elements at the 3' end of a coding
region, including a 3' untranslated region (3' UTR) and a
polyadenylation signal, can derive from the inserted gene or from a
heterologous source. Particular embodiments utilize the woodchuck
hepatitis virus posttranscriptional regulatory element (WPRE) as a
3' UTR or a rabbit beta-globin polyadenylation signal or both. 3'
control elements can also include control elements connecting to
coding regions. For example, it is possible to express multiple
LAMP-2 isoforms under the control of a single promoter by placing
an internal ribosome entry site (IRES) (see for example FIGS. 1I
and 1M) or a self-cleaving peptide sequence (such as a picornaviral
2A peptide; see for example FIGS. 1J and 1N) between the 1st and
2nd, and 2nd and 3rd (if present) isoforms. In the case of an IRES,
2 or 3 polypeptides are translated. In the case of a self-cleaving
peptide the LAMP-2 isoforms along with the intervening
self-cleaving peptide are present in the vector as a single reading
frame that is translated as a single polypeptide that is cleaved
into it substituent LAMP-2 isoforms after or during
translation.
[0081] Transcription under control of a single promoter is also
achievable when the vector is constructed to support alternative
splicing. In such constructs the first eight exons of LAMP-2 are
present as spliced together in cDNA followed by two or three
intron-exon pairs to include two or all three of the alternative
exons that give rise to the three LAMP-2 isoforms (see for example
FIGS. 1K and 1O). In various embodiments intron-exon pairs occur in
any order, either mirroring the natural order of B, A, C, or
altering it. The native introns are too long to include in vectors
that are size constrained such as AAV. In such cases the introns
are truncated by removal of central sequences while retaining the
necessary 5' and 3' splice sites and splice signals. Typically,
retention of just a few bases at the 5' end of the intron and about
100-200 bases at the 3' end of the intron is sufficient to ensure
splicing. Alternatively, heterologous introns can be substituted
for the native introns.
[0082] The AAV expression vector which harbors the DNA molecule of
interest bounded by AAV ITRs, can be constructed by directly
inserting the selected sequence (s) into an AAV genome which has
had the major AAV open reading frames ("ORFs") excised therefrom.
Other portions of the AAV genome can also be deleted, so long as a
sufficient portion of the ITRs remain to allow for replication and
packaging functions. Such constructs can be designed using
techniques well known in the art. See, e.g. U.S. Pat. Nos.
5,173,414 and 5,139,941; International Publications Nos. WO
92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4 Mar.
1993); Lebkowski et al., 1988; Vincent et al., 1990; Carter, 1992;
Muzyczka, 1992; Kotin, 1994; Shelling and Smith, 1994; and Zhou et
al., 1994, the complete contents of each of which is hereby
incorporated by reference.
[0083] Alternatively, AAV ITRs can be excised from the viral genome
or from an AAV vector containing the same and fused 5' and 3' of a
selected nucleic acid construct that is present in another vector
using standard ligation techniques. AAV vectors which contain ITRs
have been described in, e.g. U.S. Pat. No. 5,139,941, the complete
contents of which is hereby incorporated by reference. In
particular, several AAV vectors are described therein which are
available from the American Type Culture Collection ("ATCC") under
Accession Numbers 53222, 53223, 53224, 53225 and 53226.
Additionally, chimeric genes can be produced synthetically to
include AAV ITR sequences arranged 5' and 3' of one or more
selected nucleic acid sequences. Preferred codons for expression of
the chimeric gene sequence in mammalian CNS cells can be used. The
complete chimeric sequence is assembled from overlapping
oligonucleotides prepared by standard methods. See, e.g., Edge
Nature, vol, 292, 1981, page 756; Nambair et al., Science, vol.
223, 1984, page 1299; Jay et al., J. Biol. Chem. vol. 259, 1984,
page 6311, the complete contents of each of which is hereby
incorporated by reference. In order to produce AAV virions, an AAV
expression vector is introduced into a suitable host cell using
known techniques, such as by transfection. A number of transfection
techniques are generally known in the art. See, e.g. Graham et al,
Virology, 52, 456-467, (1973); Sambrook et al. (1989) Molecular
Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New
York, Davis et al. (1986) Basic Methods in Molecular Biology,
Elsevier, and Chu et al. (1981) Gene 13:197. Particularly suitable
transfection methods include calcium phosphate co-precipitation
(Graham et al., 1973), direct microinjection into cultured cells
(Capeechi, 1980), electroporation (Shigekawa et al., 1988),
liposome mediated gene transfer (Mannino et al., 1988),
lipid-mediated transduction (Feigner et al., 1987, PNAS USA, 84,
21, 7413-17), and nucleic acid delivery using high-velocity
microprojectiles (Klein et al., 1987, Endocrinology 120:2339-45).
The complete contents of each of the foregoing references are
hereby incorporated by reference in entirety.
[0084] In one embodiment, the AAV, comprises, in addition to a
LAMP-2 isoform encoding nucleic acid sequence(s), the backbone of
AAV vector with ITR derived from AAV2, the promoter, such as the
mouse PGK (phosphoglycerate kinase) gene or the
cytomegalovirus/chicken beta-actin hybrid promoter (CAG) consisting
of the enhancer from the cytomegalovirus immediate gene and the
promoter, splice donor, and intron from the chicken beta-actin
gene, the splice acceptor from rabbit .beta.-globin, or any
promoter such as PGK, CAG, MCK, EF-1 or native LAMP-2 promoter. In
varying embodiments, the viral vector is encapsulated with an
anionic liposome, e.g., as described in U.S. Patent Publ. No.
2015/0284691.
[0085] In varying embodiments structure of the expression cassette
within the vector comprises first and second (e.g., 5' and 3)
inverse terminal repeats (ITRs) from any known AAV serotype or
subgroup including scAAV, any known promoter region (e.g., a
cytomegalovirus (CMV) promoter or a chicken-beta actin promoter)
with or without any known enhancer element (e.g., a CMV enhancer),
the lysosome-associated protein 2 gene (including all known
isoforms, e.g., LAMP-2A, LAMP-2B, and LAMP-2C, and all known
polymorphisms of those isoforms), and a polyadenylation signal
(including, but not limited to, rabbit beta-globin or).
