U.S. patent application number 14/378886 was filed with the patent office on 2015-04-23 for aav vector compositions and methods for gene transfer to cells, organs and tissues.
The applicant listed for this patent is THE CHILDREN'S HOSPITAL OF PHILADELPHIA. Invention is credited to Katherine A. High, Philip Johnson, Federico Mingozzi, Junwei Sun.
Application Number | 20150111955 14/378886 |
Document ID | / |
Family ID | 48984822 |
Filed Date | 2015-04-23 |
United States Patent
Application |
20150111955 |
Kind Code |
A1 |
High; Katherine A. ; et
al. |
April 23, 2015 |
AAV VECTOR COMPOSITIONS AND METHODS FOR GENE TRANSFER TO CELLS,
ORGANS AND TISSUES
Abstract
The invention relates to adeno-associated virus (AAV) serotype
AAV-Rh74 and related AAV vectors, and AAV-Rh74 and related AAV
vector mediated gene transfer methods and uses. In particular,
AAV-Rh74 targets polynucleotides to cells, tissues or organs for
expression (transcription) of genes encoding therapeutic proteins
and peptides, and polynucleotides that function as or are
transcribed into inhibitory nucleic acid sequences.
Inventors: |
High; Katherine A.; (Merion
Station, PA) ; Mingozzi; Federico; (Paris, FR)
; Sun; Junwei; (Philadelphia, PA) ; Johnson;
Philip; (Wynnewood, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE CHILDREN'S HOSPITAL OF PHILADELPHIA |
Philadelphia |
PA |
US |
|
|
Family ID: |
48984822 |
Appl. No.: |
14/378886 |
Filed: |
February 19, 2013 |
PCT Filed: |
February 19, 2013 |
PCT NO: |
PCT/US13/26695 |
371 Date: |
August 14, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61600415 |
Feb 17, 2012 |
|
|
|
Current U.S.
Class: |
514/44R ;
435/456 |
Current CPC
Class: |
C12N 2750/14143
20130101; A61P 43/00 20180101; C12N 15/86 20130101; A61P 7/04
20180101 |
Class at
Publication: |
514/44.R ;
435/456 |
International
Class: |
C12N 15/86 20060101
C12N015/86 |
Claims
1. A method for delivering or transferring a heterologous
polynucleotide sequence into a mammal or a cell of a mammal,
comprising administering an adeno-associated virus (AAV) vector,
said vector comprising a heterologous polynucleotide sequence, to
said mammal or a cell of said mammal, thereby delivering or
transferring the heterologous polynucleotide sequence into the
mammal or cell of the mammal.
2. The method of claim 1, wherein the heterologous polynucleotide
sequence is operably linked to an expression control element
conferring transcription of said heterologous polynucleotide
sequence.
3. A method of treating a mammal deficient in protein expression or
function, comprising: (a) providing adeno-associated virus (AAV)
vector, said vector comprising a heterologous polynucleotide
encoding a polypeptide that can correct for the deficient protein
expression or function, wherein the heterologous polynucleotide
sequence is operably linked to an expression control element
conferring transcription of said heterologous polynucleotide
sequence; and (b) administering an amount of the AAV vector to the
mammal wherein said polypeptide is expressed in the mammal.
4. The method of claim 1 or 3, wherein said adeno-associated virus
(AAV) vector comprises an AAV with a VP1 sequence having 90% or
more sequence identity to: TABLE-US-00003
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLP
GYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHA
DAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKR
PVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEP
PAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV
ITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFN
RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIA
NNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLN
NGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQS
LDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNW
LPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMAT
HKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVAT
EQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKI
PHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASF
ITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTE
GTYSEPRPIGTRYLTRNL,
or 1-50 amino acid substitutions, deletions or additions thererto;
a VP2 sequence having 90% or more sequence identity to:
TABLE-US-00004 TAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDP
QPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTW
LGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGY
FDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTK
TIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLT
LNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQ
SLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNW
LPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATH
KDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQ
YGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHT
DGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQY
STGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSE
PRPIGTRYLTRNL,
or 1-50 amino acid substitutions, deletions or additions thererto;
and/or a VP3 sequence having 90% or more sequence identity to:
TABLE-US-00005 MAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALP
TYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDW
QRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFT
DSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSF
YCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQ
YLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRV
STTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPS
SGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQ
QQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSP
LMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSV
EIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGT RYLTRNL,
or 1-50 amino acid substitutions, deletions or additions
thererto.
5. The method of claim 1 or 3, wherein said adeno-associated virus
(AAV) vector comprises an AAV with a VP1, VP2, or VP3 sequence
having 95% or more identity to one or more VP1, VP2, or VP3
sequences set forth in FIG. 3.
6. The method of claim 1 or 3, wherein said adeno-associated virus
(AAV) vector comprises a VP1, VP2, or VP3 sequence having 96% or
more identity to one or more VP1, VP2, or VP3 sequences set forth
in FIG. 3.
