U.S. patent application number 17/600715 was filed with the patent office on 2022-07-21 for peptide-modified hybrid recombinant adeno-associated virus serotype between aav9 and aavrh74 with reduced liver tropism and increased muscle transduction.
This patent application is currently assigned to Genethon. The applicant listed for this patent is Association Institut de Myologie, Genethon, Institut National de la Sante et de la Recherche Medicale, Sorbonne Universite, Universite d'Evry Val d'Essonne. Invention is credited to Federico MINGOZZI, Giuseppe RONZITTI, Patrice VIDAL.
Application Number | 20220228167 17/600715 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-21 |
United States Patent
Application |
20220228167 |
Kind Code |
A1 |
RONZITTI; Giuseppe ; et
al. |
July 21, 2022 |
Peptide-Modified Hybrid Recombinant Adeno-Associated Virus Serotype
Between AAV9 and AAVrh74 with Reduced Liver Tropism and Increased
Muscle Transduction
Abstract
The invention relates to a recombinant adeno-associated virus
(AAV) capsid protein, which is a peptide-modified hybrid between
AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins
comprising at least one copy of a peptide comprising the RGD motif,
wherein said recombinant peptide-modified hybrid AAV capsid protein
has a further reduced liver tropism and an increased muscle
transduction compared to the recombinant hybrid AAV capsid protein
not having said peptide. The invention relates also to the derived
peptide-modified hybrid AAV serotype vector particles packaging a
gene of interest and their use in gene therapy, in particular for
treating neuromuscular genetic diseases, in particular muscular
genetic diseases.
Inventors: |
RONZITTI; Giuseppe;
(Fontainebleau, FR) ; VIDAL; Patrice; (Ris
Orangis, FR) ; MINGOZZI; Federico; (Philadelphia,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genethon
Institut National de la Sante et de la Recherche Medicale
Universite d'Evry Val d'Essonne
Sorbonne Universite
Association Institut de Myologie |
Evry-Courcouronnes
Paris
Evry
Paris
Paris |
|
FR
FR
FR
FR
FR |
|
|
Assignee: |
Genethon
Evry-Courcouronnes
FR
Institut National de la Sante et de la Recherche
Medicale
Paris
FR
Universite d'Evry Val d'Essonne
Evry
FR
Sorbonne Universite
Paris
FR
Association Institut de Myologie
Paris
FR
|
Appl. No.: |
17/600715 |
Filed: |
October 4, 2019 |
PCT Filed: |
October 4, 2019 |
PCT NO: |
PCT/EP2019/076958 |
371 Date: |
October 1, 2021 |
International
Class: |
C12N 15/86 20060101
C12N015/86; A61P 25/28 20060101 A61P025/28; C07K 14/075 20060101
C07K014/075; C12N 15/11 20060101 C12N015/11; C12N 9/22 20060101
C12N009/22 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 4, 2019 |
EP |
PCT/EP2019/058560 |
Claims
1. A recombinant adeno-associated virus (AAV) capsid protein, which
is a peptide-modified hybrid between AAV serotype 9 (AAV9) and AAV
serotype 74 (AAVrh74) capsid proteins comprising at least one copy
of a peptide comprising the RGD motif, wherein said recombinant
peptide-modified hybrid AAV capsid protein has a further reduced
liver tropism and an increased muscle transduction compared to the
recombinant hybrid AAV capsid protein not having said peptide.
2. The recombinant hybrid AAV capsid protein according to claim 1,
which results from the replacement of a variable region in the AAV9
or AAVrh74 capsid sequence with the corresponding variable region
of the other AAV serotype capsid sequence, wherein the variable
region of AAV9 capsid corresponds to the sequence situated from any
one of positions 331 to 493 to any one of positions 556 to 736 in
AAV9 capsid of SEQ ID NO: 1 or a fragment of at least 10, 15, 20,
25, 30, 35, 40, 45, 50, 55 or 60 consecutive amino acids of the
sequence situated from positions 493 to 556 in AAV9 capsid of SEQ
ID NO: 1, and the variable region of AAVrh74 capsid corresponds to
the sequence situated from any one of positions 332 to 495 to any
one of positions 558 to 738 in AAVrh74 capsid of SEQ ID NO: 2 or a
fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60
consecutive amino acids of the sequence situated from positions 495
to 558 in AAVrh74 capsid of SEQ ID NO: 2.
3. The recombinant hybrid AAV capsid protein according to claim 2,
which results from the replacement of the variable region
corresponding to the sequence situated from positions 449 to 609 in
AAV9 capsid of SEQ ID NO: 1 or from positions 450 to 611 in AAVrh74
capsid of SEQ ID NO: 2 with the corresponding variable region of
the other AAV serotype capsid sequence.
4. The recombinant hybrid AAV capsid protein according to claim 1,
which is derived from a hybrid AAV capsid protein comprising a
sequence selected from the group consisting of the sequences SEQ ID
NO: 3 and SEQ ID NO: 4 and the sequences having at least 85%, 90%,
95%, 97%, 98% or 99% identity with said sequences.
5-6. (canceled)
7. The recombinant hybrid AAV capsid protein according to claim 1,
wherein the peptide comprising the RGD motif is of up to 30 amino
acids, and comprises or consists of any one of: RGDLGLS (SEQ ID NO:
8), LRGDGLS (SEQ ID NO: 14), LGRGDLS (SEQ ID NO: 15), LGLRGDS (SEQ
ID NO: 16), LGLSRGD (SEQ ID NO: 17) and RGDMSRE (SEQ ID NO:
18).
8-10. (canceled)
11. The recombinant hybrid AAV capsid protein according to claim 7,
wherein the sequences SEQ ID NO: 8 and 14 to 18 are flanked by GQSG
(SEQ ID NO: 9) and AQAA (SEQ ID NO: 10), respectively at the N- and
C-terminal end of the peptide.
12. The recombinant hybrid AAV capsid protein according to claim 7,
wherein the peptide comprises or consists of SEQ ID NO: 13.
13. (canceled)
14. The recombinant hybrid AAV capsid protein according to claim 1,
wherein the at least one copy of the peptide comprising the RGD
motif is inserted into a site exposed on the AAV capsid surface
chosen from a site around any of positions 261, 383, 449, 575 or
590 according to the numbering in SEQ ID NO: 3.
15. (canceled)
16. The recombinant hybrid AAV capsid protein according to claim
14, wherein the at least one copy of the peptide comprising the RGD
motif is inserted around position 449 or 590 according to the
numbering in SEQ ID NO: 3.
17. (canceled)
18. The recombinant hybrid AAV capsid protein according to claim
16, wherein the insertion site of the at least one copy of the
peptide comprising the RGD motif is from positions 587 to 592
according to the numbering in SEQ ID NO: 3, and wherein the peptide
comprising the RGD motif replaces all the residues from positions
587 to 592 of the AAV capsid protein according to the numbering in
SEQ ID NO: 3.
19. (canceled)
20. The recombinant hybrid AAV capsid protein according to claim 1,
which comprises a sequence selected from the group consisting of
SEQ ID NO: 5 and the sequences having at least 85%, 90%, 95%, 97%,
98% or 99% identity with said sequence.
21. The recombinant hybrid AAV capsid protein according to claim 1,
which is a hybrid VP1, VP2 or VP3 protein.
22. A recombinant chimeric AAV capsid protein, which is selected
from the group consisting of: a chimeric VP1 protein comprising:
(i) a VP1-specific N-terminal region having a sequence from natural
or artificial AAV serotype other than AAV9 and AAVrh74, (ii) a
VP2-specific N-terminal region having a sequence from AAV9, AAVrh74
or natural or artificial AAV serotype other than AAV9 and AAVrh74,
and (iii) a VP3 C-terminal region having the sequence of a hybrid
VP3 protein according to claim 21, and a chimeric VP2 protein
comprising: (i) a VP2-specific N-terminal region having a sequence
from natural or artificial AAV serotype other than AAV9 and
AAVrh74, and (ii) a VP3 C-terminal region having the sequence of a
hybrid VP3 protein according to claim 21.
23. A polynucleotide encoding the recombinant hybrid AAV capsid
protein according to claim 1, in expressible form, and eventually
further encoding AAV Replicase protein in expressible form.
24. (canceled)
25. An AAV vector particle packaging a gene of interest, which
comprises the hybrid recombinant AAV capsid protein according to
claim 1, and eventually also at least one AAV capsid protein from
natural or artificial AAV serotype other than AAV9 and AAVrh74.
26. The AAV vector particle according to claim 25, wherein the gene
of interest is selected from the group consisting of: (i)
therapeutic genes; (ii) genes encoding therapeutic proteins or
peptides such as therapeutic antibodies or antibody fragments and
genome editing enzymes; and (iii) genes encoding therapeutic RNAs
such as interfering RNAs, guide RNAs for genome editing and
antisense RNAs capable of exon skipping.
27. A pharmaceutical composition comprising a therapeutically
effective amount of AAV vector particles according to claim 26.
28. A method of treating a disease by gene therapy, comprising
administering to a patient in need thereof a therapeutically
effective amount of a pharmaceutical composition comprising the
recombinant hybrid AAV capsid protein according to claim 1.
29. The method according to claim 28, wherein the gene therapy
targets a gene responsible for a neuromuscular genetic disorders
selected from the group consisting of Duchenne muscular dystrophy,
Limb-girdle muscular dystrophies, Spinal muscular atrophy,
Myotubular myopathy, Pompe disease and Glycogen storage disease
III.
30. The method according to claim 29, wherein the target gene is
selected from the group consisting of DMD, CAPN3, DYSF, FKRP, ANO5,
SMN1, MTM1, GAA and AGL genes.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a recombinant
adeno-associated virus (AAV) capsid, which is a peptide-modified
hybrid between AAV serotype 9 (AAV9) and AAV serotype rh74
(AAVrh74) capsid proteins having a further reduced liver tropism
and an increased muscle transduction compared to the hybrid AAV
capsid protein not having the peptide. The invention relates also
to the derived peptide-modified hybrid AAV serotype vector
particles packaging a gene of interest, and their use in gene
therapy, in particular for treating neuromuscular genetic diseases,
in particular muscular genetic diseases.
BACKGROUND OF THE INVENTION
[0002] Recombinant Adeno-Associated Virus (rAAV) vectors are widely
used for in vivo gene transfer. rAAV vectors are non-enveloped
vectors composed of a capsid of 20 nm of diameter and a single
strand DNA of 4.7 kb. The genome carries two genes, rep and cap,
flanked by two palindromic regions named Inverted terminal Repeats
(ITR). The cap gene codes for three structural proteins VP1, VP2
and VP3 that compose the AAV capsid. VP1, VP2 and VP3 share the
same C-terminal end which is all of VP3. Using AAV2 has a
reference, VP1 has a 735 amino acid sequence (GenBank YP_680426);
VP2 (598 amino acids) starts at the Threonine 138 (T138) and VP3
(533 amino acids) starts at the methionine 203 (M203). AAV
serotypes are defined by their capsid. Different serotypes exist,
each of them displaying its own tissue targeting specificity.
Therefore, the choice of using a serotype depends on the tissue to
transduce. Skeletal muscle and liver tissues are infected and
transduced efficiently by different serotypes of AAV vectors such
as AAV8, AAV9 and
[0003] AAV-rh74.
[0004] Chimeric or hybrid AAV serotypes have been generated by
exchanging fragments of capsid sequences between capsids of
different naturally occurring AAV serotypes, in order to increase
AAV transduction efficiency or increase AAV tropism to a cell or
tissue type of interest.
[0005] Hybrid AAV capsids were generated by combining structural
domains of capsids of AAV8 and AAV serotypes isolated from primate
brain. The resulting AAV hybrid serotypes can transduce retinal
tissue in human and mice with no increase in efficiency compared to
AAV2 and AAV5 vectors (Charbel Issa et al., PLOS ONE, 2013, 8,
e60361). However, one of the hybrid AAV serotype shows improved
transduction efficiency for fat tissue compared to AAV1, AAV8 and
AAV9 (Liu et al., Molecular Therapy, 2014, 1, 8, doi:10.1038/mtm).
WO 2015/191508 discloses recombinant hybrid AAV capsids generated
by exchanging variable regions of AAV capsids from various species
(human, primate, avian, snake, bovine), in particular AAV capsids
with central nervous system tropism to generate CNS specific
chimeric capsids.
[0006] WO 2017/096164 discloses recombinant hybrid AAV capsids
between AAV1, AAV2, AAV3b, AAV6 and AAV8 serotypes exhibiting
enhanced human skeletal muscle tropism. However, all naturally
occurring AAV serotypes and variants tested to date have a
propensity to accumulate within the liver. This causes problems, in
particular when the AAV vector is administered by the systemic
route. Firstly, a transgene aimed to be expressed in muscle may
have toxic effects on the liver. Secondly, AAV vector entry in
liver reduces the amount of vector available for skeletal muscles.
Consequently, higher doses of AAV vectors are required. This
increases the possibility to induce liver toxicity and the cost of
vector production.
[0007] Tissue-specific promoters and microRNA-based gene regulation
strategies have been used to segregate gene expression patterns
among different tissue types. However, such regulatory strategies
do not preclude sequestration of AAV vector genomes in off-target
organs such as the liver after systemic administration.
[0008] Attenuation of heparin binding by mutating the basic
residues R585 or R588 of the capsid protein was shown to abolish
heparin sulfate binding and reduce the liver tropism of
AAV2-derived vectors (Asokan et al., Nat. Biotechnol., 2010, 28,
79-82). However, this strategy can only work for serotypes like
AAV2 and AAV6 whose liver tropism is determined by basic residues
binding to heparin.
[0009] Therefore, there is a need for new AAV vectors, having a
reduced liver tropism and a concomitant increased muscle
transduction.
[0010] An AAV2 vector displaying a RGDLGLS (SEQ ID NO: 8) peptide
inserted into AAV capsid (position R588 relative to VP1 protein
numbering) efficiently transduces primary murine breast cancer
cells in vitro but fails to transduce breast cancer tumor cells in
vivo (Michelfelder et al., PLoS ONE, 2009, 4, e5122). AAV9
displaying the same surface peptide (RGDLGLS or P1) targets
efficiently human astrocytes in vitro (Kunze et al., Glia, 2018,
66, 413-427).
SUMMARY OF THE INVENTION
[0011] The inventors have generated a new peptide-modified hybrid
AAV serotype using a combination of two serotypes that infect
efficiently the muscle and liver tissues, AAV9 and AAV-rh74. The
new peptide-modified hybrid AAV serotype was generated by the
insertion of a peptide comprising the RGD motif into a variable
region of the cap gene between the AAV9 and AAVrh74 serotypes (FIG.