[0086] Translation of the virally delivered LAMP-2 isoform gene, or
a combination of various isoform genes, results in expression of
the LAMP-2 protein for that particular isoform, which is then
targeted to the lysosomal membranes of the host cell by the host
cell machinery. Restoration of LAMP-2 isoform(s) expression and
function then restores autophagic flux (potentially including, but
not necessarily limited to, all known forms of autophagy such as
macroautophagy, mitophagy, chaperone-mediated autophagy, and
DNA/RNA autophagy), which is deficient and causal in Danon
patients, allowing diseased host cells to eliminate toxic cellular
components and damaged organelles and thus improving cell function
and survival. Ultimately, restoration of LAMP-2 isoform(s)
expression serves to treat the underlying genetic deficiency that
causes Danon disease and relieves the phenotype and symptoms of the
disease. In other disorders of autophagy where LAMP-2 may be
expressed, but not at a sufficient level to create adequate
autophagic flux and thus promote normal cell function and survival,
delivery of the transgenic LAMP-2 isoform(s) by the rAAV vector
results in overexpression of the resultant LAMP-2 protein(s). This
overexpression restores autophagic flux to near normal, normal or
supra-normal levels. Restoration of autophagic flux in diseases
that cause a disruption of normal autophagy then alleviates,
mitigates, and/or reverses the disease phenotype and symptoms.
4. Vector Pharmaceutical Compositions
[0087] Provided are pharmaceutical compositions, e.g., for use in
preventing or treating a disorder characterized by deficient
autophagic flux (e.g., Danon disease) which comprises a
therapeutically effective amount of a vector which comprises a
nucleic acid sequence of a polynucleotide that encodes one or more
isoforms of LAMP-2.
[0088] It will be understood that the single dosage or the total
daily dosage of the compounds and compositions of the present
invention will be decided by the attending physician within the
scope of sound medical judgment. The specific therapeutically
effective dose level for any particular patient will depend upon a
variety of factors including the disorder being treated and the
severity of the disorder; activity of the specific compound
employed; the specific composition employed, the age, body weight,
general health, sex and diet of the patient; the time of
administration, route of administration, and rate of excretion of
the specific compound employed; the duration of the treatment;
drugs used in combination or coincidental with the specific nucleic
acid or polypeptide employed; and like factors well known in the
medical arts. For example, it is well within the skill of the art
to start doses of the compound at levels lower than those required
to achieve the desired therapeutic effect and to gradually increase
the dosage until the desired effect is achieved. However, the daily
dosage of the products may be varied over a wide range per adult
per day. The therapeutically effective amount of the vector
according to the invention that should be administered, as well as
the dosage for the treatment of a pathological condition with the
number of viral or non-viral particles and/or pharmaceutical
compositions described herein, will depend on numerous factors,
including the age and condition of the patient, the severity of the
disturbance or disorder, the method and frequency of administration
and the particular peptide to be used.
[0089] The pharmaceutical compositions that contain the vector may
be in any form that is suitable for the selected mode of
administration, for example, for intraventricular,
intramyocardarial, intracoronary, intravenous, intra-arterial,
intra-renal, intraurethral, epidural or intramuscular
administration. The gene therapy vector comprising a polynucleotide
encoding one or more LAMP-2 isoforms can be administered, as sole
active agent, or in combination with other active agents, in a unit
administration form, as a mixture with conventional pharmaceutical
supports, to animals and human beings.
[0090] In varying embodiments, the pharmaceutical compositions
contain vehicles which are pharmaceutically acceptable for a
formulation capable of being injected. These may be in particular
isotonic, sterile, saline solutions (monosodium or disodium
phosphate, sodium, potassium, calcium or magnesium chloride and the
like or mixtures of such salts), or dry, especially freeze-dried
compositions which upon addition, depending on the case, of
sterilized water or physiological saline, permit the constitution
of injectable solutions.
[0091] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases, the form must be sterile
and must be fluid. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms, such as bacteria and
fungi.
[0092] Solutions comprising the gene therapy vectors as free base
or pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0093] The gene therapy vectors can be formulated into a
composition in a neutral or salt form. Pharmaceutically acceptable
salts include the acid addition salts (formed with the free amino
groups of the protein) and which are formed with inorganic acids
such as, for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0094] The carrier can also be as solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetables oils. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminium
monostearate and gelatin.
[0095] Sterile injectable solutions are prepared by incorporating
the active polypeptides in the required amount in the appropriate
solvent with several of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
5. Methods of Treating Diseases of Autophagy
[0096] Further provided are methods of preventing, mitigating,
ameliorating, reducing, inhibiting, eliminating and/or reversing
one or more symptoms of Danon disease or another autophagy disorder
in a subject in need thereof, comprising administering to the
subject a gene therapy vector as described above and herein, e.g.,
an adeno-associated virus (AAV) vector comprising an expression
cassette comprising a polynucleotide encoding one or more isoforms
of lysosome-associated membrane protein 2 (LAMP-2). The vector is
delivered to the subject in need thereof and whereby the
polynucleotide encoding one or more LAMP-2 isoforms is expressed by
the transduced cells at a therapeutically effective level.
[0097] In one particular embodiment, the vector is an AAV9. In
another particular embodiment the vector is an AAV8. In another
particular embodiment, the vector is an AAVrH10. In a further
aspect of these embodiments, they are used specifically in the
treatment of Danon disease.
[0098] In varying embodiments, the vector is administered via a
route selected from the group consisting of intravenous,
intraarterial, intracardiac, intracoronary, intramyocardial,
intrarenal, intra-urethral, epidural, subcutaneous, and
intramuscular. In varying embodiments, the vector is delivered
directly into the myocardium by epicardiac injection via a
minithoracotomy, by intracoronary injection, by endomyocardic
injection or by another type of injection useful in the heart.
Additional routes of administration may also comprise local
application of the vector under direct visualization, e.g.,
superficial cortical application, or other nonstereotactic
application.
[0099] Viral vectors will typically provoke an immune response
which can include an antibody response that can reduce or
completely inhibit the effectiveness of a particular vector in that
individual when repeat dosing becomes necessary or desirable.
Repeat dosing may become appropriate, for example, because vector
can be lost over time due to cell proliferation, especially
episomal vectors. Tissues that are more difficult to access can
require multiple administrations of vector to transfect a
sufficient number of cells for effective treatment of the disease.
Expression of the transgene can also be lost over time. The need to
administer a gene therapy vector in the face of an inhibiting
antibody response can be addressed in various ways. For example,
antibody titers can be reduced by apheresis prior to administration
of the vector or the patient can be immunosuppressed with an
appropriate medical regimen. Alternatively, empty AAV capsid could
be administered to bind host antibodies and immune cells prior to
injection of therapeutic AAV containing the LAMP-2 transgene(s).