7. The method of claim 1 or 3, wherein said adeno-associated virus
(AAV) vector comprises a VP1, VP2, or VP3 sequence having 97% or
more identity to one or more VP1, VP2, or VP3 sequences set forth
in FIG. 3.
8. The method of claim 1 or 3, wherein said adeno-associated virus
(AAV) vector comprises a VP1, VP2, or VP3 sequence having 98% or
more identity to one or more VP1, VP2, or VP3 sequences set forth
in FIG. 3.
9. The method of claim 1 or 3, wherein said adeno-associated virus
(AAV) vector comprises a VP1, VP2, or VP3 sequence having 99% or
more identity to one or more VP1, VP2, or VP3 sequences set forth
in FIG. 3.
10. The method of claim 1 or 3, wherein said adeno-associated virus
(AAV) vector comprises a VP1, VP2, or VP3 sequence having 99.5% or
more identity to one or more VP1, VP2, or VP3 sequences set forth
in FIG. 3.
11. The method of claim 1 or 3, wherein said adeno-associated virus
(AAV) vector comprises a VP1, VP2, or VP3 sequence of AAV-Rh74 set
forth in FIG. 3, an AAV vector related to AAV-Rh74 or AAV-Rh74.
12. The method of claim 1 or 3, wherein said heterologous
polynucleotide sequence, or said protein, is expressed at levels
having a therapeutic effect on the mammal.
13. The method of claim 1 or 3, wherein said adeno-associated virus
(AAV) vector has increased tropism for hepatocytes compared to AAV2
or AAV8.
14. The method of claim 1 or 3, wherein said heterologous
polynucleotide sequence or said protein is expressed in a cell,
tissue or organ of said mammal.
15. The method of claim 14, wherein the cell comprises a secretory
cell.
16. The method of claim 14, wherein the cell comprises an endocrine
cell.
17. The method of claim 14, wherein the cell comprises hepatocyte,
a neural cell, a glial cell, a retinal cell, an epithelial cell, a
lung cell or a totipotent, pluripotent or multipotent stem
cell.
18. The method of claim 14, wherein the tissue or organ of said
mammal comprises liver, brain, central nervous system, spinal cord,
eye, retina or lung.
19. The method of claim 1 or 3, wherein the mammal produces an
insufficient amount of a protein, or a defective or aberrant
protein.
20. The method of claim 19, wherein the protein comprises CFTR
(cystic fibrosis transmembrane regulator protein), a blood
coagulation (clotting) factor (Factor XIII, Factor IX, Factor X,
Factor VIII, Factor VIIa, protein C, etc.) a gain of function blood
coagulation factor, an antibody, retinal pigment
epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL
receptor, lipoprotein lipase, ornithine transcarbamylase,
.beta.-globin, .alpha.-globin, spectrin, .alpha.-antitrypsin,
adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7),
sulfamidase, an enzyme involved in lysosomal storage disease
(ARSA), hypoxanthine guanine phosphoribosyl transferase, .beta.-25
glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase,
branched-chain keto acid dehydrogenase, a hormone, a growth factor,
insulin-like growth factor 1 or 2, platelet derived growth factor,
epidermal growth factor, nerve growth factor, neurotrophic factor-3
and -4, brain-derived neurotrophic factor, glial derived growth
factor, transforming growth factor .alpha. and .beta., a cytokine,
.alpha.-interferon, .beta.-interferon, interferon-.gamma.,
interleukin-2, interleukin-4, interleukin 12,
granulocyte-macrophage colony stimulating factor, lymphotoxin, a
suicide gene product, herpes simplex virus thymidine kinase,
cytosine deaminase, diphtheria toxin, cytochrome P450,
deoxycytidine kinase, tumor necrosis factor, a drug resistance
protein, a tumor suppressor protein (e.g., p53, Rb, Wt-1, NF1, Von
Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), a peptide
with immunomodulatory properties, a tolerogenic or immunogenic
peptide or protein Tregitope or hCDR1, insulin, glucokinase,
guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1
(Choroideremia), LCA 5 (LCA-Lebercilin), ornithine ketoacid
aminotransferase (Gyrate Atrophy), Retinoschisin 1 (X-linked
Retinoschisis), USH1C (Usher's Syndrome 1C), X-linked retinitis
pigmentosa GTPase (XLRP), MERTK (AR forms of RP: retinitis
pigmentosa), DFNB1 (Connexin 26 deafness), ACHM 2, 3 and 4
(Achromatopsia), PKD-1 or PKD-2 (Polycystic kidney disease), TPP1,
CLN2, a gene product implicated in lysosomal storage diseases
(e.g., sulfatases, N-acetylglucosamine-1-phosphate transferase,
cathepsin A, GM2-AP, NPC1, VPC2, a sphingolipid activator protein,
or one or more zinc finger nucleases for genome editing, or donor
sequences used as repair templates for genome editing.