1). The inventors have found that surprisingly, the insertion of
the peptide comprising the RGD motif on the hybrid capsid protein
increased muscle transduction and ameliorated liver detargeting
(FIG. 2).
[0012] The new peptide-modified hybrid AAV serotypes are useful in
gene therapy of diseases affecting muscle tissues, in particular
skeletal muscle tissue and/or cardiac tissue, such as neuromuscular
disorders, in particular muscular disorders, including genetic
diseases, autoimmune diseases, neurodegenerative diseases and
cancer.
[0013] Therefore, the invention encompasses a peptide-modified
hybrid recombinant AAV capsid between AAV9 and AAVrh74 capsids with
increased muscle transduction and reduced liver tropism, AAV vector
particles comprising the peptide-modified hybrid recombinant AAV
capsid, compositions comprising the peptide-modified hybrid AAV
serotype vector particles, and methods of making and using said
peptide-modified hybrid AAV serotype vector particles and
compositions, in particular in gene therapy.
DETAILED DESCRIPTION OF THE INVENTION
Peptide-Modified Recombinant Hybrid AAV Capsid Protein
[0014] One aspect of the invention relates to a recombinant
adeno-associated virus (AAV) capsid protein, which is a
peptide-modified hybrid between AAV serotype 9 (AAV9) and AAV
serotype 74 (AAVrh74) capsid proteins comprising at least one copy
of a peptide comprising the RGD motif, wherein said recombinant
peptide-modified hybrid AAV capsid protein has a further reduced
liver tropism and an increased muscle transduction compared to the
hybrid AAV capsid protein not having the peptide.
[0015] As used herein, the term "tropism" refers to the specificity
of an AAV capsid protein present in an AAV viral particle, for
infecting or transducing a particular type of cell or tissue.
[0016] The tropism of an AAV capsid for a particular type of cell
or tissue may be determined by measuring the ability of AAV vector
particles comprising the peptide-modified hybrid AAV capsid protein
to infect or to transduce a particular type of cell or tissue,
using standard assays that are well-known in the art such as those
disclosed in the examples of the present application.
[0017] As used herein, the term "liver tropism" or "hepatic
tropism" refers to the tropism for liver or hepatic tissue and
cells, including hepatocytes.
[0018] In some embodiments, the liver tropism of the
peptide-modified hybrid AAV capsid protein is further reduced by at
least 5%, 10%, 20%, 30%, 40%, 50% or more compared to the liver
tropism of the hybrid AAV capsid protein not having the
peptide.
[0019] According to the invention, the peptide-modified hybrid AAV
capsid protein has increased tropism or transduction for muscle
cells and tissues compared to the hybrid AAV capsid not having the
peptide.
[0020] Muscle tissues include in particular cardiac and skeletal
muscle tissues.
[0021] As used herein, the term "muscle cells" refers to myocytes,
myotubes, myoblasts, and/or satellite cells.
[0022] As used herein, "or" means "and/or".
[0023] In some embodiments, the muscle tropism or muscle
transduction of the peptide-modified hybrid AAV capsid protein is
increased by at least 5%, 10%, 20%, 30%, 40%, 50% or more;
preferably at least 50%, 60%, 70%, 80%, 90%, 99%, 100% or more
compared to the hybrid AAV capsid protein not having the
peptide.
[0024] In some embodiments, the peptide-modified hybrid AAV capsid
protein is a peptide-modified hybrid VP1, VP2 or VP3 protein.
[0025] In some embodiments, the peptide-modified hybrid AAV capsid
protein has tropism for at least skeletal muscle tissue. In some
preferred embodiments, the peptide-modified hybrid AAV capsid
protein has tropism for both skeletal and cardiac muscle tissues.
An example of this type of peptide-modified hybrid is the
peptide-modified hybrid AAV capsid of SEQ ID NO: 5 (named AAV-MT in
the examples). This type of peptide-modified hybrid AAV capsid is
useful for the treatment of cardiac and skeletal muscle
disorders.
[0026] The peptide-modified hybrid AAV capsid protein according to
the invention may be derived from any AAV9 and AAVrh74 capsid
protein sequences; such sequences are well-known in the art and
available in public sequence data base. For example, AAV9 capsid
protein corresponds to GenBank accession numbers: AY530579.1; SEQ
ID NO: 123 of WO 2005/033321; SEQ ID NO: 1 of WO 2012/112832; clade
F AAV of WO 2016049230; AAV9 capsid variants in which one or more
of the native residues at positions 271 (D), 446(Y), and 470 (N)
are replaced with another amino acid, preferably alanine as
disclosed in WO 2012/112832; AAV9 capsid variants at one or more of
positions K143R, T251A, S499A, S669A and S490A as disclosed in US
2014/0162319. AAVrh74 capsid protein corresponds to SEQ ID NO: 1 of
WO 2015/013313; SEQ ID NO: 6 of WO 2006/110689; SEQ ID NO: 1 of WO
2013/123503; SEQ ID NO: 4 of WO 2013/158879; and K137R, K333R,
K550R, K552R, K569R, K691R, K695R, K709R variants and combination
thereof.
[0027] In some embodiments, the peptide-modified hybrid AAV capsid
protein according to the invention is derived from the AAV9 capsid
protein of SEQ ID NO: 1 (GenBank AY530579.1) and the AAVrh74
protein of SEQ ID NO: 2.
[0028] In some embodiments, the peptide-modified hybrid AAV capsid
protein according to the invention results from the replacement of
a variable region in the AAV9 or AAVrh74 capsid sequence with the
corresponding variable region of the other AAV serotype capsid
sequence, [0029] wherein the variable region of AAV9 capsid
corresponds to the sequence situated from any one of positions 331
to 493 to any one of positions 556 to 736 in AAV9 capsid of SEQ ID
NO: 1 (reference sequence), or a fragment of at least 10, 15, 20,
25, 30, 35, 40, 45, 50, 55 or 60 consecutive amino acids of the
sequence situated from positions 493 to 556 in AAV9 capsid of SEQ
ID NO: 1, and [0030] the variable region of AAVrh74 capsid
corresponds to the sequence situated from any one of positions 332
to 495 to any one of positions 558 to 738 in AAVrh74 capsid of SEQ
ID NO: 2 (reference sequence), or a fragment of at least 10, 15,
20, 25, 30, 35, 40, 45, 50, 55 or 60 consecutive amino acids of the
sequence situated from positions 495 to 558 in AAVrh74 capsid of
SEQ ID NO: 2.
[0031] The invention encompasses peptide-modified hybrid AAV capsid
proteins derived from any AAV9 and AAVrh74 capsid protein sequences
by replacement of a variable region in the AAV9 or AAVrh74 capsid
sequence with the corresponding variable region of the other AAV
serotype capsid sequence, as defined above. According to the
invention, the variable region is defined using AAV9 capsid of SEQ
ID NO: 1 and AAVrh74 capsid of SEQ ID NO: 2 as reference. After
sequence alignment of any other AAV9 capsid sequence with SEQ ID
NO: 1 or any of other AAVrh74 capsid sequence with SEQ ID NO: 2,
using standard protein sequence alignment programs that are
well-known in the art, such as for example BLAST, FASTA, CLUSTALW,
and the like, a person skilled in the art can easily obtained the
corresponding positions of the variable region in other AAV9 or
AAVrh74 capsid sequences.
[0032] In some preferred embodiments, the peptide-modified hybrid
AAV capsid protein according to the invention results from the
replacement of the variable region corresponding to that situated
from positions 449 to 609 in the AAV9 capsid sequence of SEQ ID NO:
1 or from positions 450 to 611 in the AAVrh74 capsid sequence of
SEQ ID NO: 2 with the corresponding variable region of the other
serotype.
[0033] In some preferred embodiments, the peptide-modified hybrid
AAV capsid protein according to the invention is derived from a
hybrid AAV capsid selected from the group consisting of: SEQ ID NO:
3, SEQ ID NO: 4, and the sequences having at least 85%, 90%, 95%,
97%, 98% or 99% identity with said sequences. SEQ ID NO: 3 is
derived from AAV9 capsid protein of SEQ ID NO: 1 by replacement of
AAV9 variable region (positions 449 to 609 of SEQ ID NO: 1) with
the variable region of AAVrh74 capsid protein (positions 450 to 611
of SEQ ID NO: 2); the corresponding hybrid is named Hybrid
Cap9-rh74 in the examples. VP2 corresponds to the amino acid
sequence from T138 to the end of SEQ ID NO: 3. VP3 corresponds to
the amino acid sequence from M203 to the end of SEQ ID NO: 3. SEQ
ID NO: 4 is derived from AAVrh74 capsid protein of SEQ ID NO: 2 by
replacement of rh74 variable region (positions 450 to 611 of SEQ ID
NO: 2) with the variable region of AAV9 capsid protein (positions
449 to 609 of SEQ ID NO: 1); the corresponding hybrid is named
Hybrid Caprh74-9 in the examples. VP2 corresponds to the amino acid
sequence from T138 to the end of SEQ ID NO: 4. VP3 corresponds to
the amino acid sequence from M204 to the end of SEQ ID NO: 4.
[0034] In some preferred embodiments, the peptide-modified hybrid
AAV capsid protein according to the invention is derived from AAV9
capsid protein by replacement of a variable region of AAV9 capsid
sequence with the corresponding variable region of AAVrh74 capsid
sequence as defined above, preferably the peptide-modified hybrid
AAV capsid protein comprises the replacement of the variable region
corresponding to that situated from positions 449 to 609 in AAV9
capsid of SEQ ID NO: 1 with the variable region corresponding to
that situated from positions 450 to 611 in AAVrh74 capsid of SEQ ID
NO: 2. In some more preferred embodiments, the peptide-modified
hybrid AAV capsid protein according to the invention is derived
from a hybrid AAV capsid protein selected from the group consisting
of: of SEQ ID NO: 3 and the sequences having at least 85%, 90%,
95%, 97%, 98% or 99% identity with said sequence; preferably SEQ ID
NO: 3.
[0035] The term "identity" refers to the sequence similarity
between two polypeptide molecules or between two nucleic acid
molecules. When a position in both compared sequences is occupied
by the same base or same amino acid residue, then the respective
molecules are identical at that position. The percentage of
identity between two sequences corresponds to the number of
matching positions shared by the two sequences divided by the
number of positions compared and multiplied by 100. Generally, a
comparison is made when two sequences are aligned to give maximum
identity. The identity may be calculated by alignment using, for
example, the GCG (Genetics Computer Group, Program Manual for the
GCG Package, Version 7, Madison, Wisconsin) pileup program, or any
of sequence comparison algorithms such as BLAST, FASTA or
CLUSTALW.
[0036] The peptide comprising the RGD motif is preferably of up to
30 amino acids.
[0037] In some embodiments the peptide of up to 30 amino acids
comprises or consists of any one of: RGDLGLS (SEQ ID NO: 8),
LRGDGLS (SEQ ID NO: 14), LGRGDLS (SEQ ID NO: 15), LGLRGDS (SEQ ID
NO: 16), LGLSRGD (SEQ ID NO: 17) and RGDMSRE (SEQ ID NO: 18);
preferably SEQ ID NO: 8. The sequences SEQ ID NO: 8 and 14 to 18
may be flanked by up to five or more amino acids at their N- and/or
C-terminal ends, such as for example by GQSG (SEQ ID NO: 9) and
AQAA (SEQ ID NO: 10), respectively at the N- and C-terminal end of
the peptide.
[0038] The peptide-modified hybrid AAV capsid protein of the
invention may comprise up to 5 copies of the peptide comprising the
RGD motif, preferably 1 copy of said peptide. The peptide-modified
hybrid AAV capsid protein may comprise multiple copies of the same
peptide or one or more copies of different peptides.
[0039] The peptide-modified hybrid AAV capsid protein of the
invention comprises the one or more peptide(s) comprising the RGD
motif, inserted into a site exposed on the AAV capsid surface.
Sites on the AAV capsid which are exposed on the capsid surface and
tolerate peptide insertions, i.e. do not affect assembly and
packaging of the virus capsid, are well-known in the art and
include for example the AAV capsid surface loops or antigenic loops
(Girod et al., Nat. Med., 1999, 5, 1052-1056; Grifman et al.,
Molecular Therapy, 2001, 3, 964-975); other sites are disclosed in
Rabinowitz et al., Virology, 1999, 265, 274-285; Wu et al., J.
Virol., 2000, 74, 8635-8647.
[0040] In particular, the peptide(s) comprising the RGD motif are
inserted around any of positions 261, 383, 449, 575 or 590
according to the numbering in SEQ ID NO: 3, preferably around
position 449 or 590, more preferably around position 590. The
positions are indicated by reference to SEQ ID NO: 3; one skilled
in the art will be able to find easily the corresponding positions
in another sequence after alignment with SEQ ID NO: 3.
[0041] The insertion site is advantageously from positions 587 to
592 or 588 to 593 according to the numbering in SEQ ID NO: 3,
preferably from positions 587 to 592. The insertion of the peptide
may or may not cause the deletion of some or all of the residue(s)
from the insertion site. The peptide advantageously replaces all
the residues from positions 587 to 592 or 588 to 593 of the AAV
capsid protein according to the numbering in SEQ ID NO: 3,
preferably all of the residues from positions 587 to 592.
[0042] In some preferred embodiments, the peptide replaces all the
residues from positions 587 to 592 of the AAV capsid protein
according to the numbering in SEQ ID NO: 3. Preferably, said
peptide comprises any one of RGDLGLS (SEQ ID NO: 8), LRGDGLS (SEQ
ID NO: 14), LGRGDLS (SEQ ID NO: 15), LGLRGDS (SEQ ID NO: 16),
LGLSRGD (SEQ ID NO: 17) and RGDMSRE (SEQ ID NO: 18); preferably SEQ
ID NO: 8. A more preferred peptide is RGDLGLS (SEQ ID NO: 8)
flanked by GQSG (SEQ ID NO: 9) and AQAA (SEQ ID NO: 10),
respectively at its N- and C-terminal end, corresponding to
GQSGRGDLGLSAQAA (SEQ ID NO: 13) .