Depending on the target tissue, a direct local administration
instead of a systemic administration can be useful in overcoming or
ameliorating the inhibitory effect of anti-vector antibodies. In
yet another approach, a vector based on a different serotype of the
virus from which it was derived can be used. For example, if an
AAV9 vector was used initially and there becomes a problem with
anti-AAV antibody titer, but there is need for further gene
therapy, one can administer an AAV8 vector carrying the same (or
different) gene construct.
[0100] Upon formulation, solutions can be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed. Multiple doses can also be administered.
[0101] As appropriate, the vectors described herein may be
formulated in any suitable vehicle for delivery. For instance, they
may be placed into a pharmaceutically acceptable suspension,
solution or emulsion. Suitable mediums include saline and liposomal
preparations. More specifically, pharmaceutically acceptable
carriers may include sterile aqueous of non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate. Aqueous
carriers include but are not limited to water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Intravenous vehicles include fluid and nutrient
replenishers, electrolyte replenishers (such as those based on
Ringer's dextrose), and the like.
[0102] Preservatives and other additives may also be present such
as, for example, antimicrobials, antioxidants, chelating agents,
and inert gases and the like.
[0103] A colloidal dispersion system may also be used for targeted
gene delivery. Colloidal dispersion systems include macromolecule
complexes, nanocapsules, microspheres, beads, and lipid-based
systems including oil-in-water emulsions, micelles, mixed micelles,
and liposomes.
[0104] An appropriate regimen can be determined by a physician, and
will depend on the age, sex, weight, of the subject, and the stage
of the disease. As an example, for delivery of a nucleic acid
sequence encoding a LAMP-2 polypeptide using a viral expression
vector, each unit dosage of LAMP-2 polypeptide expressing vector
may comprise 2.5 .mu.l to 100 .mu.l of a composition including a
viral expression vector in a pharmaceutically acceptable fluid at a
concentration ranging from 10.sup.11 to 10.sup.16 viral genome per
ml, for example.
[0105] In some regimens, a vector carrying a gene for a single
LAMP-2 isoform is used, for example LAMP-2B. In other regimens, a
vector carrying a gene for two LAMP-2 isoforms is used, for example
LAMP-2B and LAMP-2A. In some embodiments, the two isoforms are
carried in different vectors preparations. In one aspect, both
vector preparations are derived from the same vector, for example
an AAV9 vector or any other particular vector described above. In
other embodiments, both polypeptides are encoded in a single
vector; see for example FIG. 1. Similarly, if the regimen comprises
administration of nucleic acid sequences encoding all three
isoforms, the isoforms can be carried in individual vectors or in a
single vector. In regimens using multiple isoforms carried by
individual vectors the multiple vectors can be administered at the
same time, individually or mixed together, or they can be
administered sequentially at intervals of hours, days, or
weeks.
6. Kits
[0106] Further provided are kits comprising a gene therapy vector
comprising a polynucleotide encoding one or more LAMP-2 isoforms,
as described above and herein. In varying embodiments, the kits
provide the gene therapy vectors prepared in one or more unitary
dosage forms ready for administration to a subject, for example in
a preloaded syringe or in an ampoule. In varying embodiments, the
gene therapy vector is provided in a lyophilized form.
Examples
Example 1. AAV9 Gene Therapy
[0107] Particular vectors, consistent with the descriptions above,
were constructed as an AAV9 vector with ITRs derived from AAV2 and
encoding either LAMP-2A or LAMP-2B under the control of a chicken
beta-actin promoter with a CMV enhancer (CAG promoter).
Additionally, these vectors incorporated the woodchuck hepatitis
virus posttranscriptional regulatory element and rabbit beta-globin
polyadenylation signal (see FIG. 1B). These vectors were obtained
from the National Heart, Lung, and Blood Institute Gene Therapy
Resource Program Preclinical Vector Core facility at the University
of Pennsylvania. The human LAMP-2 sequences were incorporated with
the expectation that they would be functional in mouse due to the
high degree of sequence identity between the homologues especially
at the C-terminal region, which forms the transmembrane and
cytoplasmic tail components of the protein (see FIG. 4).
[0108] The vector is used to deliver the LAMP-2 isoforms in vivo.
The AAV9 serotype has excellent tropism for cardiac and skeletal
muscle as well as nervous tissue, the organs most affected by Danon
disease. However, Danon is a multi-system disorder and many
different organs can potentially be involved; therefore, expression
of the transgenic LAMP-2 isoforms is under the control of a
constitutively active chicken beta-actin (CBA) promoter with a
cytomegalovirus (CMV) enhancer (CAG construct). This promoter is
ubiquitously active and will allow for LAMP-2 transgene expression
in all tissues depending on infection efficiency. An AAV9 vector
carrying an enhanced green fluorescent protein (eGFP) gene was also
obtained for use as a control reagent.
Example 2. Administration of AAV9 LAMP-2 Isoform Vectors to LAMP-2
Knock Out Mice
[0109] Lamp-2 KO mice (Nature. 2000 Aug. 24; 406(6798):902-6; Basic
Res Cardiol. 2006 July; 101(4):281-91) develop a Danon-like
syndrome, though the severity of disease is more moderate than the
human disease, likely at least in part due to the shorter life span
of mice providing less time for damage due to impaired autophagic
flux to accumulate. It is therefore desirable to wait until the
mice are at least 3 months old and preferably 5-6 months old before
initiating treatment so that there has been sufficient accumulation
of pathology that its reversal is readily detectable.
[0110] In an idealized procedure, six-month old Lamp-2 KO mice and
receive 5.times.10.sup.11, 1.times.10.sup.12, or 2.times.10.sup.12
genome copies (gc) of AAV vector via intravenous injection into the
external jugular vein. In various experiments, WT mice receive the
LAMP-2 isoform vectors and Lamp-2 KO mice receive eGFP vector as
controls. Sufficient numbers of mice are used so that
subpopulations can be sacrificed and evaluated at various time
points, for example, at 1, 2, and 6 months post-administration.