21. The method of claim 1 or 3, wherein the heterologous
polynucleotide sequence comprises a gene encoding a polypeptide,
peptide or a protein.
22. The method of claim 21, wherein the gene encodes a therapeutic
peptide or protein.
23. The method of claim 21, wherein the gene comprises or encodes
CFTR (cystic fibrosis transmembrane regulator protein), a blood
coagulation (clotting) factor (Factor XIII, Factor IX, Factor X,
Factor VII, Factor VIIa, protein C, etc.) a gain of function blood
coagulation factor, an antibody, retinal pigment
epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL
receptor, lipoprotein lipase, ornithine transcarbamylase,
.beta.-globin, .alpha.-globin, spectrin, .alpha.-antitrypsin,
adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7),
sulfamidase, an enzyme involved in lysosomal storage disease
(ARSA), hypoxanthine guanine phosphoribosyl transferase, .beta.-25
glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase,
branched-chain keto acid dehydrogenase, a hormone, a growth factor,
insulin-like growth factor 1 or 2, platelet derived growth factor,
epidermal growth factor, nerve growth factor, neurotrophic factor-3
and -4, brain-derived neurotrophic factor, glial derived growth
factor, transforming growth factor .alpha. and .beta., a cytokine,
.alpha.-interferon, .beta.-interferon, interferon-.gamma.,
interleukin-2, interleukin-4, interleukin 12,
granulocyte-macrophage colony stimulating factor, lymphotoxin, a
suicide gene product, herpes simplex virus thymidine kinase,
cytosine deaminase, diphtheria toxin, cytochrome P450,
deoxycytidine kinase, tumor necrosis factor, a drug resistance
protein, a tumor suppressor protein (e.g., p53, Rb, Wt-1, NF1, Von
Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), a peptide
with immunomodulatory properties, a tolerogenic or immunogenic
peptide or protein Tregitope or hCDR1, insulin, glucokinase,
guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1
(Choroideremia), LCA 5 (LCA-Lebercilin), ornithine ketoacid
aminotransferase (Gyrate Atrophy), Retinoschisin 1 (X-linked
Retinoschisis), USH1C (Usher's Syndrome 1C), X-linked retinitis
pigmentosa GTPase (XLRP), MERTK (AR forms of RP: retinitis
pigmentosa), DFNB1 (Connexin 26 deafness), ACHM 2, 3 and 4
(Achromatopsia), PKD-1 or PKD-2 (Polycystic kidney disease), TPP1,
CLN2, a gene product implicated in lysosomal storage diseases
(e.g., sulfatases, N-acetylglucosamine-1-phosphate transferase,
cathepsin A, GM2-AP, NPC1, VPC2, a sphingolipid activator protein
or one or more zinc finger nucleases for genome editing, or donor
sequences used as repair templates for genome editing.
24. The method of claim 1, wherein the heterologous polynucleotide
sequence comprises an inhibitory nucleic acid.
25. The method of claim 24, wherein the inhibitory nucleic acid
comprises micro-RNA (miRNA), siRNA, shRNA, trans-splicing RNA,
antisense RNA or triplex forming RNA.
26. The method of claim 24, wherein the inhibitory nucleic acid
binds to a pathogenic gene, a transcript of a pathogenic gene, or a
gene transcript associated with a polynucleotide repeat disease, a
huntingtin (HTT) gene, a gene associated with
dentatorubropallidolusyan atropy (atrophin 1, ATN1), androgen
receptor on the X chromosome in spinobulbar muscular atrophy, human
Ataxin-1, -2, -3, and -7, Ca.sub.v2.1 P/Q voltage-dependent calcium
channel is encoded by the (CACNA1A), TATA-binding protein, Ataxin 8
opposite strand, also known as ATXN8OS, Serine/threonine-protein
phosphatase 2A 55 kDa regulatory subunit B beta isoform in
spinocerebellar ataxia (type 1, 2, 3, 6, 7, 8, 12 17), FMR1
(fragile X mental retardation 1) in fragile X syndrome, FMR1
(fragile X mental retardation 1) in fragile X-associated
tremor/ataxia syndrome, FMR1 (fragile X mental retardation 2) or
AF4/FMR2 family member 2 in fragile XE mental retardation;
Myotonin-protein kinase (MT-PK) in myotonic dystrophy; Frataxin in
Friedreich's ataxia; a mutant of superoxide dismutase 1 (SOD1) gene
in amyotrophic lateral sclerosis; a gene involved in pathogenesis
of Parkinson's disease and/or Alzheimer's disease; apolipoprotein B
(APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9),
hypercoloesterolemia; HIV Tat, human immunodeficiency virus
transactivator of transcription gene, in HIV infection; HIV TAR,
HIV TAR, human immunodeficiency virus transactivator response
element gene, in HIV infection; C--C chemokine receptor (CCR5) in
HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV
infection, liver-specific microRNA (miR-122) in hepatitis C virus