[0043] In some preferred embodiment, the peptide-modified hybrid
AAV capsid protein comprises or consists of a sequence selected
from the group consisting of SEQ ID NO: 5, the sequences having at
least 85%, 90%, 95%, 97%, 98% or 99% identity with said sequence
and the fragment thereof corresponding to VP2 or VP3 capsid
protein. VP2 corresponds to the amino acid sequence from T138 to
the end of SEQ ID NO: 5. VP3 corresponds to the amino acid sequence
from M203 to the end of SEQ ID NO: 5. In some preferred
embodiments, said peptide-modified hybrid AAV capsid protein
comprises the sequence SEQ ID NO: 5 or a fragment thereof
corresponding to VP2 or VP3 capsid protein. SEQ ID NO: 5 is derived
from the hybrid Cap9-rh74 of SEQ ID NO: 3 by the insertion of the
peptide of SEQ ID NO: 8.
[0044] In some preferred embodiments, said peptide-modified hybrid
AAV capsid protein comprises a sequence selected from the group
consisting of the sequence SEQ ID NO: 5, the sequences having at
least 85%, 90%, 95%, 97%, 98% or 99% identity with said sequence,
and the fragment thereof corresponding to VP2 or VP3 capsid
protein. VP2 corresponds to the amino acid sequence from T138 to
the end of SEQ ID NO: 5. VP3 corresponds to the amino acid sequence
from M203 to the end of SEQ ID NO: 5. In some preferred
embodiments, said peptide-modified hybrid AAV capsid protein
comprises the sequence SEQ ID NO: 5 or a fragment thereof
corresponding to VP2 or VP3 capsid protein.
[0045] The invention encompasses also AAV VP1 and VP2 chimeric
capsid proteins derived from the peptide-modified AAV9/rh74 hybrid
VP3 capsid protein according to the invention, wherein the
VP1-specific N-terminal region and/or VP2-specific N-terminal
region are from a natural or artificial AAV serotype other than
AAV9 and AAVrh74.
[0046] In some embodiments, the peptide-modified AAV VP1 chimeric
capsid protein comprises: [0047] (i) a VP1-specific N-terminal
region having a sequence from natural or artificial AAV serotype
other than AAV9 and AAVrh74, [0048] (ii) a VP2-specific N-terminal
region having a sequence from AAV9, AAVrh74 or natural or
artificial AAV serotype other than AAV9 and AAVrh74, and [0049]
(iii) a VP3 C-terminal region having the sequence of a
peptide-modified hybrid VP3 protein according to the invention.
[0050] In some embodiments, the AAV VP2 chimeric capsid proteins
comprises [0051] (i) a VP2-specific N-terminal region having a
sequence from natural or artificial AAV serotype other than AAV9
and AAVrh74, and [0052] (ii) a VP3 C-terminal region having the
sequence of a peptide-modified hybrid VP3 protein according to the
invention.
Polynucleotide, Vector, and Use for AAV Vector Production
[0053] Another aspect of the invention is a polynucleotide encoding
the recombinant peptide-modified hybrid AAV capsid protein in
expressible form. The polynucleotide may be DNA, RNA or a synthetic
or semi-synthetic nucleic acid.
[0054] In some embodiments, the polynucleotide is a AAV9/rh74
hybrid cap gene encoding peptide-modified hybrid VP1, VP2 and VP3
capsid proteins according to the invention. In some preferred
embodiments, the polynucleotide comprises the sequence SEQ ID NO: 6
(encoding the peptide-modified hybrid AAV capsid protein of SEQ ID
NO: 5).
[0055] In some other embodiments, the polynucleotide is a chimeric
cap gene which encodes for a peptide-modified AAV9/rh74 hybrid VP3
capsid protein according to the invention and a chimeric VP1 capsid
protein, and maybe also a chimeric VP2 capsid protein wherein the
VP1-specific N-terminal region, and maybe also the VP2-specific
N-terminal region, are from a natural or artificial AAV serotype
other than AAV9 and AAVrh74. Such chimeric cap gene may be
generated by any suitable technique, using the coding sequence for
a peptide-modified AAV9/rh74 hybrid VP3 capsid protein according to
the invention in combination with heterologous sequences which may
be obtained from different selected AAV serotypes, non-contiguous
portions of the same AAV serotypes, from a non-viral AAV source or
from a non-viral source.
[0056] In some embodiments, the polynucleotide further encodes AAV
Replicase (Rep) protein in expressible form, preferably Rep from
AAV2.
[0057] The polynucleotide is advantageously inserted into a
recombinant vector, which includes, in a non-limiting manner,
linear or circular DNA or RNA molecules consisting of chromosomal,
non-chromosomal, synthetic or semi-synthetic nucleic acids, such as
in particular viral vectors, plasmid or RNA vectors.
[0058] Numerous vectors into which a nucleic acid molecule of
interest can be inserted in order to introduce it into and maintain
it in a eukaryotic host cell are known per se; the choice of an
appropriate vector depends on the use envisioned for this vector
(for example, replication of the sequence of interest, expression
of this sequence, maintaining of this sequence in extrachromosomal
form, or else integration into the chromosomal material of the
host), and also on the nature of the host cell.
[0059] In some embodiments, the vector is a plasmid.
[0060] The recombinant vector for use in the present invention is
an expression vector comprising appropriate means for expression of
the peptide-modified hybrid AAV capsid protein, and maybe also AAV
Rep protein. Usually, each coding sequence (hybrid AAV Cap and AAV
Rep) is inserted in a separate expression cassette either in the
same vector or separately. Each expression cassette comprises the
coding sequence (open reading frame or ORF) functionally linked to
the regulatory sequences which allow the expression of the
corresponding protein in AAV producer cells, such as in particular
promoter, promoter/enhancer, initiation codon (ATG), stop codon,
transcription termination signal. Alternatively, the hybrid AAV Cap
and the AAV Rep proteins may be expressed from a unique expression
cassette using an Internal Ribosome Entry Site (IRES) inserted
between the two coding sequences or a viral 2A peptide. In
addition, the codon sequences encoding the hybrid AAV Cap, and AAV
Rep if present, are advantageously optimized for expression in AAV
producer cells, in particular human producer cells.
[0061] The vector, preferably a recombinant plasmid, is useful for
producing hybrid AAV vectors comprising the peptide-modified hybrid
AAV capsid protein of the invention, using standard AAV production
methods that are well-known in the art (Review in Aponte-Ubillus et
al., Applied Microbiology and Biotechnology, 2018, 102:
1045-1054).
[0062] Following co-transfection, the cells are incubated for a
time sufficient to allow the production of AAV vector particles,
the cells are then harvested, lysed, and AAV vector particles are
purified by standard purification methods such as affinity
chromatography or Cesium Chloride density gradient
ultracentrifugation.
AAV Particle, Pharmaceutical Composition and Therapeutic Uses
[0063] Another aspect of the invention is an AAV particle
comprising the peptide-modified hybrid recombinant AAV capsid
protein of the invention. The AAV particle may comprise
peptide-modified hybrid VP1, VP2 and VP3 capsid proteins encoded by
a hybrid cap gene according to the invention. Alternatively or
additionally, the AAV particle may comprise chimeric VP1 and VP2
capsid proteins and a peptide-modified hybrid VP3 protein encoded
by a chimeric cap gene according to the invention.
[0064] In some embodiments, the AAV particle is a mosaic AAV
particle further comprising another AAV capsid protein from a
natural or artificial AAV serotype other than AAV9 and AAVrh74
serotype, wherein the mosaic AAV particle has a reduced liver
tropism compared to AAV9 and AAVrh74 serotypes. An artificial AAV
serotype may be with no limitation, a chimeric AAV capsid, a
recombinant AAV capsid, or a humanized AAV capsid. Such an
artificial capsid may be generated by any suitable technique, using
a selected AAV sequence (e.g. a fragment of a VP1 capsid protein)
in combination with heterologous sequences which may be obtained
from a different selected AAV serotype, non-contiguous portions of
the same AAV serotype, from a non-viral AAV source or from a
non-viral source.
[0065] Preferably, the AAV particle is an AAV vector particle. The
genome of the AAV vector may either be a single-stranded or
self-complementary double-stranded genome (McCarty et al, Gene
Therapy, 2003, Dec., 10(26), 2112-2118). Self-complementary vectors
are generated by deleting the terminal resolution site (trs) from
one of the AAV terminal repeats. These modified vectors, whose
replicating genome is half the length of the wild-type AAV genome
have the tendency to package DNA dimers. The AAV genome is flanked
by ITRs. In particular embodiments, the AAV vector is a pseudotyped
vector, i.e. its genome and capsid are derived from AAVs of
different serotypes. In some preferred embodiments, the genome of
the pseudotyped vector is derived from AAV2.
[0066] In some preferred embodiments, the AAV vector particle is
packaging a gene of interest.
[0067] The AAV particle may be obtained using the method of
producing recombinant AAV vector particles of the invention.
[0068] By "gene of interest", it is meant a gene useful for a
particular application, such as with no limitation, diagnosis,
reporting, modifying, therapy and genome editing.
[0069] For example, the gene of interest may be a therapeutic gene,
a reporter gene or a genome-editing enzyme.
[0070] By "gene of interest for therapy", "gene of therapeutic
interest", or "heterologous gene of interest", it is meant a
therapeutic gene or a gene encoding a therapeutic protein, peptide
or RNA.
[0071] The gene of interest is any nucleic acid sequence capable of
modifying a target gene or target cellular pathway, in particular
in muscle cells. For example, the gene may modify the expression,
sequence or regulation of the target gene or cellular pathway. In
some embodiments, the gene of interest is a functional version of a
gene or a fragment thereof. The functional version of said gene
includes the wild-type gene, a variant gene such as variants
belonging to the same family and others, or a truncated version,
which preserves the functionality of the encoded protein at least
partially. A functional version of a gene is useful for replacement
or additive gene therapy to replace a gene, which is deficient or
non-functional in a patient. In other embodiments, the gene of
interest is a gene which inactivates a dominant allele causing an
autosomal dominant genetic disease. A fragment of a gene is useful
as recombination template for use in combination with a genome
editing enzyme.
[0072] Alternatively, the gene of interest may encode a protein of
interest for a particular application, (for example an antibody or
antibody fragment, a genome-editing enzyme) or a RNA. In some
embodiments, the protein is a therapeutic protein including a
therapeutic antibody or antibody fragment, or a genome-editing
enzyme. In some embodiments, the RNA is a therapeutic RNA. The gene
of interest is a functional gene able to produce the encoded
protein, peptide or RNA in the target cells of the disease, in
particular muscle cells.
[0073] The AAV viral vector comprises the gene of interest in a
form expressible in muscle cells, including cardiac and skeletal
muscle cells. In particular, the gene of interest is operatively
linked to a ubiquitous, tissue-specific or inducible promoter which
is functional in muscle cells. The gene of interest may be inserted
in an expression cassette further comprising polyA sequences.
[0074] The RNA is advantageously complementary to a target DNA or
RNA sequence or binds to a target protein. For example, the RNA is
an interfering RNA such as a shRNA, a microRNA, a guide RNA (gRNA)
for use in combination with a Cas enzyme or similar enzyme for
genome editing, an antisense RNA capable of exon skipping such as a
modified small nuclear RNA (snRNA) or a long non-coding RNA. The
interfering RNA or microRNA may be used to regulate the expression
of a target gene involved in muscle disease. The guide RNA in
complex with a Cas enzyme or similar enzyme for genome editing may
be used to modify the sequence of a target gene, in particular to
correct the sequence of a mutated/deficient gene or to modify the
expression of a target gene involved in a disease, in particular a
neuromuscular disease. The antisense RNA capable of exon skipping
is used in particular to correct a reading frame and restore
expression of a deficient gene having a disrupted reading frame. In
some embodiments, the RNA is a therapeutic RNA.
[0075] The genome-editing enzyme according to the invention is any
enzyme or enzyme complex capable of modifying a target gene or
target cellular pathway, in particular in muscle cells. For
example, the genome-editing enzyme may modify the expression,
sequence or regulation of the target gene or cellular pathway. The
genome-editing enzyme is advantageously an engineered nuclease,
such as with no limitations, a meganuclease, zinc finger nuclease
(ZFN), transcription activator-like effector-based nuclease
(TALENs), Cas enzyme from clustered regularly interspaced
palindromic repeats (CRISPR)-Cas system and similar enzymes. The
genome-editing enzyme, in particular an engineered nuclease such as
Cas enzyme and similar enzymes, may be a functional nuclease which
generates a double-strand break (DSB) in the target genomic locus
and is used for site-specific genome editing applications,
including with no limitations: gene correction, gene replacement,
gene knock-in, gene knock-out, mutagenesis, chromosome
translocation, chromosome deletion, and the like. For site-specific
genome editing applications, the genome-editing enzyme, in
particular an engineered nuclease such as Cas enzyme and similar
enzymes may be used in combination with a homologous recombination
(HR) matrix or template (also named DNA donor template) which
modifies the target genomic locus by double-strand break
(DSB)-induced homologous recombination. In particular, the HR
template may introduce a transgene of interest into the target
genomic locus or repair a mutation in the target genomic locus,
preferably in an abnormal or deficient gene causing a neuromuscular
disease. Alternatively, the genome-editing enzyme, such as Cas
enzyme and similar enzymes may be engineered to become
nuclease-deficient and used as DNA-binding protein for various
genome engineering applications such as with no limitation:
transcriptional activation, transcriptional repression, epigenome
modification, genome imaging, DNA or RNA pull-down and the
like.
[0076] Another aspect of the invention is a pharmaceutical
composition comprising a therapeutically effective amount of AAV
particles comprising the peptide-modified hybrid recombinant AAV
capsid protein of the invention, preferably AAV vector particles
packaging a therapeutic gene of interest.
[0077] In some embodiments of the invention, the pharmaceutical
composition of the invention is for use as a medicament, in
particular in gene therapy. The invention encompasses the use of
the pharmaceutical composition of the invention as a medicament, in
particular for the treatment of a disease by gene therapy.
[0078] Gene therapy can be performed by gene transfer, gene
editing, exon skipping, RNA-interference, trans-splicing or any
other genetic modification of any coding or regulatory sequences in
the cell, including those included in the nucleus, mitochondria or
as commensal nucleic acid such as with no limitation viral
sequences contained in cells.
[0079] The two main types of gene therapy are the following: [0080]
a therapy aiming to provide a functional replacement gene for a
deficient/abnormal gene: this is replacement or additive gene
therapy; [0081] a therapy aiming at gene or genome editing: in such
a case, the purpose is to provide to a cell the necessary tools to
correct the sequence or modify the expression or regulation of a
deficient/abnormal gene so that a functional gene is expressed or
an abnormal gene is suppressed (inactivated): this is gene editing
therapy.