Example 3. Evaluation of LAMP-2 Isoform Gene Transcription
Following Vector Administration
[0111] Lamp-2 KO mice, approximately 3-4 month of age, received
increasing doses of the vectors AAV9.LAMP-2B and AAV9.LAMP-2A (see
FIG. 1B and descriptions above) at 5.times.10.sup.11,
1.times.10.sup.12, and 2.times.10.sup.12 gc/mouse. One mouse per
condition was sacrificed and RT-qPCR was performed, in triplicate,
on digested heart tissue to assess mRNA expression (gene
transcription) of the human transgenes. An untreated WT mouse was
used to demonstrate no expression of the human LAMP-2 isoforms. The
data show a dose-dependent increase in expression of the human
transgenes (see FIG. 5).
Example 4. Evaluation of LAMP-2 Isoform Protein Expression
Following Vector Administration
[0112] Using the same mice as in Example 3, immunoblotting was
performed on digested heart tissue to assess protein expression of
the human transgenes relative to GAPDH. A control Lamp-2 KO mouse
treated with AAV9.EGFP demonstrated no significant LAMP-2 protein
expression. The data show a dose-dependent increase in expression
of the human transgenes in mice receiving the viral vectors (see
FIG. 6).
Example 5. Subcellular Localization of Transgenic LAMP-2B Following
Vector Administration
[0113] Heart sections from a Lamp-2 KO mouse that had received
2.times.10.sup.12 gc/mouse of either AAV9.LAMP-2B or AAV9.EGFP
sacrificed 1 month post-delivery were stained with DAPI (which
binds to AT-rich DNA and thus makes cell nuclei visible) and
fluorescently-tagged anti-LAMP-2 antibody (see FIG. 7). The pattern
of LAMP-2 staining indicates localization of the human transgenic
LAMP-2B protein to intracellular vacuoles and is similar to the
staining seen in a WT mouse control for the mouse Lamp-2 protein.
No human LAMP-2 staining is seen in the Lamp-2 KO mouse receiving
the AAV9.EGFP vector control. These data demonstrate that treatment
with the AAV9.LAMP-2B vector leads to expression of the human
LAMP-2B protein in a physiologically appropriate location.
[0114] In a similar, experiment human LAMP-2 staining was
maintained at 2 months post-delivery of AAV9.LAMP-2B at a dose of
5.times.10.sup.11 gc/mouse in a test mouse. Though consistent with
Example 4, less staining was seen with this lower vector dosage. By
comparison, 3 months post-delivery of 5.times.10.sup.11 gc/mouse of
the AAV9.EGFP vector revealed no human LAMP-2 staining. See FIG.
8.
Example 6. The CAG-RFP-EGFP-LC3B Autophagy Reporter System
[0115] The CAG-RFP-EGFP-LC3B Autophagy Reporter System allows
assessment of macroautophagic flux. Microtubule-associated protein
1 light chain 3 (LC3), is expressed on the surface of
autophagosomes. A gene fusion is constructed to express LC3 fused
to both red fluorescent protein (RFP) and eGFP. When this fused
protein is expressed the, RFP component fluoresces in both
autophagosomes and lysosomes whereas the eGFP component fluoresces
in autophagosomes, but is quenched by the acidic environment of the
lysosomes. As a result, in a merged image autophagosomes are yellow
and lysosomes are red (see FIG. 9A). When expressed in a WT
background, one sees more red puncta than green puncta in separate
red and green images and a mixture of red and yellow puncta in
merged images (see FIGS. 9B-D'). Lamp-2 is required for normal
fusion of autophagosomes with lysosomes to form autolysosomes.
Thus, when this construct is expressed in a Lamp-2 KO mouse
approximately equal numbers of red and green puncta are seen in
separate red and green images with almost entirely yellow puncta in
the merged images (see FIGS. 9E-G'). The accumulation of
autophagosomes and near absence of autolysosomes, along with an
overall greater number of autophagic vacuoles (AVs), reflects a
defect in autophagic flux due to the absence of Lamp-2 (see FIG.
9H).
Example 7. Administration of Vector Carrying Either LAMP-2A or
LAMP-2B Restores Autophagic Flux
[0116] Lamp-2 KO mice expressing the CAG-RFP-EGFP-LC3B construct
(Lamp-2 KO/CAG-RFP-EGFP-LC3B reporter mice) were administered
either AAV9.LAMP-2B or AAV9.LAMP-2A and compared to Lamp-2 KO mice
expressing the CAG-RFP-EGFP-LC3B autophagy reporter system
(CAG-RFP-EGFP-LC3B reporter mice). One month post vector delivery,
the mice were sacrificed and heart sections assessed by fluorescent
microscopy to assess autophagic flux. The untreated Lamp-2
KO/reporter mice continued to show almost only yellow autophagic
vacuoles, that is autophagosomes (see FIG. 10B and FIG. 9G'). In
contrast, the Lamp-2 KO/reporter mice receiving either gene therapy
vector, AAV9.LAMP-2B or AAV9.LAMP-2A, showed many red autophagic
vacuoles, that is autolysosomes, in a similar proportion to a
control WT reporter mouse (see FIGS. 10 A, C and D and exemplary
puncta therein indicated with arrows). Quantitation of AVs
demonstrated that the proportion of immature autophagosomes to
mature autolysosomes was similar in treated Lamp-2 KO mice compared
to WT mice (see FIG. 10E). Thus, gene therapy with either
AAV9.LAMP-2B or AAV9.LAMP-2A restored normal autophagosome-lysosome
fusion in the cardiomyocytes of Lamp-2 KO mice.
Example 8. Restoration of Cardiomyocyte Ultrastructure as Assessed
by Electron Microscopy
[0117] A Lamp-2 KO mouse was injected intravenously with
5.times.10.sup.11 gc/mouse of AAV9.LAMP-2B and compared to an
age-matched historical Lamp-2 KO mouse and a WT mouse that were not
treated. One month post vector delivery the mice were sacrificed
and heart sections analyzed by electron microscopy. As compared to
WT (see FIGS. 11A, A'), the untreated Lamp-2 KO mouse showed an
accumulation and increase in size of AVs (see FIGS. 11B, B', yellow
arrows) and an increase in the number of abnormal mitochondria (see
FIG. 11B, red arrows). In contrast, electron micrographs of the
Lamp-2 KO mouse treated with AAV9.LAMP-2B more closely resembled
the ultrastructure of the untreated WT mouse (see FIGS. 11C, C').
Thus, treatment with the gene therapy vector restores the
ultrastructure of cardiomyocytes.