infection; p53, acute kidney injury or delayed graft function
kidney transplant or kidney injury acute renal failure; protein
kinase N3 (PKN3) in advance recurrent or metastatic solid
malignancies; LMP2, LMP2 also known as proteasome subunit beta-type
9 (PSMB 9), metastatic melanoma; LMP7, also known as proteasome
subunit beta-type 8 (PSMB 8), metastatic melanoma; MECL1 also known
as proteasome subunit beta-type 10 (PSMB 10), metastatic melanoma;
vascular endothelial growth factor (VEGF) in solid tumors; kinesin
spindle protein in solid tumors, apoptosis suppressor B-cell
CLL/lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide
reductase M2 (RRM2) in solid tumors; Furin in solid tumors;
polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol
acyltransferase 1 (DGAT1) in hepatitis C infection, beta-catenin in
familial adenomatous polyposis; beta2 adrenergic receptor,
glaucoma; RTP801/Redd1 also known as DAN damage-inducible
transcript 4 protein, in diabetic macular oedma (DME) or
age-related macular degeneration; vascular endothelial growth
factor receptor I (VEGFR1) in age-related macular degeneration or
choroidal neivascularization, caspase 2 in non-arteritic ischaemic
optic neuropathy; Keratin 6A N17K mutant protein in pachyonychia
congenital; influenza A virus genome/gene sequences in influenza
infection; severe acute respiratory syndrome (SARS) coronavirus
genome/gene sequences in SARS infection; respiratory syncytial
virus genome/gene sequences in respiratory syncytial virus
infection; Ebola filovirus genome/gene sequence in Ebola infection;
hepatitis B and C virus genome/gene sequences in hepatitis B and C
infection; herpes simplex virus (HSV) genome/gene sequences in HSV
infection, coxsackievirus B3 genome/gene sequences in
coxsackievirus B3 infection; silencing of a pathogenic allele of a
gene (allele-specific silencing) like torsin A (TOR1A) in primary
dystonia, pan-class I and HLA-allele specific in transplant; or
mutant rhodopsin gene (RHO) in autosomal dominantly inherited
retinitis pigmentosa (adRP).
27. The method of claim 1 or 3, wherein the mammal has a lung
disease (e.g., cystic fibrosis), a bleeding disorder (e.g.,
hemophilia A or hemophilia B with or without inhibitors),
thalassemia, a blood disorder (e.g., anemia), Alzheimer's disease,
Parkinson's disease, Huntington's disease, amyotrophic lateral
sclerosis (ALS), epilepsy, lysosomal storage diseases, a copper or
iron accumulation disorders (e.g., Wilson's or Menkes disease)
lysosomal acid lipase deficiency, a neurological or
neurodegenerative disorder, cancer, type 1 or type 2 diabetes,
Gaucher's disease, Hurler's disease, adenosine deaminase
deficiency, a metabolic defect (e.g., glycogen storage diseases), a
retinal degenerative disease (such as RPE65 deficiency,
choroideremia, and other diseases of the eye), a disease of solid
organs (e.g., brain, liver, kidney, heart), or an infectious viral
(e.g., hepatitis B and C, HIV, etc.), bacterial or fungal
disease.
28. The method of claim 1 or 3, wherein the expression control
element comprises a constitutive or regulatable control
element.
29. The method of claim 1 or 3, wherein the expression control
element comprises a tissue-specific expression control element or
promoter.
30. The method of claim 1 or 3, wherein the AAV vector is delivered
intravenously, intraarterially, intramuscularly, subcutaneously,
orally, by intubation, via catheter, dermally, ultra-cranially, via
inhalation, intra-cavity, or mucosally.
31. The method of claim 1 or 3, wherein the mammal is human.
32. The method of claim 1 or 3, wherein the mammal is sero-positive
for an AAV serotype other than AAV-Rh74.
33. The method of claim 1 or 3, wherein the mammal is sero-positive
for an AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,
AAV9, AAV10 or AAV11.
34. The method of claim 1 or 3, wherein the mammal is sero-negative
for AAV-Rh74.
35. The method of claim 1 or 3, further comprising administering
empty capsid AAV.
36. The method of claim 1 or 3, further comprising administering
empty capsid AAV-Rh74.
37. The method of claim 4, wherein the VP1, VP2 or VP3 sequence has
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, conservative, non-conservative,
or conservative and non-conservative amino acid substitutions.
38. The method of claim 1 or 3, wherein the AAV comprises a
pharmaceutical composition.
39. The method of claim 38, wherein the pharmaceutical composition
comprises empty capsid AAV.
40. The method of claim 38, wherein the pharmaceutical composition
comprises empty capsid AAV-Rh74.