[0082] In additive gene therapy, the gene of interest may be a
functional version of a gene, which is deficient or mutated in a
patient, as is the case for example in a genetic disease. In such a
case, the gene of interest will restore the expression of a
functional gene.
[0083] Gene or genome editing uses one or more gene(s) of interest,
such as: [0084] (i) a gene encoding a therapeutic RNA as defined
above such as an interfering RNA like a shRNA or a microRNA, a
guide RNA (gRNA) for use in combination with a Cas enzyme or
similar enzyme, or an antisense RNA capable of exon skipping such
as a modified small nuclear RNA (snRNA); and [0085] (ii) a gene
encoding a genome-editing enzyme as defined above such as an
engineered nuclease like a meganuclease, zinc finger nuclease
(ZFN), transcription activator-like effector-based nuclease
(TALENs), Cas enzyme or similar enzymes; or a combination of such
genes, and maybe also a fragment of a functional version of a gene
for use as recombination template, as defined above.
[0086] Gene therapy is used for treating various diseases,
including with no limitations, genetic diseases, in particular
neuromuscular genetic disorders such as muscular genetic disorders;
cancer; neurodegenerative diseases and auto-immune diseases.
[0087] In some embodiments, gene therapy is used for treating
diseases affecting muscle tissues, in particular skeletal muscle
tissue and/or cardiac tissue, such as with no-limitations:
neuromuscular genetic disorders, in particular muscular genetic
disorders such as.
[0088] Examples of mutated genes in neuromuscular genetic
disorders, including muscular genetic disorders that can be
targeted by gene therapy using the pharmaceutical composition of
the invention are listed in the following tables:
Muscular Dystrophies
TABLE-US-00001 [0089] Gene Protein DMD Dystrophin EMD Emerin FHL1
Four and a half LIM domain 1 LMNA Lamin A/C SYNE1 Spectrin repeat
containing, nuclear envelope 1 (nesprin 1) SYNE2 Spectrin repeat
containing, nuclear envelope 2 (nesprin 2) TMEM43 Transmembrane
protein 43 TOR1AIP1 Torsin A interacting protein 1 DUX4 Double
homeobox 4 SMCHD1 Structural maintenance of chromosomes flexible
hinge domain containing 1 PTRF Polymerase I and transcript release
factor MYOT Myotilin CAV3 Caveolin 3 DNAJB6 HSP-40 homologue,
subfamily B, number 6 DES Desmin TNPO3 Transportin 3 HNRNPDL
Heterogeneous nuclear ribonucleoprotein D-like CAPN3 Calpain 3 DYSF
Dysferlin SGCG Gamma sarcoglycan SGCA Alpha sarcoglycan SGCB Beta
sarcoglycan SGCD Delta-sarcoglycan TCAP Telethonin TRIM32
Tripartite motif-containing 32 FKRP Fukutin-related protein TTN
Titin POMT1 Protein-O-mannosyltransferase 1 ANO5 Anoctamin 5 FKTN
Fukutin POMT2 Protein-O-mannosyltransferase 2 POMGNT1 O-linked
mannose beta1,2-N-acetylglucosaminyltransferase PLEC Plectin
TRAPPC11 trafficking protein particle complex 11 GMPPB GDP-mannose
pyrophosphorylase B DAG1 Dystroglycan1 DPM3 Dolichyl-phosphate
mannosyltransferase polypeptide 3 ISPD Isoprenoid synthase domain
containing VCP Valosin-containing protein LIMS2 LIM and senescent
cell antigen-like domains 2 GAA Glucosidase alpha, acid
Congenital Muscular Dystrophies
TABLE-US-00002 [0090] Gene Protein LAMA2 Laminin alpha 2 chain of
merosin COL6A1 Alpha 1 type VI collagen COL6A2 Alpha 2 type VI
collagen COL6A3 Alpha 3 type VI collagen SEPN1 Selenoprotein N1
FHL1 Four and a half LIM domain 1 ITGA7 Integrin alpha 7 precursor
DNM2 Dynamin 2 TCAP Telethonin LMNA Lamin A/C FKTN Fukutin POMT1
Protein-O-mannosyltransferase 1 POMT2 Protein-O-mannosyltransferase
2 FKRP Fukutin-related protein POMGNT1 O-linked mannose
beta1,2-N-acetylglucosaminyltransferase ISPD Isoprenoid synthase
domain containing POMGNT2 protein O-linked mannose
N-acetylglucosaminyltransferase 2 B3GNT1 UDP-GlcNAc:betaGal
beta-1,3-N-acetylglucosaminyl- transferase 1 GMPPB GDP-mannose
pyrophosphorylase B LARGE Like-glycosyltransferase DPM1
Dolichyl-phosphate mannosyltransferase 1, catalytic subunit DPM2
Dolichyl-phosphate mannosyltransferase polypeptide 2, regulatory
subunit ALG13 UDP-N-acetylglucosami-nyltransferase B3GALNT2
Beta-1,3-N-acetylgalacto-saminyltransferase 2 TMEM5 Transmembrane
protein 5 POMK Protein-O-mannose kinase CHKB Choline kinase beta
ACTA1 Alpha actin, skeletal muscle TRAPPC11 trafficking protein
particle complex 11
Congenital Myopathies
TABLE-US-00003 [0091] Gene Protein TPM3 Tropomyosin 3 NEB Nebulin
ACTA1 Alpha actin, skeletal muscle TPM2 Tropomyosin 2 (beta) TNNT1
Slow troponin T KBTBD13 Kelch repeat and BTB (POZ) domain
containing 13 CFL2 Cofilin 2 (muscle) KLHL40 Kelch-like family
member 40 KLHL41 Kelch-like family member 41 LMOD3 Leiomodin 3
(fetal) SEPN1 Selenoprotein N1 RYR1 Ryanodine receptor 1 (skeletal)
MYH7 Myosin, heavy polypeptide 7, cardiac muscle, beta MTM1
Myotubularin DNM2 Dynamin 2 BIN1 Amphiphysin TTN Titin SPEG SPEG
complex locus MEGF10 Multiple EGF-like-domains 10 MYH2 Myosin,
heavy polypeptide 2, skeletal muscle MYBPC3 Cardiac myosin binding
protein-C CNTN1 Contactin-1 TRIM32 Tripartite motif-containing 32
PTPLA Protein tyrosine phosphatase-like (3-Hydroxyacyl-CoA
dehydratase CACNA1S Calcium channel, voltage-dependent, L type,
alpha 1S subunit
Distal Myopathies
TABLE-US-00004 [0092] Gene symbol protein DYSF Dysferlin TTN Titin
GNE UDP-N-acetylglucosamine-2- epimerase/N-acetylmannosamine kinase
MYH7 Myosin, heavy polypeptide 7, cardiac muscle, beta MATR3 Matrin
3 TIA1 Cytotoxic granuleassociated RNA binding protein MYOT
Myotilin NEB Nebulin CAV3 Caveolin 3 LDB3 LIM domain binding 3 ANO5
Anoctamin 5 DNM2 Dynamin 2 KLHL9 Kelch-like homologue 9 FLNC
Filamin C, gamma (actin-binding protein - 280) VCP
Valosin-containing protein
Other Myopathies
TABLE-US-00005 [0093] Gene symbol protein ISCU Iron-sulfur cluster
scaffold homolog (E. coli) MSTN Myostatin FHL1 Four and a half LIM
domain 1 BAG3 BCL2-associated athanogene 3 ACVR1 Activin A
receptor, type II-like kinase 2 MYOT Myotilin FLNC Filamin C, gamma
(actin-binding protein - 280) LDB3 LIM domain binding 3 LAMP2
Lysosomal-associated membrane protein 2 precursor VCP
Valosin-containing protein CAV3 Caveolin 3 SEPN1 Selenoprotein N1
CRYAB Crystallin, alpha B DES Desmin VMA21 VMA21 Vacuolar H+-ATPase
Homolog (S. Cerevisiae) PLEC plectin PABPN1 Poly(A) binding
protein, nuclear 1 TTN Titin RYR1 Ryanodine receptor 1 (skeletal)
CLN3 Ceroid-lipofuscinosis, neuronal 3 (=battenin) TRIM54 TRIM63
Tripartite motif containing 63, E3 ubiquitin protein ligase
Myotonic Syndromes
TABLE-US-00006 [0094] Gene protein DMPK Myotonic dystrophy protein
kinase CNPB Cellular nucleic acid-binding protein CLCN1 Chloride
channel 1, skeletal muscle (Thomsen disease, autosomal dominant)
CAV3 Caveolin 3 HSPG2 Perlecan ATP2A1 ATPase, Ca++ transporting,
fast twitch 1
Ion Channel Muscle Diseases
TABLE-US-00007 [0095] Gene protein CLCN1 Chloride channel 1,
skeletal muscle (Thomsen disease, autosomal dominant) SCN4A Sodium
channel, voltage-gated, type IV, alpha SCN5A Voltage-gated sodium
channel type V alpha CACNA1S Calcium channel, voltage-dependent, L
type, alpha 1S subunit CACNA1A Calcium channel, voltage-dependent,
P/Q type, alpha 1A subunit KCNE3 Potassium voltage-gated channel,
Isk-related family, member 3 KCNA1 Potassium voltage-gated channel,
shaker-related subfamily, member 1 KCNJ18 Kir2.6 (inwardly
rectifying potassium channel 2.6) KCNJ2 Potassium
inwardly-rectifying channel J2 KCNH2 Voltage-gated potassium
channel, subfamily H, member 2 KCNQ1 Potassium voltage-gated
channel, KQT-like subfamily, member 1 KCNE2 Potassium voltage-gated
channel, Isk-related family, member 2 KCNE1 Potassium voltage-gated
channel, Isk-related family, member 1
Malignant Hyperthermia
TABLE-US-00008 [0096] Gene protein RYR1 Ryanodine receptor 1
(skeletal) CACNA1S Calcium channel, voltage-dependent, L type,
alpha 1S subunit
Metabolic Myopathies
TABLE-US-00009 [0097] Gene protein GAA Acid alpha-glucosidase
preproprotein AGL Amylo-1,6-glucosidase, 4-alpha-glucanotransferase
GBE1 Glucan (1,4-alpha-), branching enzyme 1 (glycogen branching
enzyme, Andersen disease, glycogen storage disease type IV) PYGM
Glycogen phosphorylase PFKM Phosphofructokinase, muscle PHKA1
Phosphorylase b kinase, alpha submit PGM1 Phosphoglucomutase 1 GYG1
Glycogenin 1 GYS1 Glycogen synthase 3 glycogen synthase 1 (muscle)
glycogen synthase 1 (muscle) PRKAG2 Protein kinase, AMP-activated,
gamma 2 non-catalytic subunit RBCK1 RanBP-type and C3HC4-type zinc
finger containing 1 (heme-oxidized IRP2 ubiquitin ligase 1) PGK1
Phosphoglycerate kinase 1 PGAM2 Phosphoglycerate mutase 2 (muscle)
LDHA Lactate dehydrogenase A ENO3 Enolase 3, beta muscle specific
CPT2 Carnitine palmitoyltransferase II SLC22A5 Solute carrier
family 22 member 5 SLC25A20 Carnitine-acylcarnitine translocase
ETFA Electron-transfer-flavoprotein, alpha polypeptide ETFB
Electron-transfer-flavoprotein, beta polypeptide ETFDH
Electron-transferring-flavoprotein dehydrogenase ACADVL
Acyl-Coenzyme A dehydrogenase, very long chain ABHD5 Abhydrolase
domain containing 5 PNPLA2 Adipose triglyceride lipase (desnutrin)
LPIN1 Lipin 1 (phosphatidic acid phosphatase 1) PNPLA8 Patatin-like
phospholipase domain containing 8
Hereditary Cardiomyopathies
TABLE-US-00010 [0098] Gene protein MYH6 Myosin heavy chain 6 MYH7
Myosin, heavy polypeptide 7, cardiac muscle, beta TNNT2 Troponin
T2, cardiac TPM1 Tropomyosin 1 (alpha) MYBPC3 Cardiac myosin
binding protein-C PRKAG2 Protein kinase, AMP-activated, gamma 2
non-catalytic subunit TNNI3 Troponin I, cardiac MYL3 Myosin light
chain 3 TTN Titin MYL2 Myosin light chain 2 ACTC1 Actin, alpha,
cardiac muscle precursor CSRP3 Cysteine and glycine-rich protein 3
(cardiac LIM protein) TNNC1 Slow troponin C VCL Vinculin MYLK2
Myosin light chain kinase 2 CAV3 Caveolin 3 MYOZ2 Myozenin 2, or
calsarcin 1, a Z disk protein JPH2 Junctophilin-2 PLN Phospholamban
NEXN Nexilin(F-actin binding protein) ANKRD1 Ankyrin repeat domain
1 (cardiac muscle) ACTN2 Actinin alpha2 NDUFAF1 NADH-ubiquinone
oxidoreductase 1 alpha subcomplex TSFM Ts translation elongation
factor, mitochondrial AARS2 Alanyl-tRNA synthetase 2, mitochondrial
MRPL3 Mitochondrial ribosomal protein L3 COX15 COX15 homolog,
cytochrome c oxidase assembly protein (yeast) MTO1 Mitochondrial
tRNA translation optimization 1 MRPL44 Mitochondrial ribosomal
protein L44 LMNA Lamin A/C LDB3 LIM domain binding 3 SCN5A
Voltage-gated sodium channel type V alpha DES Desmin EYA4 Eyes
absent 4 SGCD Delta-sarcoglycan TCAP Telethonin ABCC9 ATP-binding
cassette, sub-family C (member 9) TMPO Lamina-associated
polypeptide 2 PSEN2 Presenilin 2 CRYAB Crystallin, alpha B FKTN
Fukutin TAZ Tafazzin DMD Dystrophin LAMA4 Laminin alpha 4 ILK
Integrin-linked kinase MYPN Myopalladin RBM20 RNA binding motif
protein 20 SYNE1 Spectrin repeat containing, nuclear envelope 1
(nesprin 1) MURC Muscle-related coiled-coil protein DOLK Dolichol
kinase GATAD1 GATA zinc finger domain containing 1 SDHA succinate
dehydrogenase complex, subunit A, flavoprotein (Fp) GAA Acid