[0118] In summary, these examples show that gene therapy vectors
based on adeno-associated virus encoding the LAMP-2A and LAMP-2B
isoforms can be administered intravenously to successfully achieve
transgene expression in heart tissue. Additionally, such expression
leads to the reversal of defects in autophagic flux and
cardiomyocyte ultrastructure, defects that are also associated with
Danon disease. These data support the use of such vectors for gene
therapy in the treatment of Danon disease and other disorders
related to defects in autophagic flux.
[0119] In closing, it is to be understood that although aspects of
the present specification are highlighted by referring to specific
embodiments, one skilled in the art will readily appreciate that
these disclosed embodiments are only illustrative of the principles
of the subject matter disclosed herein. Therefore, it should be
understood that the disclosed subject matter is in no way limited
to a particular methodology, protocol, and/or reagent, etc.,
described herein. As such, various modifications or changes to or
alternative configurations of the disclosed subject matter can be
made in accordance with the teachings herein without departing from
the spirit of the present specification. Lastly, the terminology
used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention, which is defined solely by the claims. Accordingly, the
present invention is not limited to that precisely as shown and
described.
[0120] Certain embodiments of the present invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Of course, variations on these described
embodiments will become apparent to those of ordinary skill in the
art upon reading the foregoing description. The inventor expects
skilled artisans to employ such variations as appropriate, and the
inventors intend for the present invention to be practiced
otherwise than specifically described herein. Accordingly, this
invention includes all modifications and equivalents of the subject
matter recited in the claims appended hereto as permitted by
applicable law. Moreover, any combination of the above-described
embodiments in all possible variations thereof is encompassed by
the invention unless otherwise indicated herein or otherwise
clearly contradicted by context.
[0121] Groupings of alternative embodiments, elements, or steps of
the present invention are not to be construed as limitations. Each
group member may be referred to and claimed individually or in any
combination with other group members disclosed herein. It is
anticipated that one or more members of a group may be included in,
or deleted from, a group for reasons of convenience and/or
patentability. When any such inclusion or deletion occurs, the
specification is deemed to contain the group as modified thus
fulfilling the written description of all Markush groups used in
the appended claims.
[0122] Unless otherwise indicated, all numbers expressing a
characteristic, item, quantity, parameter, property, term, and so
forth used in the present specification and claims are to be
understood as being modified in all instances by the term "about."
As used herein, the term "about" means that the characteristic,
item, quantity, parameter, property, or term so qualified
encompasses a range of plus or minus ten percent above and below
the value of the stated characteristic, item, quantity, parameter,
property, or term. Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the specification and
attached claims are approximations that may vary. At the very
least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of the claims, each numerical
indication should at least be construed in light of the number of
reported significant digits and by applying ordinary rounding
techniques. Notwithstanding that the numerical ranges and values
setting forth the broad scope of the invention are approximations,
the numerical ranges and values set forth in the specific examples
are reported as precisely as possible. Any numerical range or
value, however, inherently contains certain errors necessarily
resulting from the standard deviation found in their respective
testing measurements. Recitation of numerical ranges of values
herein is merely intended to serve as a shorthand method of
referring individually to each separate numerical value falling
within the range. Unless otherwise indicated herein, each
individual value of a numerical range is incorporated into the
present specification as if it were individually recited
herein.
[0123] The terms "a," "an," "the" and similar referents used in the
context of describing the present invention (especially in the
context of the following claims) are to be construed to cover both
the singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. All methods described herein can