Description
RELATED APPLICATION INFORMATION
[0001] This application is the National Phase of International
Application No. PCT/US2013/026695, filed Feb. 19, 2013, which
designated the U.S. and that International Application was
published under PCT Article 21(2) in English, which claims priority
to Application Ser. No. 61/600,415, filed Feb. 17, 2012, all of
which applications are expressly incorporated herein by reference
in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 14, 2014, is named CHOP.sub.--0433686_ST25.txt and is
19,644 bytes in size.
INTRODUCTION
[0003] Genetic disorders, caused by absence or a defect in a
desirable gene (loss of function) or expression of an undesirable
or defective gene or (gain of function) lead to a variety of
diseases. One example of a loss of function genetic disorder is
hemophilia, an inherited bleeding disorder caused by deficiency in
either coagulation factor VIII (FVIII, hemophilia A) or factor IX
(FIX, hemophilia B). One example of a gain of function genetic
disorder is Huntington's disease, a disease caused by a pathologic
"HTT" gene (encodes the huntingtin protein) that encodes a mutated
protein that accumulates within and leads to gradual destruction of
neurons, particularly in the basal ganglia and the cerebral
cortex.
[0004] Current treatment for hemophilia consists in the intravenous
administration of recombinant clotting factor either on demand, in
case a bleeding occurs, or prophylactically. However, this
therapeutic approach has several drawbacks such as the need for
repeated infusions, the cost of the treatment, the risk of
developing anti-therapeutic factor immune responses, and the risk
of potentially fatal bleedings. These limitations have prompted the
development of gene-based therapies for hemophilia. To this end,
hemophilia is ideal for gene transfer based therapy as 1) the
therapeutic window is very wide, as levels just above 1% of normal
already can result in a change in phenotype from severe to
moderate, and levels of 100% are not associated to any side
effects; 2) tissue specific expression of the therapeutic transgene
is not strictly required; and 3) there is a considerable experience
in measuring the endpoints of therapeutic efficacy. Furthermore,
liver expression of clotting factor has been demonstrated to induce
immunological tolerance to the clotting factor itself, reducing the
likelihood of potentially harmful immune responses against clotting
factor.
[0005] Currently, adeno-associated virus (AAV) vectors are
recognized as the gene transfer vectors of choice since they have
the best safety and efficacy profile for the delivery of genes in
vivo. Of the AAV serotypes isolated so far, AAV2 and AAV8 have been
used to target the liver of humans affected by severe hemophilia B.
Both vectors worked efficiently and in the case of AAV8 long-term
expression of the therapeutic transgene was documented. Recent data
in humans showed that targeting the liver with an AAV vector
achieves long-term expression of the FIX transgene at therapeutic
levels.
[0006] While these data are promising, the identification of AAV
serotypes with high tropism for liver and low seroprevalence in
humans (a natural host for wild type AAV) is fundamental for 1)
achieving therapeutic levels of transgene expression in liver at
the lowest vector dose possible to decrease risk of triggering
anti-AAV capsid immune responses; and 2) alternate AAV serotypes
with unique seroprevalence will allow to treat those patients
populations otherwise not eligible for AAV gene transfer due to
pre-existing humoral immunity to AAVs. The invention addresses
these needs and provides additional benefits.
SUMMARY
[0007] The invention provides adeno-associated virus (AAV) serotype
AAV-Rh74 vector, and related AAV vectors. Such vectors include
AAV-Rh74 which target hepatocyte cells of the liver, among other
cell types. As a vector for polynucleotide sequence delivery,
AAV-Rh74 and related AAV vectors drive expression of the
polynucleotide in cells. Polynucleotides that encode proteins, such
as proteins for therapeutic applications are able to be expressed
at therapeutic levels after administration. Furthermore, AAV-Rh74
and related AAV vector mediated polynucleotide transfer produced
protein expression levels that were significantly higher than
several other serotypes currently studied in preclinical and
clinical settings (see, e.g., FIGS. 1 and 2). In particular,
AAV-Rh74 could target polynucleotides to the liver with efficiency
at least comparable or superior to the gold standard for liver
transduction, AAV8, both in mice and in hemophilia B dogs. Thus,
AAV-Rh74 can be used to deliver polynucleotides, such as gene
coding sequences, to express proteins that provide a desirable or
therapeutic benefit, as well as for inhibitory nucleotides that
reduce or inhibit expression of an undesirable or defective gene,
thereby treating a variety of diseases. For example, AAV-Rh74 can
be used to deliver therapeutic genes (e.g., FIX, FVIII) to treat
hemophilia A, B, and to deliver genes for a wide range of other
metabolic or plasma protein deficiencies, or to deliver genes for
other therapeutic purposes, such as but not limited to genes
encoding zinc finger nucleases to carry out genome editing in the
liver, and for local (liver) delivery of immunomodulatory agents
such as alpha-interferon for treatment of hepatitis virus
infections, or to treat virtually any disease that requires either
liver transduction or presence of the therapeutic transgene product
in the bloodstream (which can be achieved by targeting the
transgenes for liver expression).