alpha-glucosidase preproprotein DTNA Dystrobrevin, alpha FLNA
Filamin A, alpha (actin binding protein 280) TGFB3 Transforming
growth factor, beta 3 RYR2 Ryanodine receptor 2 TMEM43
Transmembrane protein 43 DSP Desmoplakin PKP2 Plakophilin 2 DSG2
Desmoglein 2 DSC2 Desmocollin 2 JUP Junction plakoglobin CASQ2
Calsequestrin 2 (cardiac muscle) KCNQ1 Potassium voltage-gated
channel, KQT-like subfamily, member 1 KCNH2 Voltage-gated potassium
channel, subfamily H, member 2 ANK2 Ankyrin 2 KCNE1 Potassium
voltage-gated channel, Isk-related family, member 1 KCNE2 Potassium
voltage-gated channel, Isk-related family, member 2 KCNJ2 Potassium
inwardly-rectifying channel J2 CACNA1C Calcium channel,
voltage-dependent, L type, alpha 1C subunit SCN4B Sodium channel,
voltage-gated, type IV, beta subunit AKAP9 A kinase (PRKA) anchor
protein (yotiao) 9 SNTA1 Syntrophin, alpha 1 KCNJ5 Potassium
inwardly-rectifying channel, subfamily J, member 5 NPPA Natriuretic
peptide precursor A KCNA5 Potassium voltage-gated channel,
shaker-related subfamily, member 5 GJA5 Connexin 40 SCN1B Sodium
channel, voltage-gated, type I, beta subunit SCN2B Sodium channel,
voltage-gated, type II, beta subunit NUP155 Nucleoporin 155 kDa
GPD1L Glycerol-3-phosphate dehydrogenase 1-like CACNB2 Calcium
channel, voltage-dependent, beta 2 subunit KCNE3 Potassium
voltage-gated channel, Isk-related family, member 3 SCN3B Sodium
channel, voltage-gated, type III, beta subunit HCN4
Hyperpolarization activated cyclic nucleotide-gated potassium
channel 4
Congenital Myasthenic Syndromes
TABLE-US-00011 [0099] Gene protein CHRNA1 Cholinergic receptor,
nicotinic, alpha polypeptide 1 CHRNB1 Cholinergic receptor,
nicotinic, beta 1 muscle CHRND Cholinergic receptor, nicotinic,
delta CHRNE Cholinergic receptor, nicotinic, epsilon RAPSN Rapsyn
CHAT Choline acetyltransferase isoform COLQ Acetylcholinesterase
collagen-like tail subunit MUSK muscle, skeletal, receptor tyrosine
kinase DOK7 Docking protein 7 AGRN Agrin GFPT1
Glutamine-fructose-6-phosphate transaminase 1 DPAGT1
Dolichyl-phosphate (UDP-N-acetylglucosamine) N-
acetylglucosaminephosphotransferase 1 (GlcNAc-1-P transferase)
LAMB2 Laminin, beta 2 (laminin S) SCN4A Sodium channel,
voltage-gated, type IV, alpha CHRNG Cholinergic receptor,
nicotinic, gamma polypeptide PLEC plectin ALG2
Alpha-1,3/1,6-mannosyltransferase ALG14
UDP-N-acetylglucosaminyltransferase SYT2 Synaptotagmin II PREPL
Prolyl endopeptidase-like
Motor Neuron Diseases
TABLE-US-00012 [0100] Gene protein SMN1 Survival of motor neuron 1,
telomeric IGHMBP2 Immunoglobulin mu binding protein 2 PLEKHG5
Pleckstrin homology domain containing, family G (with RhoGef
domain) member 5 HSPB8 Heat shock 27 kDa protein 8 HSPB1 Heat shock
27 kDa protein 1 HSPB3 Heat shock 27 kDa protein 3 AARS Alanyl-tRNA
synthetase GARS Glycyl-tRNA synthetase BSCL2 Seipin REEP1 Receptor
accessory protein 1 SLC5A7 Solute carrier family 5 (sodium/choline
cotransporter), member 7 DCTN1 Dynactin 1 UBA1 Ubiquitin-activating
enzyme 1 ATP7A ATPase, Cu++ transporting, alpha polypeptide DNAJB2
DnaJ (Hsp40) homolog, subfamily B, member 2 TRPV4 Transient
receptor potential cation channel, subfamily V, member 4 DYNC1H1
Dynein, cytoplasmic 1, heavy chain 1 BICD2 Bicaudal D homolog 2
(Drosophila) FBXO38 F-box protein 38 ASAH1 N-acylsphingosine
amidohydrolase (acid ceramidase) 1 VAPB Vesicle-associated membrane
protein-associated protein B and C EXOSC8 Exosome component 8 SOD1
Superoxide dismutase 1, soluble ALS2 Alsin SETX Senataxin FUS
Fusion (involved in t(12; 16) in malignant liposarcoma) ANG
Angiogenin TARDBP TAR DNA binding protein FIG4 Sac
domain-containing inositol phosphatase 3 OPTN Optineurin ATXN2
Ataxin 2 VCP Valosin-containing protein UBQLN2 Ubiquilin 2 SIGMAR1
Sigma non-opioid intracellular receptor 1 CHMP2B Charged
multivesicular body protein 2B PFN1 Profilin 1 MATR3 Matrin 3 NEFH
Neurofilament, heavy polypeptide PRPH Peripherin C9orf72 Chromosome
9 open reading frame 72 CHCHD10 Coiled-coil-helix-coiled-coil-helix
domain containing 10 SQSTM1 Sequestosome 1 AR Androgen receptor
GLE1 GLE1 RNA export mediator homolog (yeast) ERBB3 V-erb-b2
erythroblastic leukemia viral oncogene homolog 3 (avian) PIP5K1C
Phosphatidylinositol-4-phosphate 5-kinase, type I, gamma EXOSC3
Exosome component 3 VRK1 Vaccinia related kinase 1 SLC52A3 Solute
carrier family 52, riboflavin transporter, member 3 SLC52A2 Solute
carrier family 52, riboflavin transporter, member 2 HEXB
Hexosaminidase B
Hereditary Motor and Sensory Neuropathies
TABLE-US-00013 [0101] Gene Protein PMP22 Peripheral myelin protein
22 MPZ Myelin protein zero LITAF Lipopolysaccharide-induced TNF
factor EGR2 Early growth response 2 protein NEFL Neurofilament,
light polypeptide 68 kDa HOXD10 Homeobox D10 ARHGEF10 Rho guanine
nucleotide exchange factor 10 FBLN5 Fibulin 5 (extra-cellular
matrix) DNM2 Dynamin 2 YARS Tyrosyl-tRNA synthetase INF2 Inverted
formin 2 GNB4 Guanine nucleotidebinding protein, beta-4 GDAP1
Ganglioside-induced differentiation-associated protein 1 MTMR2
Myotubularin-related protein 2 SBF2 SET binding factor 2 SBF1 SET
binding factor 1 SH3TC2 KIAA1985 protein NDRG1 N-myc downstream
regulated gene 1 PRX Periaxin HK1 Hexokinase 1 FGD4 Actin-filament
binding protein Frabin FIG4 Sac domain-containing inositol
phosphatase 3 SURF1 surfeit 1 GJB1 Gap junction protein, beta 1, 32
kDa (connexin 32) AIFM1 Apoptosis-inducing factor,
mitochondrionassociated 1 PRPS1 Phosphoribosyl pyrophosphate
synthetase 1 PDK3 Pyruvate dehydrogenase kinase, isoenzyme 3 KIF1B
Kinesin family member 1B MFN2 Mitofusin 2 RAB7A RAB7, member RAS
oncogene family TRPV4 Transient receptor potential cation channel,
subfamily V, member 4 GARS Glycyl-tRNA synthetase HSPB1 Heat shock
27 kDa protein 1 HSPB8 Heat shock 27 kDa protein 8 AARS Alanyl-tRNA
synthetase DYNC1H1 Dynein, cytoplasmic 1, heavy chain 1 LRSAM1
leucine rich repeat and sterile alpha motif containing 1 DHTKD1
dehydrogenase E1 and transketolase domain containing 1 TRIM2
Tripartite motif containing 2 TFG TRK-fused gene MARS
methionyl-tRNA synthetase KIF5A Kinesin family member 5A LMNA Lamin
A/C MED25 Mediator complex subunit 25 DNAJB2 DnaJ (Hsp40) homolog,
subfamily B, member 2 HINT1 Histidine triad nucleotide binding
protein 1 KARS Lysyl-tRNA synthetase PLEKHG5 Pleckstrin homology
domain containing, family G (with RhoGef domain) member 5 COX6A1
Cytochrome c oxidase subunit VIa polypeptide 1 IGHMBP2
Immunoglobulin mu binding protein 2 SPTLC1 Serine
palmitoyltransferase subunit 1 SPTLC2 Serine palmitoyltransferase
long chain base subunit 2 ATL1 Atlastin GTPase 1 KIF1A Kinesin
family member 1A WNK1 WNK lysine deficient protein kinase 1 IKBKAP
Inhibitor of kappa light polypeptide gene enhancer in B-cells,
kinase complex-associated protein NGF Nerve growth factor (beta
polypeptide) DNMT1 DNA (cytosine-5)-methyltransferase 1 SLC12A6
Potassium chloride cotransporter KCC3 GJB3 Gap junction protein,
beta 3, 31 kDa (=connexin 31) sept-09 Septin 9 GAN Gigaxonin CTDP1
CTD phosphatase subunit 1 VRK1 Vaccinia related kinase 1
Hereditary Paraplegia
TABLE-US-00014 [0102] Gene symbol protein ATL1 Atlastin SPAST
Spastin NIPA1 Non-imprinted in Prader-Willi/Angelman syndrome 1
KIAA0196 Strumpellin KIF5A Kinesin family member 5A RTN2 Reticulon
2 HSPD1 Heat shock 60 kDa protein 1 (chaperonin) BSCL2 Seipin REEP1
Receptor accessory protein 1 ZFYVE27 Protrudin SLC33A1 Solute
carrier family 33 (acetyl- CoA transporter) CYP7B1 Cytochrome P450,
family 7, subfamily B, polypeptide 1 SPG7 Paraplegin SPG11
Spatacsin ZFYVE26 Spastizin ERLIN2 ER lipid raft associated 2 SPG20
Spartin SPG21 Maspardin B4GALNT1 beta-1,4-N-acetyl-galactosaminyl
transferase 1 DDHD1 DDHD domain containing 1 KIF1A Kinesin family
member 1A FA2H Fatty acid 2-hydroxylase PNPLA6 Patatin-like
phospholipase domain containing 6 C19orf12 chromosome 19 open
reading frame 12 GJC2 gap junction protein, gamma 2, 47 kDa NT5C2
5'-nucleotidase, cytosolic II GBA2 glucosidase, beta (bile acid) 2
AP4B1 adaptor-related protein complex 4, beta 1 subunit AP5Z1
Hypothetical protein LOC9907 TECPR2 tectonin beta-propeller repeat
containing 2 AP4M1 Adaptor-related protein complex 4, mu 1 subunit
AP4E1 Adaptor-related protein complex 5, zeta 1 subunit AP4S1
adaptor-related protein complex 4, sigma 1 subunit DDHD2 DDHD
domain containing 2 C12orf65 adaptor-related protein complex 4,
sigma 1 subunit CYP2U1 cytochrome P450, family 2, subfamily U,
polypeptide 1 ARL6IP1 ADP-ribosylation factor-like 6 interacting
protein 1 AMPD2 adenosine monophosphate deaminase 2 ENTPD1
ectonucleoside triphosphate diphosphohydrolase 1 ALDH3A2 Aldehyde
dehydrogenase 3A2 ALS2 Alsin L1CAM L1 cell adhesion molecule PLP1
Proteolipid protein 1 MTPAP mitochondrial poly(A) polymerase AFG3L2
AFG3 ATPase family gene 3-like 2 (S. cerevisiae) 1 SACS Sacsin
Other Neuromuscular Disorders
TABLE-US-00015 [0103] Gene protein TOR1A Torsin A SGCE Sarcoglycan,
epsilon IKBKAP Inhibitor of kappa light polypeptide gene enhancer
in B- cells, kinase complex-associated protein TTR Transthyretin
(prealbumin, amyloidosis type I) KIF21A Kinesin family member 21A
PHOX2A Paired-like aristaless homeobox protein 2A TUBB3 Tubulin,
beta 3 TPM2 Tropomyosin 2 (beta) MYH3 Myosine, heavy chain 3,
skeletal muscle, embryonic TNNI2 Troponin I, type 2 TNNT3 Troponin
T3, skeletal SYNE1 Spectrin repeat containing, nuclear envelope 1
(nesprin 1) MYH8 Myosin heavy chain, 8, skeletal muscle, perinatal
POLG Polymerase (DNA directed), gamma SLC25A4 Mitochondrial
carrier; adenine nucleotide translocator C10orf2 chromosome 10 open
reading frame 2 POLG2 Mitochondrial DNA polymerase, accessory
subunit RRM2B Ribonucleotide reductase M2 B (TP53 inducible) TK2
Thymidine kinase 2, mitochondrial SUCLA2 Succinate-CoA ligase,
ADP-forming, beta subunit OPA1 optic atrophy 1 STIM1 Stromal
interaction molecule 1 ORAI1 ORAI calcium release-activated calcium
modulator 1 PUS1 Pseudouridylate synthase 1 CHCHD10
Coiled-coil-helix-coiled-coil-helix domain containing 10 CASQ1
Calsequestrin 1 (fast-twitch, skeletal muscle) YARS2 tyrosyl-tRNA
synthetase 2, mitochondrial
[0104] Any one of the above listed genes may be targeted in
replacement gene therapy, wherein the gene of interest is a
functional version of the deficient or mutated gene.
[0105] Alternatively, the above listed genes may be used as target
for gene editing. Gene editing is used to correct the sequence of a
mutated gene or modify the expression or regulation of a
deficient/abnormal gene so that a functional gene is expressed in
muscle cells. In such cases, the gene of interest is chosen from
those encoding therapeutic RNAs such as interfering RNAs, guide
RNAs for genome editing and antisense RNAs capable of exon
skipping, wherein the therapeutic RNAs target the preceding list of
genes. Tools such as CRISPR/Cas9 may be used for that purpose.
[0106] Thus, by gene editing or gene replacement a correct version
of this gene is provided in muscle cells of affected patients, this
may contribute to effective therapies against this disease.