be performed in any suitable order unless otherwise indicated
herein or otherwise clearly contradicted by context. The use of any
and all examples, or exemplary language (e.g., "such as") provided
herein is intended merely to better illuminate the present
invention and does not pose a limitation on the scope of the
invention otherwise claimed. No language in the present
specification should be construed as indicating any non-claimed
element essential to the practice of the invention.
[0124] Specific embodiments disclosed herein may be further limited
in the claims using consisting of or consisting essentially of
language. When used in the claims, whether as filed or added per
amendment, the transition term "consisting of" excludes any
element, step, or ingredient not specified in the claims. The
transition term "consisting essentially of" limits the scope of a
claim to the specified materials or steps and those that do not
materially affect the basic and novel characteristic(s).
Embodiments of the present invention so claimed are inherently or
expressly described and enabled herein.
[0125] All patents, patent publications, and other publications
referenced and identified in the present specification are
individually and expressly incorporated herein by reference in
their entirety for the purpose of describing and disclosing, for
example, the compositions and methodologies described in such
publications that might be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
Sequence CWU 1
1
1211233DNAHomo sapiens 1atggtgtgct tccgcctctt cccggttccg ggctcagggc
tcgttctggt ctgcctagtc 60ctgggagctg tgcggtctta tgcattggaa cttaatttga
cagattcaga aaatgccact 120tgcctttatg caaaatggca gatgaatttc
acagttcgct atgaaactac aaataaaact 180tataaaactg taaccatttc
agaccatggc actgtgacat ataatggaag catttgtggg 240gatgatcaga
atggtcccaa aatagcagtg cagttcggac ctggcttttc ctggattgcg
300aattttacca aggcagcatc tacttattca attgacagcg tctcattttc
ctacaacact 360ggtgataaca caacatttcc tgatgctgaa gataaaggaa
ttcttactgt tgatgaactt 420ttggccatca gaattccatt gaatgacctt
tttagatgca atagtttatc aactttggaa 480aagaatgatg ttgtccaaca
ctactgggat gttcttgtac aagcttttgt ccaaaatggc 540acagtgagca
caaatgagtt cctgtgtgat aaagacaaaa cttcaacagt ggcacccacc
600atacacacca ctgtgccatc tcctactaca acacctactc caaaggaaaa
accagaagct 660ggaacctatt cagttaataa tggcaatgat acttgtctgc
tggctaccat ggggctgcag 720ctgaacatca ctcaggataa ggttgcttca
gttattaaca tcaaccccaa tacaactcac 780tccacaggca gctgccgttc
tcacactgct ctacttagac tcaatagcag caccattaag 840tatctagact
ttgtctttgc tgtgaaaaat gaaaaccgat tttatctgaa ggaagtgaac
900atcagcatgt atttggttaa tggctccgtt ttcagcattg caaataacaa
tctcagctac 960tgggatgccc ccctgggaag ttcttatatg tgcaacaaag
agcagactgt ttcagtgtct 1020ggagcatttc agataaatac ctttgatcta
agggttcagc ctttcaatgt gacacaagga 1080aagtattcta cagctcaaga
ctgcagtgca gatgacgaca acttcctagt gcccatagcg 1140gtgggagctg
ccttggcagg agtacttatt ctagtgttgc tggcttattt tattggtctc
1200aagcaccatc atgctggata tgagcaattt tag 123321233DNAHomo sapiens
2atggtgtgct tccgcctctt cccggttccg ggctcagggc tcgttctggt ctgcctagtc
60ctgggagctg tgcggtctta tgcattggaa cttaatttga cagattcaga aaatgccact
120tgcctttatg caaaatggca gatgaatttc acagttcgct atgaaactac
aaataaaact 180tataaaactg taaccatttc agaccatggc actgtgacat
ataatggaag catttgtggg 240gatgatcaga atggtcccaa aatagcagtg
cagttcggac ctggcttttc ctggattgcg 300aattttacca aggcagcatc
tacttattca attgacagcg tctcattttc ctacaacact 360ggtgataaca
caacatttcc tgatgctgaa gataaaggaa ttcttactgt tgatgaactt
420ttggccatca gaattccatt gaatgacctt tttagatgca atagtttatc
aactttggaa 480aagaatgatg ttgtccaaca ctactgggat gttcttgtac
aagcttttgt ccaaaatggc 540acagtgagca caaatgagtt cctgtgtgat
aaagacaaaa cttcaacagt ggcacccacc 600atacacacca ctgtgccatc
tcctactaca acacctactc caaaggaaaa accagaagct 660ggaacctatt
cagttaataa tggcaatgat acttgtctgc tggctaccat ggggctgcag
720ctgaacatca ctcaggataa ggttgcttca gttattaaca tcaaccccaa
tacaactcac 780tccacaggca gctgccgttc tcacactgct ctacttagac
tcaatagcag caccattaag 840tatctagact ttgtctttgc tgtgaaaaat
gaaaaccgat tttatctgaa ggaagtgaac 900atcagcatgt atttggttaa
tggctccgtt ttcagcattg caaataacaa tctcagctac 960tgggatgccc
ccctgggaag ttcttatatg tgcaacaaag agcagactgt ttcagtgtct
1020ggagcatttc agataaatac ctttgatcta agggttcagc ctttcaatgt
gacacaagga 1080aagtattcta cagcccaaga gtgttcgctg gatgatgaca
ccattctaat cccaattata 1140gttggtgctg gtctttcagg cttgattatc
gttatagtga ttgcttacgt aattggcaga 1200agaaaaagtt atgctggata
tcagactctg taa 123331236DNAHomo sapiens 3atggtgtgct tccgcctctt
cccggttccg ggctcagggc tcgttctggt ctgcctagtc 60ctgggagctg tgcggtctta
tgcattggaa cttaatttga cagattcaga aaatgccact 120tgcctttatg
caaaatggca gatgaatttc acagttcgct atgaaactac aaataaaact
180tataaaactg taaccatttc agaccatggc actgtgacat ataatggaag
catttgtggg 240gatgatcaga atggtctcaa aatagcagtg cagttcggac