[0008] In addition to efficient delivery of polynucleotides by
AAV-Rh74 and related vectors into cells in vitro, ex vivo and in
vivo, prevalence of anti-AAV-Rh74 antibodies in humans is lower
than anti-AAV2 antibodies, and differs from that of anti-AAV8
antibodies (Table 1). Owing to low seroprevealence, AAV-Rh74 and
related vectors can be used in a greater percentage of humans which
otherwise would not be eligible for gene transfer, for example,
humans that may be sero-positive for other AAV serotypes (e.g.,
AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,
etc.). In addition, AAV-Rh74 can be efficiently produced at high
titers (Table 2). Thus, AAV-Rh74 and related vectors can be
produced in large amounts for more prevalent clinical diseases.
[0009] In accordance with the invention, there are provided methods
for delivering or transferring a heterologous polynucleotide
sequence into a mammal or a cell of a mammal. In one embodiment, a
method includes administering an adeno-associated virus (AAV)
vector that includes a heterologous polynucleotide sequence to a
mammal or a cell of a mammal under suitable conditions to deliver
or transfer the heterologous polynucleotide sequence into the
mammal or the cell of a mammal, thereby delivering or transferring
the heterologous polynucleotide. In one aspect, the method allows
transfer/delivery of the heterologous polynucleotide into the
mammal and/or cell. In another aspect, the method allows
transfer/delivery of the heterologous polynucleotide into the
mammal and/or cell, and subsequent transcription of the
heterologous polynucleotide thereby forming a transcript. In a
further aspect, the method allows transfer/delivery of the
heterologous polynucleotide into the cell, subsequent transcription
to form a transcript and subsequent translation to form a gene
product (protein). In particular, for example, in the latter two
aspects a heterologous polynucleotide sequence is operably linked
to an expression control element conferring transcription of the
heterologous polynucleotide sequence, and optionally subsequent
translation of the transcript.
DESCRIPTION OF DRAWINGS
[0010] FIG. 1 shows human factor IX (FIX) plasma levels in C57BL/6
mice (n=5 per group) injected via the tail vein with AAV vectors
expressing the FIX transgene under the control of a liver-specific
promoter. Vector dose 2.5.sup.10 vector genomes per mouse. FIX
transgene product (FIX protein) plasma levels were measured by
ELISA at weeks 1, 2, and 4 post gene transfer. AAV-Rh74 conferred
the highest levels of FIX transgene expression.
[0011] FIG. 2 shows canine FIX plasma levels in hemophilia B dogs
after the delivery of 3.sup.12 vector genomes per kilogram (kg) of
weight. AAV vectors were infused intravenously (IV) though the
saphenous vein and FIX levels were monitored by ELISA. Expression
of the therapeutic FIX transgene was driven by a liver specific
promoter. AAV8 and AAV-Rh74 vectors performed roughly equally in
hemophilia B dogs and were both superior to AAV6.
[0012] FIG. 3 shows AAV-Rh74 VP1, VP2, and VP3 amino acid sequences
and, for VP1, polynucleotide (DNA) sequence (SEQ ID NOs:1-4).
[0013] FIG. 4 shows administration of AAV8 and AAVrh74 vector
expressing human Factor IX (FIX) (under the control of a
liver-specific promoter) to rhesus macaques, a non-human primate,
and expression of FIX in the animals. Animals receiving the
AAVrh74-FIX vectors (last two bars towards the right margin)
expressed the FIX transgene at higher levels compared to the other
groups of animals injected at the same dose.
DETAILED DESCRIPTION
[0014] The invention is based, at least in part, on data indicating
that adeno-associated virus (AAV) serotype AAV-Rh74 has a high
tropism for hepatocytes, which are cells of the liver. As a vector
for polynucleotide (e.g. genes, inhibitory nucleic acid, etc.)
transfer/delivery into cells, AAV-Rh74 can drive therapeutic levels
of expression in liver after intravenous administration.
Furthermore, AAV-Rh74 mediated gene transfer/delivery produced
protein expression levels that were significantly higher than
several other serotypes (see, e.g., FIGS. 1 and 2). In particular,
AAV-Rh74 targets genes for delivery to the liver with efficiency at
least comparable or superior to the gold standard for liver
transduction, AAV8, both in mice and in hemophilia B dogs. Thus,
AAV-Rh74 can be used to transfer/deliver polynucleotides, such as
coding sequences (genes) for proteins that provide a desirable or
therapeutic benefit, as well as inhibitory (e.g., anti-sense)
nucleic acid that reduce or inhibit expression of an undesirable or
defective (e.g., pathologic) gene, thereby treating a variety of
diseases. For example, AAV-Rh74 can be used to transfer/deliver
therapeutic genes to treat hemophilia A, B, and to transfer/deliver
genes for a wide range of other metabolic or plasma protein
deficiencies, or for other therapeutic purposes, such as but not
limited to genes encoding zinc finger nucleases to carry out genome
editing in the liver, and for local (liver) delivery of
immunomodulatory agents such as alpha-interferon for treatment of
hepatitis virus infections, and to treat virtually any disease that
requires either liver transduction or presence of the therapeutic
transgene product in the bloodstream (which can be achieved by
targeting the transgenes for liver expression).