[0107] In some embodiments, the target gene for gene therapy
(additive gene therapy or gene editing) is a gene responsible for
one of the neuromuscular diseases listed above, preferably selected
from the group comprising Duchenne muscular dystrophy (DMD gene),
Limb-girdle muscular dystrophies (LGMDs) (CAPN3, DYSF, FKRP, ANO5
genes and others), Spinal muscular atrophy (SMNI gene), myotubular
myopathy (MTMI gene), Pompe disease (GAA gene) and Glycogen storage
disease III (GSD3) (AGL gene).
[0108] Dystrophinopathies are a spectrum of X-linked muscle
diseases caused by pathogenic variants in DMD gene, which encodes
the protein dystrophin. Dystrophinopathies comprises Duchenne
muscular dystrophy (DMD), Becker muscular dystrophy (BMD) and
DMD-associated dilated cardiomyopathy.
[0109] The Limb-girdle muscular dystrophies (LGMDs) are a group of
disorders that are clinically similar to DMD but occur in both
sexes as a result of autosomal recessive and autosomal dominant
inheritance. Limb-girdle dystrophies are caused by mutation of
genes that encode sarcoglycans and other proteins associated with
the muscle cell membrane, which interact with dystrophin. The term
LGMD1 refers to genetic types showing dominant inheritance
(autosomal dominant), whereas LGMD2 refers to types with autosomal
recessive inheritance. Pathogenic variants at more than 50 loci
have been reported (LGMD1A to LGMD1G; LGMD2A to
LGMD2W).Calpainopathy (LGMD2A) is caused by mutation of the gene
CAPN3 with more than 450 pathogenic variants described.
Contributing genes to LGMD phenotype include: anoctamin 5 (ANO5),
blood vessel epicardial substance (BVES), calpain 3 (CAPN3),
caveolin 3 (CAV3), CDP-L-ribitol pyrophosphorylase A (CRPPA),
dystroglycan 1 (DAG1), desmin (DES), DnaJ heat shock protein family
(Hsp40) homolog, subfamily B, member 6 (DNAJB6), dysferlin (DYSF),
fukutin related protein (FKRP), fukutin (FKT), GDP-mannose
pyrophosphorylase B (GMPPB), heterogeneous nuclear
ribonucleoprotein D like (HNRNPDL), LIM zinc finger domain
containing 2 (LIMS2), lain A:C (LMNA), myotilin (MYOT), plectin
(PLEC), protein O-glucosyltransferase 1 (PLOGLUT1), protein
O-linked mannose N-acetylglucosaminyltransferase 1 (beta 1,2-)
[0110] (POMGNT1), protein O-mannose kinase (POMK), protein
O-mannosyltransferase 1 (POMT1), protein O-mannosyltransferase 2
(POMT2), sarcoglycan alpha (SGCA), sarcoglycan beta (SGCB),
sarcoglycan delta (SGCD), sarcoglycan gamma (SGCG), titin-cap
(TCAP), transportin 3 (TNPO3), torsin 1A interacting protein
(TOR1AIP1), trafficking protein particle complex 11 (TRAPPC11),
tripartite motif containing 32 (TRIM 32) and titin (TTN). Major
contributing genes to LGMD phenotype include CAPN3, DYSF, FKRP and
ANO5 (Babi Ramesh Reddy Nallamilli et al., Annals of Clinical and
Translational Neurology, 2018, 5, 1574-1587.
[0111] Spinal muscular atrophy is a genetic disorder caused by
mutations in the Survival Motor Neuron 1 (SMN1) gene which is
characterized by weakness and wasting (atrophy) in muscles used for
movement.
[0112] X-linked myotubular myopathy is a genetic disorder caused by
mutations in the myotubularin (MTM1) gene which affects muscles
used for movement (skeletal muscles) and occurs almost exclusively
in males. This condition is characterized by muscle weakness
(myopathy) and decreased muscle tone (hypotonia).
[0113] Pompe disease is a genetic disorder caused by mutations in
the acid alpha-glucosidase (GAA) gene. Mutations in the GAA gene
prevent acid alpha-glucosidase from breaking down glycogen
effectively, which allows this sugar to build up to toxic levels in
lysosomes. This buildup damages organs and tissues throughout the
body, particularly the muscles, leading to the progressive signs
and symptoms of Pompe disease.
[0114] Glycogen storage disease III (GSD3) is an autosomal
recessive metabolic disorder caused by homozygous or compound
heterozygous mutation in the Amylo-Alpha-1, 6-Glucosidase,
4-Alpha-Glucanotransferase (AGL) gene which encodes the glycogen
debrancher enzyme and associated with an accumulation of abnormal
glycogen with short outer chains. Clinically, patients with GSD III
present in infancy or early childhood with hepatomegaly,
hypoglycemia, and growth retardation. Muscle weakness in those with
Ma is minimal in childhood but can become more severe in adults;
some patients develop cardiomyopathy.
[0115] In some embodiments, the pharmaceutical composition of the
invention is for use for treating muscular diseases (i.e.,
myopathies) or muscular injuries, in particular neuromuscular
genetic disorders, with no liver damage, such as for example:
Muscular dystrophies, Congenital muscular dystrophies, Congenital
myopathies, Distal myopathies, Other myopathies, Myotonic
syndromes, Ion Channel muscle diseases, Malignant hyperthermia,
Metabolic myopathies, Hereditary Cardiomyopathies, Congenital
myasthenic syndromes, Motor Neuron diseases, Hereditary paraplegia,
Hereditary motor and sensory neuropathies and other neuromuscular
disorders. In some preferred embodiments, the pharmaceutical
composition of the invention is for use for treating muscular
diseases (i.e., myopathies) or muscular injuries, in particular
muscular genetic disorders, with no liver damage, such as for
example: Muscular dystrophies, Congenital muscular dystrophies,
Congenital myopathies, Distal myopathies, Other myopathies,
Myotonic syndromes, Ion Channel muscle diseases, Malignant
hyperthermia, Metabolic myopathies, Hereditary Cardiomyopathies and
Congenital myasthenic syndromes as defined above; more particularly
Muscular dystrophies, Congenital muscular dystrophies, Congenital
myopathies, Distal myopathies, Ion Channel muscle diseases,
Malignant hyperthermia, Metabolic myopathies and Hereditary
Cardiomyopathies as defined above.
[0116] Replacement or additive gene therapy may be used to treat
cancer, in particular rhabdomyosarcomas. Genes of interest in
cancer could regulate the cell cycle or the metabolism and
migration of the tumor cells, or induce tumor cell death. For
instance, inducible caspase-9 could be expressed in muscle cells to
trigger cell death, preferably in combination therapy to elicit
durable anti-tumor immune responses.
[0117] Gene editing may be used to modify gene expression in muscle
cells, in the case of auto-immunity or cancer, or to perturb the
cycle of viruses in such cells. In such cases, preferably, the gene
of interest is chosen from those encoding guide RNA (gRNA),
site-specific endonucleases (TALEN, meganucleases, zinc finger
nucleases, Cas nuclease), DNA templates and RNAi components, such
as shRNA and microRNA. Tools such as CRISPR/Cas9 may be used for
this purpose.
[0118] In some embodiments, gene therapy is used for treating
diseases affecting other tissues, by expression of a therapeutic
gene in muscle tissue. This is useful to avoid expression of the
therapeutic gene in the liver, in particular in patients having a
concurrent hepatic disorder such as hepatitis. The therapeutic gene
encodes preferably a therapeutic protein, peptide or antibody which
is secreted from the muscle cells into the blood stream where it
can be delivered to other target tissues such as for example the
liver. Examples of therapeutic genes include with no limitation:
Factor VIII, Factor IX and GAA genes.
[0119] The pharmaceutical composition of the invention which
comprises AAV vector particles with reduced liver tropism may be
administered to patients having concurrent liver degeneration such
as fibrosis, non-alcoholic fatty liver disease, non-alcoholic
steatohepatitis, viral or toxic hepatitis or underlying genetic
disorders inducing liver degeneration.
[0120] In the context of the invention, a therapeutically effective
amount refers to a dose sufficient for reversing, alleviating or
inhibiting the progress of the disorder or condition to which such
term applies, or reversing, alleviating or inhibiting the progress
of one or more symptoms of the disorder or condition to which such
term applies.
[0121] The effective dose is determined and adjusted depending on
factors such as the composition used, the route of administration,
the physical characteristics of the individual under consideration
such as sex, age and weight, concurrent medication, and other
factors, that those skilled in the medical arts will recognize.
[0122] In the various embodiments of the present invention, the
pharmaceutical composition comprises a pharmaceutically acceptable
carrier and/or vehicle.
[0123] A "pharmaceutically acceptable carrier" refers to a vehicle
that does not produce an adverse, allergic or other untoward
reaction when administered to a mammal, especially a human, as
appropriate. A pharmaceutically acceptable carrier or excipient
refers to a non-toxic solid, semi-solid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type.
[0124] Preferably, the pharmaceutical composition contains
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.
[0125] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or suspensions. The solution or
suspension may comprise additives which are compatible with viral
vectors and do not prevent viral vector particle entry into target
cells. In all cases, the form must be sterile and must be fluid to
the extent that easy syringe ability exists. 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. An example of an appropriate solution is a
buffer, such as phosphate buffered saline (PBS) or Ringer
lactate.
[0126] The invention provides also a method for treating a disease
affecting muscle tissue in particular skeletal muscle tissue and/or
cardiac tissue, comprising: administering to a patient a
therapeutically effective amount of the pharmaceutical composition
as described above.
[0127] The invention provides also a method for treating a disease
by expression of a therapeutic gene in muscle tissue, comprising:
administering to a patient a therapeutically effective amount of
the pharmaceutical composition as described above.
[0128] As used herein, the term "patient" or "individual" denotes a
mammal. Preferably, a patient or individual according to the
invention is a human.
[0129] In the context of the invention, the term "treating" or
"treatment", as used herein, means reversing, alleviating or
inhibiting the progress of the disorder or condition to which such
term applies, or reversing, alleviating or inhibiting the progress
of one or more symptoms of the disorder or condition to which such
term applies.
[0130] The pharmaceutical composition of the present invention, is
generally administered according to known procedures, at dosages
and for periods of time effective to induce a therapeutic effect in
the patient.
[0131] The administration may be parenteral, oral, local, or
loco-regional. The parenteral administration is advantageously by
injection or perfusion, such as e subcutaneous (SC), intramuscular
(IM), intravascular such as intravenous (IV), intraperitoneal (IP),
intradermal (ID) or else. Preferably, the administration produces a
systemic effect in the whole body, i.e., all the muscles of the
patient, including the diaphragm and the heart. Preferably, the
administration is systemic, more preferably parenteral.
[0132] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques, which are within the
skill of the art. Such techniques are explained fully in the
literature.
[0133] The invention will now be exemplified with the following
examples, which are not limitative, with reference to the attached
drawings in which:
FIGURE LEGENDS
[0134] FIG. 1: Design of peptide insertion in the hybrid AAV
serotype between AAV9 and AAVrh74.
[0135] A. Schematic representation of the Cap genes (VP1) of AAV9,
AAVrh74 and the AAV9-rh74 hybrid capsid highlighting the sequences
of the variable region. B. The VP1 protein of the AAV-MT capsid
derives from the insertion of 15 amino acid (15-mer) between amino
acid 586 and 593 of the VP1 of the AAV9-rh74 capsid.
[0136] FIG. 2: Comparative biodistribution of AAV9-RH74 and AAV-MT
in GSDIII mice. GSDIII mice were tail vein injected with
2.times.10.sup.12 vector genome/mouse of the indicated AAV vectors.
Three months after injection, tissues were collected. PBS-injected
mice were used as controls. (A-F) Vector genome copy number per
cell (VGCN/cell) measured in liver (A); heart (B); diaphragm (C);
quadriceps (D); extensor digitorum longus (EDL) (E) and
gastrocnemius (F). Data were presented as mean .+-.standard
deviation. Statistical analyses were performed by ANOVA
(*=p<0.05 vs. PBS; .sctn.=p<0.05 vs. ALL; n=4-5 mice per
group).
EXAMPLE
Peptide-Modified Hybrid AAV9-rh74 Serotype Vector
1. Material and Methods
Plasmid Construction for New Serotypes
[0137] To construct the plasmid containing AAV2 Rep sequence and
Hybrid Cap 9-rh74, a fragment of 1029 nt, containing the highly
variable part of AAV-rh74 Cap flanked with AAV9 Cap sequence
fragments and restriction sites BsiWI in 5' and Eco47III in 3', was
synthesized (GENEWIZ). This fragment was then inserted using the
mentioned restriction sites in the plasmid pAAV2-9, which contains
AAV2 Rep and AAV9 Cap, to replace the AAV9 Cap corresponding
sequence. Peptide engraftment was performed by replacing the QQNAAP
hexapeptide (SEQ ID NO: 11) in the AAV9-rh74 capsid with the
GQSGRGDLGLSAQAA (SEQ ID NO: 13) amino acid sequence.
AAV Production
[0138] HEK293T cells were grown in suspension in 250 mL of
serum-free medium. The cells were transfected with 3 plasmids: i) a
transgene plasmid, containing AAV2 ITRs flanking an expression
cassette ii) the helper plasmid pXX6, containing adenoviral
sequences necessary for AAV production, and iii) a plasmid
containing AAV Rep and Cap genes, defining the serotype of AAV. Two
days after transfection, the cells were lysed to release the AAV
particles.
[0139] The viral lysate was purified by affinity chromatography.
Viral genomes were quantified by a TaqMan real-time PCR assay using
primers and probes corresponding to the ITRs of the AAV vector
genome (Rohr et al., J. Virol. Methods, 2002, 106, 81-88).
In Vivo Studies
[0140] All mouse studies were performed according to the French and
European legislation on animal care and experimentation
(2010/63/EU) and approved by the local institutional ethical
committee (protocol no. 2016-002C). AAV vectors were administered
intravenously via the tail vein to three month-old male GDE
knockout mice. PBS-injected littermates were used as controls.
Three months after vector injections, tissues were harvested and
homogenized in DNAse/RNAse free water using Fastprep tubes (6.5
m/s; 60 seconds).
Vector Genome Copy Number (VGCN) Quantification
[0141] For vector genome copy number (VGCN) quantification in
samples, DNA was extracted from samples using MagNA Pure 96
Instrument (Roche). Real-time PCR was performed on 1.mu.L of DNA,
using the protocol for AAV vectors titration described above. Exon
Mex5 of titin gene was used as genomic DNA loading control.