ctggcttttc ctggattgcg 300aattttacca aggcagcatc tacttattca
attgacagcg tctcattttc ctacaacact 360ggtgataaca caacatttcc
tgatgctgaa gataaaggaa ttcttactgt tgatgaactt 420ttggccatca
gaattccatt gaatgacctt tttagatgca atagtttatc aactttggaa
480aagaatgatg ttgtccaaca ctactgggat gttcttgtac aagcttttgt
ccaaaatggc 540acagtgagca caaatgagtt cctgtgtgat aaagacaaaa
cttcaacagt ggcacccacc 600atacacacca ctgtgccatc tcctactaca
acacctactc caaaggaaaa accagaagct 660ggaacctatt cagttaataa
tggcaatgat acttgtctgc tggctaccat ggggctgcag 720ctgaacatca
ctcaggataa ggttgcttca gttattaaca tcaaccccaa tacaactcac
780tccacaggca gctgccgttc tcacactgct ctacttagac tcaatagcag
caccattaag 840tatctagact ttgtctttgc tgtgaaaaat gaaaaccgat
tttatctgaa ggaagtgaac 900atcagcatgt atttggttaa tggctccgtt
ttcagcattg caaataacaa tctcagctac 960tgggatgccc ccctgggaag
ttcttatatg tgcaacaaag agcagactgt ttcagtgtct 1020ggagcatttc
agataaatac ctttgatcta agggttcagc ctttcaatgt gacacaagga
1080aagtattcta cagctgaaga atgttctgct gactctgacc tcaactttct
tattcctgtt 1140gcagtgggtg tggccttggg cttccttata attgttgtct
ttatctctta tatgattgga 1200agaaggaaaa gtcgtactgg ttatcagtct gtgtaa
1236490PRTHomo sapiens 4Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys
Asn Lys Glu Gln Thr1 5 10 15Val Ser Val Ser Gly Ala Phe Gln Ile Asn
Thr Phe Asp Leu Arg Val 20 25 30Gln Pro Phe Asn Val Thr Gln Gly Lys
Tyr Ser Thr Ala Gln Asp Cys 35 40 45Ser Ala Asp Asp Asp Asn Phe Leu
Val Pro Ile Ala Val Gly Ala Ala 50 55 60Leu Ala Gly Val Leu Ile Leu
Val Leu Leu Ala Tyr Phe Ile Gly Leu65 70 75 80Lys His His His Ala
Gly Tyr Glu Gln Phe 85 90590PRTMus musculus 5Trp Asp Ala Pro Leu
Gly Ser Ser Tyr Met Cys Asn Lys Glu Gln Val1 5 10 15Leu Ser Val Ser
Arg Ala Phe Gln Ile Asn Thr Phe Asn Leu Lys Val 20 25 30Gln Pro Phe
Asn Val Thr Lys Gly Gln Tyr Ser Thr Ala Gln Asp Cys 35 40 45Ser Ala
Asp Glu Asp Asn Phe Leu Val Pro Ile Ala Val Gly Ala Ala 50 55 60Leu
Gly Gly Val Leu Ile Leu Val Leu Leu Ala Tyr Phe Ile Gly Leu65 70 75
80Lys Arg His His Thr Gly Tyr Glu Gln Phe 85 90690PRTHomo sapiens
6Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys Asn Lys Glu Gln Thr1 5
10 15Val Ser Val Ser Gly Ala Phe Gln Ile Asn Thr Phe Asp Leu Arg
Val 20 25 30Gln Pro Phe Asn Val Thr Gln Gly Lys Tyr Ser Thr Ala Gln
Glu Cys 35 40 45Ser Leu Asp Asp Asp Thr Ile Leu Ile Pro Ile Ile Val
Gly Ala Gly 50 55 60Leu Ser Gly Leu Ile Ile Val Ile Val Ile Ala Tyr
Val Ile Gly Arg65 70 75 80Arg Lys Ser Tyr Ala Gly Tyr Gln Thr Leu
85 90790PRTMus musculus 7Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met
Cys Asn Lys Glu Gln Val1 5 10 15Leu Ser Val Ser Arg Ala Phe Gln Ile
Asn Thr Phe Asn Leu Lys Val 20 25 30Gln Pro Phe Asn Val Thr Lys Gly
Gln Tyr Ser Thr Ala Gln Glu Cys 35 40 45Ser Leu Asp Asp Asp Thr Ile
Leu Ile Pro Ile Ile Val Gly Ala Gly 50 55 60Leu Ser Gly Leu Ile Ile
Val Ile Val Ile Ala Tyr Leu Ile Gly Arg65 70 75 80Arg Lys Thr Tyr
Ala Gly Tyr Gln Thr Leu 85 90891PRTHomo sapiens 8Trp Asp Ala Pro
Leu Gly Ser Ser Tyr Met Cys Asn Lys Glu Gln Thr1 5 10 15Val Ser Val
Ser Gly Ala Phe Gln Ile Asn Thr Phe Asp Leu Arg Val 20 25 30Gln Pro
Phe Asn Val Thr Gln Gly Lys Tyr Ser Thr Ala Glu Glu Cys 35 40 45Ser
Ala Asp Ser Asp Leu Asn Phe Leu Ile Pro Val Ala Val Gly Val 50 55
60Ala Leu Gly Phe Leu Ile Ile Val Val Phe Ile Ser Tyr Met Ile Gly65
70 75 80Arg Arg Lys Ser Arg Thr Gly Tyr Gln Ser Val 85 90991PRTMus
musculus 9Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys Asn Lys Glu
Gln Val1 5 10 15Leu Ser Val Ser Arg Ala Phe Gln Ile Asn Thr Phe Asn
Leu Lys Val 20 25 30Gln Pro Phe Asn Val Thr Lys Gly Gln Tyr Ser Thr
Ala Glu Glu Cys 35 40 45Ala Ala Asp Ser Asp Leu Asn Phe Leu Ile Pro
Val Ala Val Gly Val 50 55 60Ala Leu Gly Phe Leu Ile Ile Ala Val Phe
Ile Ser Tyr Met Ile Gly65 70 75 80Arg Arg Lys Ser Arg Thr Gly Tyr
Gln Ser Val 85 9010410PRTHomo sapiens 10Met Val Cys Phe Arg Leu Phe
Pro Val Pro Gly Ser Gly Leu Val Leu1 5 10 15Val Cys Leu Val Leu Gly
Ala Val Arg Ser Tyr Ala Leu Glu Leu Asn 20 25 30Leu Thr Asp Ser Glu
Asn Ala Thr Cys Leu Tyr Ala Lys Trp Gln Met 35 40 45Asn Phe Thr Val
Arg Tyr Glu Thr Thr Asn Lys Thr Tyr Lys Thr Val 50 55 60Thr Ile Ser
Asp His Gly Thr Val Thr Tyr Asn Gly Ser Ile Cys Gly65 70 75 80Asp
Asp Gln Asn Gly Pro Lys Ile Ala Val Gln Phe Gly Pro Gly Phe 85 90
95Ser Trp Ile Ala Asn Phe Thr Lys Ala Ala Ser Thr Tyr Ser Ile Asp
100 105 110Ser Val Ser Phe Ser Tyr Asn Thr Gly Asp Asn Thr Thr Phe
Pro Asp 115 120 125Ala Glu Asp Lys Gly Ile Leu Thr Val Asp Glu Leu
Leu Ala Ile Arg 130 135 140Ile Pro Leu Asn Asp Leu Phe Arg Cys Asn
Ser Leu Ser Thr Leu Glu145 150 155 160Lys Asn Asp Val Val Gln His
Tyr Trp Asp Val Leu Val Gln Ala Phe 165 170 175Val Gln Asn Gly Thr
Val Ser Thr Asn Glu Phe Leu Cys Asp Lys Asp 180 185 190Lys Thr Ser
Thr Val Ala Pro Thr Ile His Thr Thr Val Pro Ser Pro 195 200 205Thr
Thr Thr Pro Thr Pro Lys Glu Lys Pro Glu Ala Gly Thr Tyr Ser 210 215