[0015] As set forth herein, adeno-associated virus (AAV) serotype
AAV-Rh74 and related AAV vectors provide delivery of polynucleotide
sequences to cells ex vivo, in vitro and in vivo. Such
polynucleotide sequences can encode proteins such that the cells
into which the polynucleotides are delivered express the encoded
proteins. For example, AAV-Rh74 and related AAV vectors can include
polynucleotides encoding a desired protein or peptide, or a
polynucleotide that when transcribed comprises an inhibitory
sequence (e.g., RNA), for example, a sequence that targets a gene
for inhibition of expression. Vector delivery or administration to
a subject (e.g., mammal) therefore provides not only
polynucleotides encoding proteins and peptides to the subject, but
also inhibitory nucleic acids that target genes for inhibition of
expression or function in the subject.
[0016] Thus, in accordance with the invention adeno-associated
virus (AAV) serotype AAV-Rh74, and related AAV vectors, including
polynucleotide sequences encoding peptides and proteins, as well as
polynucleotide sequences which directly or when transcribed
comprise inhibitory nucleic acids that target genes for inhibition
of expression or function, are provided. Such AAV-Rh74 and related
AAV vector serotypes (e.g., VP1, VP2, and/or VP3 sequences) are
distinct from other AAV serotypes, including, for example,
AAV1-AAV11 or Rh10 (e.g., distinct from VP1, VP2, and/or VP3
sequences of any of AAV1-AAV11, or Rh10 serotypes).
[0017] As used herein, the term "serotype" is a distinction used to
refer to an AAV having a capsid that is serologically distinct from
other AAV serotypes. Serologic distinctiveness is determined on the
basis of the lack of cross-reactivity between antibodies to one AAV
as compared to another AAV. Such cross-reactivity differences are
usually due to differences in capsid protein sequences/antigenic
determinants (e.g., due to VP1, VP2, and/or VP3 sequence
differences of AAV serotypes).
[0018] AAV-Rh74 has gene/protein sequences identical to sequences
characteristic for AAV-Rh74 (see, e.g., VP1, VP2, VP3 of FIG. 3).
As used herein, an "AAV vector related to AAV-Rh74" and grammatical
variations thereof refers to one or more AAV proteins (e.g., VP1,
VP2, and/or VP3 sequences) that has substantial sequence identity
to one or more polynucleotides or polypeptide sequences that
comprise AAV-Rh74. Such AAV vectors related to AAV-Rh74 can
therefore have one or more distinct sequences from AAV-Rh74, but
can exhibit substantial sequence identity to one or more genes
and/or have one or more functional characteristics of AAV-Rh74
(e.g., such as cell/tissue tropism). Exemplary AAV-Rh74 sequences
include VP1, VP2, and/or VP3 set forth in FIG. 3. In one
non-limiting exemplary embodiment, an AAV vector related to
AAV-Rh74 has a polynucleotide, polypeptide or subsequence thereof
that includes or consists of a sequence at least 80% or more (e.g.,
85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or
more AAV-Rh74 VP1, VP2, and/or VP3 sequences set forth in FIG.
3.
[0019] In accordance with the invention, methods and uses include
AAV-Rh74 sequences (polypeptides and nucleotides) and subsequences
thereof that exhibit less than 100% sequence identity to a
reference AAV-Rh74 gene or protein sequence (e.g., VP1, VP2, and/or
VP3 sequences set forth in FIG. 3), but are distinct from and not
identical to known AAV genes or proteins, such as AAV1-AAV11,
AAV-Rh10, genes or proteins, etc. In one embodiment, an AAV-Rh74
polypeptide or subsequence thereof includes or consists of a
sequence at least 80% or more identical, e.g., 85%, 85%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.,
i.e. up to 100% identical to any reference AAV-Rh74 sequence or
subsequence thereof (e.g., VP1, VP2 and/or VP3 sequences set forth
in FIG. 3).
[0020] AAV vectors, including AAV-Rh74, and AAV-Rh74 related
vectors, can be constructed using recombinant techniques that are
known to the skilled artisan, to include one or more heterologous
polynucleotide sequences flanked with functional AAV ITRs.
Incorporation of a heterologous polynucleotide defines the AAV as a
recombinant vector, or an "rAAV vector." Such vectors can have one
or more of the wild type AAV genes deleted in whole or in part, for
example, a rep and/or cap gene, but retain at least one functional
flanking ITR sequence, as necessary for the rescue, replication,
and packaging of the AAV particle. Thus, an AAV vector includes
sequences required in cis for viral replication and packaging
(e.g., functional ITRs).