2. Results
[0142] The AAV9-rh74 hybrid capsid was engineered by inserting a
peptide in the common region between VP1, VP2 and VP3 between Q at
positions 586 and I at position 593 of SEQ ID NO: 3 (FIG. 1). AAV
capsid modification was performed according to Kienle EC
(Dissertation for the degree of Doctor of natural Sciences,
Combined Faculties for the Natural Sciences and for Mathematics of
the Ruperto-Carola University of Heidelberg, Germany, 2014) using a
peptide as disclosed in Michelfelder et al. (PLoS ONE, 2009, 4,
e5122). Briefly, the hexapeptide QQNAAP (SEQ ID NO: 11) present in
the common region between VP1, VP2 and VP3 of the hybrid Cap9-rh74
(positions 587 to 592 of SEQ ID NO: 3) is mutated to the
octapeptide GQSGAQAA (SEQ ID NO: 12) and peptide P1 (RGDLGLS; SEQ
ID NO: 8) is inserted between glycine at position 4 and alanine at
position 5. The peptide-modified hybrid Cap9-rh74 having the
insertion of the peptide P1 has the amino acid sequence SEQ ID NO:
5 and the corresponding coding sequence is SEQ ID NO: 6. Vectors
are produced by triple transfection in HEK293 cells grown in
suspension and purified by affinity chromatography as described in
example 1. The resulting AAV Vector called AAV-Muscle Transducer
(AAV-MT) was injected in GDE knockout mice, an animal model of
GSDIII, in parallel with AAV9-rh74 at the dose of 2.times.10.sup.12
vg/mouse. Tissues were obtained three months post-injection and
vector genome copy number (VGCN) was measured by quantitative PCR
to assess tissue targeting (FIG. 2). In the liver, injection of
AAV9-rh74 resulted in significantly increased vector genome copies
compared to PBS injected mice (p<0.05; FIG. 2A) while liver
transduced with AAV-MT did not showed a significant increase in
VGCN compared to the PBS group. In general, AAV-MT showed better
transduction efficacy in muscles when compared to the parental
capsid AAV9-rh74 (FIG. 2B-F) Importantly, in diaphragm, quadriceps
and gastrocnemius, the AAV-MT outperformed the parental capsid
(p<0.05 vs. AAV9-rh74 group). Finally, in heart and extensor
digitorum longus (EDL) a significantly higher VGCN were measured
after injection with the AAV-MT capsid but not with the AAV9-rh74
when compared with PBS injected mice. These data indicate that the
AAV-MT capsid outperforms the AAV9-rh74 in muscle transduction and
maintains or even ameliorates the pronounced liver detargeting
observed for the parental capsid.
Sequence CWU 1
1
181736PRTadeno-associated virus 9 1Met Ala Ala Asp Gly Tyr Leu Pro
Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp Trp
Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30Lys Ala Asn Gln Gln His
Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu
Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala Ala
Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln Gln
Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95Asp
Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105
110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro
115 120 125Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys
Lys Arg 130 135 140Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser
Ala Gly Ile Gly145 150 155 160Lys Ser Gly Ala Gln Pro Ala Lys Lys
Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly Asp Thr Glu Ser Val Pro
Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190Ala Ala Pro Ser Gly
Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205Ala Pro Val
Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220Ser
Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile225 230
235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
Leu 245 250 255Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser
Asn Asp Asn 260 265 270Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr
Phe Asp Phe Asn Arg 275 280 285Phe His Cys His Phe Ser Pro Arg Asp
Trp Gln Arg Leu Ile Asn Asn 290 295 300Asn Trp Gly Phe Arg Pro Lys
Arg Leu Asn Phe Lys Leu Phe Asn Ile305 310 315 320Gln Val Lys Glu
Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn 325 330 335Asn Leu
Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu 340 345
350Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro
355 360 365Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu
Asn Asp 370 375 380Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys
Leu Glu Tyr Phe385 390 395 400Pro Ser Gln Met Leu Arg Thr Gly Asn
Asn Phe Gln Phe Ser Tyr Glu 405 410 415Phe Glu Asn Val Pro Phe His
Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430Asp Arg Leu Met Asn
Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445Lys Thr Ile
Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser 450 455 460Val
Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro465 470
475 480Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln
Asn 485 490 495Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp
Ala Leu Asn 500 505 510Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala
Met Ala Ser His Lys 515 520 525Glu Gly Glu Asp Arg Phe Phe Pro Leu
Ser Gly Ser Leu Ile Phe Gly 530 535 540Lys Gln Gly Thr Gly Arg Asp
Asn Val Asp Ala Asp Lys Val Met Ile545 550 555 560Thr Asn Glu Glu
Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser 565 570 575Tyr Gly
Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln 580 585
590Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln
595 600 605Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile
Pro His 610 615 620Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly
Gly Phe Gly Met625 630 635 640Lys His Pro Pro Pro Gln Ile Leu Ile
Lys Asn Thr Pro Val Pro Ala 645 650 655Asp Pro Pro Thr Ala Phe Asn
Lys Asp Lys Leu Asn Ser Phe Ile Thr 660 665 670Gln Tyr Ser Thr Gly
Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685Lys Glu Asn
Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700Tyr
Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val705 710
715 720Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn
Leu 725 730 7352738PRTadeno-associated virus rh74 2Met Ala Ala Asp
Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile
Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala
Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro 35 40 45Gly
Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55
60Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65
70 75 80Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His
Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe
Gly Gly 100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg
Val Leu Glu Pro 115 120 125Leu Gly Leu Val Glu Ser Pro Val Lys Thr
Ala Pro Gly Lys Lys Arg 130 135 140Pro Val Glu Pro Ser Pro Gln Arg
Ser Pro Asp Ser Ser Thr Gly Ile145 150 155 160Gly Lys Lys Gly Gln
Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln 165 170 175Thr Gly Asp
Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro 180 185 190Pro
Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly 195 200
205Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser
210 215 220Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp
Arg Val225 230 235 240Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro
Thr Tyr Asn Asn His 245 250 255Leu Tyr Lys Gln Ile Ser Asn Gly Thr
Ser Gly Gly Ser Thr Asn Asp 260 265 270Asn Thr Tyr Phe Gly Tyr Ser
Thr Pro Trp Gly Tyr Phe Asp Phe Asn 275 280 285Arg Phe His Cys His
Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn 290 295 300Asn Asn Trp
Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn305 310 315
320Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala
325 330 335Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu
Tyr Gln 340 345 350Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys
Leu Pro Pro Phe 355 360 365Pro Ala Asp Val Phe Met Ile Pro Gln Tyr
Gly Tyr Leu Thr Leu Asn 370 375 380Asn Gly Ser Gln Ala Val Gly Arg
Ser Ser Phe Tyr Cys Leu Glu Tyr385 390 395 400Phe Pro Ser Gln Met
Leu Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr 405 410 415Asn Phe Glu
Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser 420 425 430Leu
Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu 435 440
445Ser Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Gln Gln Leu Leu
450 455 460Phe Ser Gln Ala Gly Pro Asn Asn Met Ser Ala Gln Ala Lys
Asn Trp465 470 475 480Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val
Ser Thr Thr Leu Ser 485 490 495Gln Asn Asn Asn Ser Asn Phe Ala Trp
Thr Gly Ala Thr Lys Tyr His 500 505 510Leu Asn Gly Arg Asp Ser Leu
Val Asn Pro Gly Val Ala Met Ala Thr 515 520 525His Lys Asp Asp Glu
Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met 530 535 540Phe Gly Lys
Gln Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Ser Val545 550 555
560Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr
565 570 575Glu Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gln Gln Asn
Ala Ala 580 585 590Pro Ile Val Gly Ala Val Asn Ser Gln Gly Ala Leu
Pro Gly Met Val 595 600 605Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly
Pro Ile Trp Ala Lys Ile 610 615 620Pro His Thr Asp Gly Asn Phe His
Pro Ser Pro Leu Met Gly Gly Phe625 630 635 640Gly Leu Lys His Pro
Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val 645 650 655Pro Ala Asp
Pro Pro Thr Thr Phe Asn Gln Ala Lys Leu Ala Ser Phe 660 665 670Ile
Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu 675 680
685Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr
690 695 700Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn
Thr Glu705 710 715 720Gly Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr
Arg Tyr Leu Thr Arg 725 730 735Asn Leu3737PRTartificial
sequencesynthetic polypeptide AAV9-rh74 hybrid capsid 3Met Ala Ala
Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly
Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30Lys
Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40
45Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr
Asp65 70 75 80Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr
Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr
Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys
Lys Arg Leu Leu Glu Pro 115 120 125Leu Gly Leu Val Glu Glu Ala Ala
Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140Pro Val Glu Gln Ser Pro
Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly145 150 155 160Lys Ser Gly
Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly
Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185
190Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly
195 200 205Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly
Ser Ser 210 215 220Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly
Asp Arg Val Ile225 230 235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu
Pro Thr Tyr Asn Asn His Leu 245 250 255Tyr Lys Gln Ile Ser Asn Ser
Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270Ala Tyr Phe Gly Tyr
Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285Phe His Cys
His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300Asn
Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile305 310
315 320Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala
Asn 325 330 335Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp
Tyr Gln Leu 340 345 350Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys
Leu Pro Pro Phe Pro 355 360 365Ala Asp Val Phe Met Ile Pro Gln Tyr
Gly Tyr Leu Thr Leu Asn Asp 370 375 380Gly Ser Gln Ala Val Gly Arg
Ser Ser Phe Tyr Cys Leu Glu Tyr Phe385 390 395 400Pro Ser Gln Met
Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu 405 410 415Phe Glu
Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425
430Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser
435 440 445Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Gln Gln Leu
Leu Phe 450 455 460Ser Gln Ala Gly Pro Asn Asn Met Ser Ala Gln Ala
Lys Asn Trp Leu465 470 475 480Pro Gly Pro Cys Tyr Arg Gln Gln Arg
Val Ser Thr Thr Leu Ser Gln 485 490 495Asn Asn Asn Ser Asn Phe Ala
Trp Thr Gly Ala Thr Lys Tyr His Leu 500 505 510Asn Gly Arg Asp Ser
Leu Val Asn Pro Gly Val Ala Met Ala Thr His 515 520 525Lys Asp Asp
Glu Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met Phe 530 535 540Gly
Lys Gln Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Ser Val Met545 550
555 560Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr
Glu 565 570 575Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gln Gln Asn
Ala Ala Pro 580 585 590Ile Val Gly Ala Val Asn Ser Gln Gly Ala Leu
Pro Gly Met Val Trp 595 600 605Gln Asn Arg Asp Val Tyr Leu Gln Gly
Pro Ile Trp Ala Lys Ile Pro 610 615 620His Thr Asp Gly Asn Phe His
Pro Ser Pro Leu Met Gly Gly Phe Gly625 630 635 640Met Lys His Pro
Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro 645 650 655Ala Asp
Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile 660 665
670Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu
675 680 685Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr
Thr Ser 690 695 700Asn Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val
Asn Thr Glu Gly705 710 715 720Val Tyr Ser Glu Pro Arg Pro Ile Gly
Thr Arg Tyr Leu Thr Arg Asn 725 730 735Leu4737PRTartificial
sequencesynthetic polypeptide AAVrh74-9 hybrid capsid 4Met Ala Ala
Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly
Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys
Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro 35 40
45Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr
Asp65 70 75 80Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr
Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr
Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys
Lys Arg Val Leu Glu Pro 115 120 125Leu Gly Leu Val Glu Ser Pro Val
Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140Pro Val Glu Pro Ser Pro
Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile145 150 155 160Gly Lys Lys
Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln 165 170 175Thr
Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro 180 185
190Pro Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly
195 200 205Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val
Gly Ser 210 215 220Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu
Gly Asp Arg Val225 230
235 240Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn
His 245 250 255Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ser
Thr Asn Asp 260 265 270Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly
Tyr Phe Asp Phe Asn 275 280 285Arg Phe His Cys His Phe Ser Pro Arg
Asp Trp Gln Arg Leu Ile Asn 290 295 300Asn Asn Trp Gly Phe Arg Pro
Lys Arg Leu Asn Phe Lys Leu Phe Asn305 310 315 320Ile Gln Val Lys
Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala 325 330 335Asn Asn
Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln 340 345
350Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe
355 360 365Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr
Leu Asn 370 375 380Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr
Cys Leu Glu Tyr385 390 395 400Phe Pro Ser Gln Met Leu Arg Thr Gly
Asn Asn Phe Glu Phe Ser Tyr 405 410 415Asn Phe Glu Asp Val Pro Phe
His Ser Ser Tyr Ala His Ser Gln Ser 420 425 430Leu Asp Arg Leu Met
Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu 435 440 445Ser Lys Thr
Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe 450 455 460Ser
Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile465 470
475 480Pro Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr
Gln 485 490 495Asn Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser
Trp Ala Leu 500 505 510Asn Gly Arg Asn Ser Leu Met Asn Pro Gly Pro
Ala Met Ala Ser His 515 520 525Lys Glu Gly Glu Asp Arg Phe Phe Pro
Leu Ser Gly Ser Leu Ile Phe 530 535 540Gly Lys Gln Gly Thr Gly Arg
Asp Asn Val Asp Ala Asp Lys Val Met545 550 555 560Ile Thr Asn Glu
Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu 565 570 575Ser Tyr
Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala 580 585
590Gln Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp
595 600 605Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys
Ile Pro 610 615 620His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met
Gly Gly Phe Gly625 630 635 640Leu Lys His Pro Pro Pro Gln Ile Leu
Ile Lys Asn Thr Pro Val Pro 645 650 655Ala Asp Pro Pro Thr Thr Phe
Asn Gln Ala Lys Leu Ala Ser Phe Ile 660 665 670Thr Gln Tyr Ser Thr
Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu 675 680 685Gln Lys Glu
Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser 690 695 700Asn
Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Glu Gly705 710
715 720Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg
Asn 725 730 735Leu5746PRTartificial sequencepeptide-modified
AAV9-rh74 hybrid capsid 5Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp
Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp Trp Ala Leu
Lys Pro Gly Ala Pro Gln Pro 20 25 30Lys Ala Asn Gln Gln His Gln Asp
Asn Ala Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly Pro
Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala Ala Asp Ala
Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln Gln Leu Lys
Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95Asp Ala Glu
Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110Asn
Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120
125Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly
Ile Gly145 150 155 160Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu
Asn Phe Gly Gln Thr 165 170 175Gly Asp Thr Glu Ser Val Pro Asp Pro
Gln Pro Ile Gly Glu Pro Pro 180 185 190Ala Ala Pro Ser Gly Val Gly
Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205Ala Pro Val Ala Asp
Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220Ser Gly Asn
Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile225 230 235
240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu
245 250 255Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn
Asp Asn 260 265 270Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe
Asp Phe Asn Arg 275 280 285Phe His Cys His Phe Ser Pro Arg Asp Trp
Gln Arg Leu Ile Asn Asn 290 295 300Asn Trp Gly Phe Arg Pro Lys Arg
Leu Asn Phe Lys Leu Phe Asn Ile305 310 315 320Gln Val Lys Glu Val
Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn 325 330 335Asn Leu Thr
Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu 340 345 350Pro
Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro 355 360
365Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp
370 375 380Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu
Tyr Phe385 390 395 400Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe
Gln Phe Ser Tyr Glu 405 410 415Phe Glu Asn Val Pro Phe His Ser Ser
Tyr Ala His Ser Gln Ser Leu 420 425 430Asp Arg Leu Met Asn Pro Leu
Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445Arg Thr Gln Ser Thr
Gly Gly Thr Ala Gly Thr Gln Gln Leu Leu Phe 450 455 460Ser Gln Ala
Gly Pro Asn Asn Met Ser Ala Gln Ala Lys Asn Trp Leu465 470 475
480Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Leu Ser Gln
485 490 495Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr
His Leu 500 505 510Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Val Ala
Met Ala Thr His 515 520 525Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser
Ser Gly Val Leu Met Phe 530 535 540Gly Lys Gln Gly Ala Gly Lys Asp
Asn Val Asp Tyr Ser Ser Val Met545 550 555 560Leu Thr Ser Glu Glu
Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu 565 570 575Gln Tyr Gly
Val Val Ala Asp Asn Leu Gln Gly Gln Ser Gly Arg Gly 580 585 590Asp
Leu Gly Leu Ser Ala Gln Ala Ala Ile Val Gly Ala Val Asn Ser 595 600
605Gln Gly Ala Leu Pro Gly Met Val Trp Gln Asn Arg Asp Val Tyr Leu
610 615 620Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn
Phe His625 630 635 640Pro Ser Pro Leu Met Gly Gly Phe Gly Met Lys
His Pro Pro Pro Gln 645 650 655Ile Leu Ile Lys Asn Thr Pro Val Pro
Ala Asp Pro Pro Thr Ala Phe 660 665 670Asn Lys Asp Lys Leu Asn Ser
Phe Ile Thr Gln Tyr Ser Thr Gly Gln 675 680 685Val Ser Val Glu Ile
Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg 690 695 700Trp Asn Pro
Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Asn Asn705 710 715
720Val Glu Phe Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro
725 730 735Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 740
74562241DNAartificial sequencesynthetic polynucleotide encoding
peptide- modified AAV9-rh74 hybrid capsidCDS(1)..(2241) 6atg gct
gcc gat ggt tat ctt cca gat tgg ctc gag gac aac ctt agt 48Met Ala
Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15gaa
gga att cgc gag tgg tgg gct ttg aaa cct gga gcc cct caa ccc 96Glu
Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25
30aag gca aat caa caa cat caa gac aac gct cga ggt ctt gtg ctt ccg
144Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro
35 40 45ggt tac aaa tac ctt gga ccc ggc aac gga ctc gac aag ggg gag
ccg 192Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu
Pro 50 55 60gtc aac gca gca gac gcg gcg gcc ctc gag cac gac aag gcc
tac gac 240Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala
Tyr Asp65 70 75 80cag cag ctc aag gcc gga gac aac ccg tac ctc aag
tac aac cac gcc 288Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys
Tyr Asn His Ala 85 90 95gac gcc gag ttc cag gag cgg ctc aaa gaa gat
acg tct ttt ggg ggc 336Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp
Thr Ser Phe Gly Gly 100 105 110aac ctc ggg cga gca gtc ttc cag gcc
aaa aag agg ctt ctt gaa cct 384Asn Leu Gly Arg Ala Val Phe Gln Ala
Lys Lys Arg Leu Leu Glu Pro 115 120 125ctt ggt ctg gtt gag gaa gcg
gct aag acg gct cct gga aag aag agg 432Leu Gly Leu Val Glu Glu Ala
Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140cct gta gag cag tct
cct cag gaa ccg gac tcc tcc gcg ggt att ggc 480Pro Val Glu Gln Ser
Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly145 150 155 160aaa tcg
ggt gca cag ccc gct aaa aag aga ctc aat ttc ggt cag act 528Lys Ser
Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170
175ggc gac aca gag tca gtc cca gac cct caa cca atc gga gaa cct ccc
576Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro
180 185 190gca gcc ccc tca ggt gtg gga tct ctt aca atg gct tca ggt
ggt ggc 624Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly
Gly Gly 195 200 205gca cca gtg gca gac aat aac gaa ggt gcc gat gga
gtg ggt agt tcc 672Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly
Val Gly Ser Ser 210 215 220tcg gga aat tgg cat tgc gat tcc caa tgg
ctg ggg gac aga gtc atc 720Ser Gly Asn Trp His Cys Asp Ser Gln Trp
Leu Gly Asp Arg Val Ile225 230 235 240acc acc agc acc cga acc tgg
gcc ctg ccc acc tac aac aat cac ctc 768Thr Thr Ser Thr Arg Thr Trp
Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255tac aag caa atc tcc
aac agc aca tct gga gga tct tca aat gac aac 816Tyr Lys Gln Ile Ser
Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270gcc tac ttc
ggc tac agc acc ccc tgg ggg tat ttt gac ttc aac aga 864Ala Tyr Phe
Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285ttc
cac tgc cac ttc tca cca cgt gac tgg cag cga ctc atc aac aac 912Phe
His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295
300aac tgg gga ttc cgg cct aag cga ctc aac ttc aag ctc ttc aac att
960Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn
Ile305 310 315 320cag gtc aaa gag gtt acg gac aac aat gga gtc aag
acc atc gcc aat 1008Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys
Thr Ile Ala Asn 325 330 335aac ctt acc agc acg gtc cag gtc ttc acg
gac tca gac tat cag ctc 1056Asn Leu Thr Ser Thr Val Gln Val Phe Thr
Asp Ser Asp Tyr Gln Leu 340 345 350ccg tac gtg ctc ggg tcg gct cac
gag ggc tgc ctc ccg ccg ttc cca 1104Pro Tyr Val Leu Gly Ser Ala His
Glu Gly Cys Leu Pro Pro Phe Pro 355 360 365gcg gac gtt ttc atg att
cct cag tac ggg tat ctg acg ctt aat gat 1152Ala Asp Val Phe Met Ile
Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp 370 375 380gga agc cag gcc
gtg ggt cgt tcg tcc ttt tac tgc ctg gaa tat ttc 1200Gly Ser Gln Ala
Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe385 390 395 400ccg
tcg caa atg cta aga acg ggt aac aac ttc cag ttc agc tac gag 1248Pro
Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu 405 410
415ttt gag aac gta cct ttc cat agc agc tac gct cac agc caa agc ctg
1296Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu
420 425 430gac cga cta atg aat cca ctc atc gac caa tac ttg tac tat
ctc tca 1344Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr
Leu Ser 435 440 445cgg act caa agc acg ggc ggt act gca gga act cag
cag ttg cta ttt 1392Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Gln
Gln Leu Leu Phe 450 455 460tct cag gcc ggg cct aac aac atg tcg gct
cag gcc aag aac tgg cta 1440Ser Gln Ala Gly Pro Asn Asn Met Ser Ala
Gln Ala Lys Asn Trp Leu465 470 475 480ccc ggt ccc tgc tac cgg cag
caa cgc gtc tcc acg aca ctg tcg cag 1488Pro Gly Pro Cys Tyr Arg Gln
Gln Arg Val Ser Thr Thr Leu Ser Gln 485 490 495aac aac aac agc aac
ttt gcc tgg acg ggt gcc acc aag tat cat ctg 1536Asn Asn Asn Ser Asn
Phe Ala Trp Thr Gly Ala Thr Lys Tyr His Leu 500 505 510aat ggc aga
gac tct ctg gtg aat cct ggc gtt gcc atg gct acc cac 1584Asn Gly Arg
Asp Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr His 515 520 525aag
gac gac gaa gag cga ttt ttt cca tcc agc gga gtc tta atg ttt 1632Lys
Asp Asp Glu Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met Phe 530 535
540ggg aaa cag gga gct gga aaa gac aac gtg gac tat agc agc gtg atg
1680Gly Lys Gln Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Ser Val
Met545 550 555 560cta acc agc gag gaa gaa ata aag acc acc aac cca
gtg gcc aca gaa 1728Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro
Val Ala Thr Glu 565 570 575cag tac ggc gtg gtg gcc gat aac ctg caa
ggc cag agt ggc cgc ggc 1776Gln Tyr Gly Val Val Ala Asp Asn Leu Gln
Gly Gln Ser Gly Arg Gly 580 585 590gat ctg ggc ctg agc gcc cag gcg
gcc att gta ggg gcc gtc aat agt 1824Asp Leu Gly Leu Ser Ala Gln Ala
Ala Ile Val Gly Ala Val Asn Ser 595 600 605caa gga gcc tta cct ggc
atg gtg tgg cag aac aga gat gtg tac ctg 1872Gln Gly Ala Leu Pro Gly
Met Val Trp Gln Asn Arg Asp Val Tyr Leu 610 615 620caa gga ccc att
tgg gcc aaa att cct cac acg gac ggc aac ttt cac 1920Gln Gly Pro Ile
Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His625 630 635 640cct
tct ccg ctg atg gga ggg ttt gga atg aag cac ccg cct cct cag 1968Pro
Ser Pro Leu Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln 645 650
655atc ctc atc aaa aac aca cct gta cct gcg gat cct cca acg gcc ttc
2016Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Ala Phe
660 665 670aac aag gac aag ctg aac tct ttc atc acc cag tat tct act
ggc caa 2064Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr
Gly Gln 675 680 685gtc agc gtg gag atc gag tgg gag ctg cag aag gaa
aac agc aag cgc 2112Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu
Asn Ser Lys Arg 690 695 700tgg aac ccg gag atc cag tac act tcc aac
tat tac aag tct aat aat 2160Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn
Tyr Tyr Lys Ser Asn Asn705 710 715 720gtt gaa ttt gct gtt aat act
gaa ggt gta tat agt gaa ccc cgc ccc 2208Val Glu Phe Ala Val Asn Thr
Glu Gly Val Tyr Ser Glu Pro Arg Pro 725 730 735att ggc acc aga tac
ctg act cgt aat ctg taa 2241Ile Gly Thr Arg Tyr Leu Thr Arg Asn
Leu
740 7457746PRTartificial sequenceSynthetic Construct 7Met Ala Ala
Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly
Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30Lys
Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40
45Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr
Asp65 70 75 80Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr
Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr
Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys
Lys Arg Leu Leu Glu Pro 115 120 125Leu Gly Leu Val Glu Glu Ala Ala
Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140Pro Val Glu Gln Ser Pro
Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly145 150 155 160Lys Ser Gly
Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly
Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185
190Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly
195 200 205Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly
Ser Ser 210 215 220Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly
Asp Arg Val Ile225 230 235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu
Pro Thr Tyr Asn Asn His Leu 245 250 255Tyr Lys Gln Ile Ser Asn Ser
Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270Ala Tyr Phe Gly Tyr
Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285Phe His Cys
His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300Asn
Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile305 310
315 320Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala
Asn 325 330 335Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp
Tyr Gln Leu 340 345 350Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys
Leu Pro Pro Phe Pro 355 360 365Ala Asp Val Phe Met Ile Pro Gln Tyr
Gly Tyr Leu Thr Leu Asn Asp 370 375 380Gly Ser Gln Ala Val Gly Arg
Ser Ser Phe Tyr Cys Leu Glu Tyr Phe385 390 395 400Pro Ser Gln Met
Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu 405 410 415Phe Glu
Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425
430Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser
435 440 445Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Gln Gln Leu
Leu Phe 450 455 460Ser Gln Ala Gly Pro Asn Asn Met Ser Ala Gln Ala
Lys Asn Trp Leu465 470 475 480Pro Gly Pro Cys Tyr Arg Gln Gln Arg
Val Ser Thr Thr Leu Ser Gln 485 490 495Asn Asn Asn Ser Asn Phe Ala
Trp Thr Gly Ala Thr Lys Tyr His Leu 500 505 510Asn Gly Arg Asp Ser
Leu Val Asn Pro Gly Val Ala Met Ala Thr His 515 520 525Lys Asp Asp
Glu Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met Phe 530 535 540Gly
Lys Gln Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Ser Val Met545 550
555 560Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr
Glu 565 570 575Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gly Gln Ser
Gly Arg Gly 580 585 590Asp Leu Gly Leu Ser Ala Gln Ala Ala Ile Val
Gly Ala Val Asn Ser 595 600 605Gln Gly Ala Leu Pro Gly Met Val Trp
Gln Asn Arg Asp Val Tyr Leu 610 615 620Gln Gly Pro Ile Trp Ala Lys
Ile Pro His Thr Asp Gly Asn Phe His625 630 635 640Pro Ser Pro Leu
Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln 645 650 655Ile Leu
Ile Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Ala Phe 660 665
670Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln
675 680 685Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser
Lys Arg 690 695 700Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr
Lys Ser Asn Asn705 710 715 720Val Glu Phe Ala Val Asn Thr Glu Gly
Val Tyr Ser Glu Pro Arg Pro 725 730 735Ile Gly Thr Arg Tyr Leu Thr
Arg Asn Leu 740 74587PRTartificial sequencesynthetic peptide 8Arg
Gly Asp Leu Gly Leu Ser1 594PRTartificial sequencesynthetic peptide
9Gly Gln Ser Gly1104PRTartificial sequencesynthetic peptide 10Ala
Gln Ala Ala1116PRTartificial sequencesynthetic peptide 11Gln Gln
Asn Ala Ala Pro1 5128PRTartificial sequencesynthetic peptide 12Gly
Gln Ser Gly Ala Gln Ala Ala1 51315PRTartificial sequencesynthetic
peptide 13Gly Gln Ser Gly Arg Gly Asp Leu Gly Leu Ser Ala Gln Ala
Ala1 5 10 15147PRTartificial sequencesynthetic peptide 14Leu Arg
Gly Asp Gly Leu Ser1 5157PRTartificial sequencesynthetic peptide
15Leu Gly Arg Gly Asp Leu Ser1 5167PRTartificial sequencesynthetic
peptide 16Leu Gly Leu Arg Gly Asp Ser1 5177PRTartificial
sequencesynthetic peptide 17Leu Gly Leu Ser Arg Gly Asp1
5187PRTartificial sequencesynthetic peptide 18Arg Gly Asp Met Ser
Arg Glu1 5
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