220Val Asn Asn Gly Asn Asp Thr Cys Leu Leu Ala Thr Met Gly Leu
Gln225 230 235 240Leu Asn Ile Thr Gln Asp Lys Val Ala Ser Val Ile
Asn Ile Asn Pro 245 250 255Asn Thr Thr His Ser Thr Gly Ser Cys Arg
Ser His Thr Ala Leu Leu 260 265 270Arg Leu Asn Ser Ser Thr Ile Lys
Tyr Leu Asp Phe Val Phe Ala Val 275 280 285Lys Asn Glu Asn Arg Phe
Tyr Leu Lys Glu Val Asn Ile Ser Met Tyr 290 295 300Leu Val Asn Gly
Ser Val Phe Ser Ile Ala Asn Asn Asn Leu Ser Tyr305 310 315 320Trp
Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys Asn Lys Glu Gln Thr 325 330
335Val Ser Val Ser Gly Ala Phe Gln Ile Asn Thr Phe Asp Leu Arg Val
340 345 350Gln Pro Phe Asn Val Thr Gln Gly Lys Tyr Ser Thr Ala Gln
Asp Cys 355 360 365Ser Ala Asp Asp Asp Asn Phe Leu Val Pro Ile Ala
Val Gly Ala Ala 370 375 380Leu Ala Gly Val Leu Ile Leu Val Leu Leu
Ala Tyr Phe Ile Gly Leu385 390 395 400Lys His His His Ala Gly Tyr
Glu Gln Phe 405 41011410PRTHomo sapiens 11Met Val Cys Phe Arg Leu
Phe Pro Val Pro Gly Ser Gly Leu Val Leu1 5 10 15Val Cys Leu Val Leu
Gly Ala Val Arg Ser Tyr Ala Leu Glu Leu Asn 20 25 30Leu Thr Asp Ser
Glu Asn Ala Thr Cys Leu Tyr Ala Lys Trp Gln Met 35 40 45Asn Phe Thr
Val Arg Tyr Glu Thr Thr Asn Lys Thr Tyr Lys Thr Val 50 55 60Thr Ile
Ser Asp His Gly Thr Val Thr Tyr Asn Gly Ser Ile Cys Gly65 70 75
80Asp Asp Gln Asn Gly Pro Lys Ile Ala Val Gln Phe Gly Pro Gly Phe
85 90 95Ser Trp Ile Ala Asn Phe Thr Lys Ala Ala Ser Thr Tyr Ser Ile
Asp 100 105 110Ser Val Ser Phe Ser Tyr Asn Thr Gly Asp Asn Thr Thr
Phe Pro Asp 115 120 125Ala Glu Asp Lys Gly Ile Leu Thr Val Asp Glu
Leu Leu Ala Ile Arg 130 135 140Ile Pro Leu Asn Asp Leu Phe Arg Cys
Asn Ser Leu Ser Thr Leu Glu145 150 155 160Lys Asn Asp Val Val Gln
His Tyr Trp Asp Val Leu Val Gln Ala Phe 165 170 175Val Gln Asn Gly
Thr Val Ser Thr Asn Glu Phe Leu Cys Asp Lys Asp 180 185 190Lys Thr
Ser Thr Val Ala Pro Thr Ile His Thr Thr Val Pro Ser Pro 195 200
205Thr Thr Thr Pro Thr Pro Lys Glu Lys Pro Glu Ala Gly Thr Tyr Ser
210 215 220Val Asn Asn Gly Asn Asp Thr Cys Leu Leu Ala Thr Met Gly
Leu Gln225 230 235 240Leu Asn Ile Thr Gln Asp Lys Val Ala Ser Val
Ile Asn Ile Asn Pro 245 250 255Asn Thr Thr His Ser Thr Gly Ser Cys
Arg Ser His Thr Ala Leu Leu 260 265 270Arg Leu Asn Ser Ser Thr Ile
Lys Tyr Leu Asp Phe Val Phe Ala Val 275 280 285Lys Asn Glu Asn Arg
Phe Tyr Leu Lys Glu Val Asn Ile Ser Met Tyr 290 295 300Leu Val Asn
Gly Ser Val Phe Ser Ile Ala Asn Asn Asn Leu Ser Tyr305 310 315
320Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys Asn Lys Glu Gln Thr
325 330 335Val Ser Val Ser Gly Ala Phe Gln Ile Asn Thr Phe Asp Leu
Arg Val 340 345 350Gln Pro Phe Asn Val Thr Gln Gly Lys Tyr Ser Thr
Ala Gln Glu Cys 355 360 365Ser Leu Asp Asp Asp Thr Ile Leu Ile Pro
Ile Ile Val Gly Ala Gly 370 375 380Leu Ser Gly Leu Ile Ile Val Ile
Val Ile Ala Tyr Val Ile Gly Arg385 390 395 400Arg Lys Ser Tyr Ala
Gly Tyr Gln Thr Leu 405 41012411PRTHomo sapiens 12Met Val Cys Phe
Arg Leu Phe Pro Val Pro Gly Ser Gly Leu Val Leu1 5 10 15Val Cys Leu
Val Leu Gly Ala Val Arg Ser Tyr Ala Leu Glu Leu Asn 20 25 30Leu Thr
Asp Ser Glu Asn Ala Thr Cys Leu Tyr Ala Lys Trp Gln Met 35 40 45Asn
Phe Thr Val Arg Tyr Glu Thr Thr Asn Lys Thr Tyr Lys Thr Val 50 55
60Thr Ile Ser Asp His Gly Thr Val Thr Tyr Asn Gly Ser Ile Cys Gly65
70 75 80Asp Asp Gln Asn Gly Pro Lys Ile Ala Val Gln Phe Gly Pro Gly
Phe 85 90 95Ser Trp Ile Ala Asn Phe Thr Lys Ala Ala Ser Thr Tyr Ser
Ile Asp 100 105 110Ser Val Ser Phe Ser Tyr Asn Thr Gly Asp Asn Thr
Thr Phe Pro Asp 115 120 125Ala Glu Asp Lys Gly Ile Leu Thr Val Asp
Glu Leu Leu Ala Ile Arg 130 135 140Ile Pro Leu Asn Asp Leu Phe Arg
Cys Asn Ser Leu Ser Thr Leu Glu145 150 155 160Lys Asn Asp Val Val
Gln His Tyr Trp Asp Val Leu Val Gln Ala Phe 165 170 175Val Gln Asn
Gly Thr Val Ser Thr Asn Glu Phe Leu Cys Asp Lys Asp 180 185 190Lys
Thr Ser Thr Val Ala Pro Thr Ile His Thr Thr Val Pro Ser Pro 195 200
205Thr Thr Thr Pro Thr Pro Lys Glu Lys Pro Glu Ala Gly Thr Tyr Ser
210 215 220Val Asn Asn Gly Asn Asp Thr Cys Leu Leu Ala Thr Met Gly
Leu Gln225 230 235 240Leu Asn Ile Thr Gln Asp Lys Val Ala Ser Val
Ile Asn Ile Asn Pro 245 250 255Asn Thr Thr His Ser Thr Gly Ser Cys
Arg Ser His Thr Ala Leu Leu 260 265 270Arg Leu Asn Ser Ser Thr Ile
Lys Tyr Leu Asp Phe Val Phe Ala Val 275 280 285Lys Asn Glu Asn Arg
Phe Tyr Leu Lys Glu Val Asn Ile Ser Met Tyr 290 295 300Leu Val Asn
Gly Ser Val Phe Ser Ile Ala Asn Asn Asn Leu Ser Tyr305 310 315
320Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys Asn Lys Glu Gln Thr
325 330 335Val Ser Val Ser Gly Ala Phe Gln Ile Asn Thr Phe Asp Leu
Arg Val 340 345 350Gln Pro Phe Asn Val Thr Gln Gly Lys Tyr Ser Thr
Ala Glu Glu Cys 355 360 365Ser Ala Asp Ser Asp Leu Asn Phe Leu Ile
Pro Val Ala Val Gly Val 370 375 380Ala Leu Gly Phe Leu Ile Ile Val
Val Phe Ile Ser Tyr Met Ile Gly385 390 395 400Arg Arg Lys Ser Arg
Thr Gly Tyr Gln Ser Val 405 410
* * * * *