[0021] The terms "polynucleotide" and "nucleic acid" are used
interchangeably herein to refer to all forms of nucleic acid,
oligonucleotides, including deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA). Polynucleotides include genomic DNA, cDNA
and antisense DNA, and spliced or unspliced mRNA, rRNA tRNA and
inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA,
microRNA, small or short interfering (si)RNA, trans-splicing RNA,
or antisense RNA). Polynucleotides include naturally occurring,
synthetic, and intentionally altered or modified polynucleotides as
well as analogues and derivatives. Polynucleotides can be single,
double, or triplex, linear or circular, and can be of any
length.
[0022] A "heterologous" polynucleotide merely refers to a
polynucleotide inserted into AAV for purposes of AAV mediated
transfer/delivery of the polynucleotide into a cell. Heterologous
polynucleotides are typically distinct from AAV nucleic acid. Once
transferred/delivered into the cell, a heterologous polynucleotide,
contained within the rAAV virion, can be expressed (e.g.,
transcribed, and translated if appropriate). Alternatively, a
transferred/delivered heterologous polynucleotide in a cell,
contained within the rAAV virion, need not be expressed. Although
the term "heterologous" is not always used herein in reference to
polynucleotides, reference to a polynucleotide even in the absence
of the modifier "heterologous" includes heterologous
polynucleotides in spite of the omission.
[0023] The "polypeptides," "proteins" and "peptides" encoded by the
"polynucleotide sequences," include full-length native sequences,
as with naturally occurring proteins, as well as functional
subsequences, modified forms or sequence variants so long as the
subsequence, modified form or variant retains some degree of
functionality of the native full-length protein. In methods and
uses of the invention, such polypeptides, proteins and peptides
encoded by the polynucleotide sequences can be but are not required
to be identical to the endogenous protein that is defective, or
whose expression is insufficient, or deficient in the treated
mammal.
[0024] Invention adeno-associated virus (AAV) serotype AAV-Rh74,
and related AAV vectors can be used to introduce/deliver
polynucleotides stably or transiently into cells and progeny
thereof. The term "transgene" is used to conveniently refer to such
a heterologous polynucleotide that has been introduced into a cell
or organism. Transgenes include any polynucleotide, such as a gene
that encodes a polypeptide or protein, a polynucleotide that is
transcribed into an inhibitory polynucleotide, or a polynucleotide
that is not transcribed (e.g., lacks a expression control element,
such as a promoter that drives transcription). For example, in a
cell having a transgene, the transgene has been
introduced/transferred by AAV "transformation" of the cell. A cell
or progeny thereof into which the transgene has been introduced is
referred to as a "transformed cell" or "transformant." Typically, a
transgene is included in progeny of the transformant or becomes a
part of the organism that develops from the cell. Accordingly, a
"transformed" or "transfected" cell (e.g., in a mammal, such as a
cell or tissue or organ cell), means a genetic change in a cell
following incorporation of an exogenous molecule, for example, a
polynucleotide or protein (e.g., a transgene) into the cell. Thus,
a "transfected" or "transformed" cell is a cell into which, or a
progeny thereof in which an exogenous molecule has been introduced,
for example. The cell(s) can be propagated and the introduced
protein expressed, or nucleic acid transcribed.
[0025] Particular non-limiting examples of polynucleotides encoding
gene products (proteins) which are useful in accordance with the
invention include, but are not limited to: genes that comprise or
encode CFTR (cystic fibrosis transmembrane regulator protein), a
blood coagulation (clotting) factor (Factor XIII, Factor IX, Factor
X, Factor VIII, Factor VIIa, protein C etc.) including gain of
function blood coagulation factors, an antibody, retinal pigment
epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL
receptor, lipoprotein lipase, ornithine transcarbamylase,
.beta.-globin, .alpha.-globin, spectrin, .alpha.-antitrypsin,
adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7),
sulfamidase, an enzyme involved in lysosomal storage disease
(ARSA), hypoxanthine guanine phosphoribosyl transferase, .beta.-25
glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase,
branched-chain keto acid dehydrogenase, a hormone, a growth factor
(e.g., insulin-like growth factors 1 and 2, platelet derived growth
factor, epidermal growth factor, nerve growth factor, neurotrophic
factor-3 and -4, brain-derived neurotrophic factor, glial derived
growth factor, transforming growth factor .alpha. and .beta.,
etc.), a cytokine (e.g., .alpha.-interferon, .beta.-interferon,
interferon-.gamma., interleukin-2, interleukin-4, interleukin 12,
granulocyte-macrophage colony stimulating factor, lymphotoxin,
etc.), a suicide gene product (e.g., herpes simplex virus thymidine
kinase, cytosine deaminase, diphtheria toxin, cytochrome P450,
deoxycytidine kinase, tumor necrosis factor, etc.), a drug
resistance protein (e.g, that provides resistance to a drug used in
cancer therapy), a tumor suppressor protein (e.g., p53, Rb, Wt-1,
NF1, Von Hippel-Lindau (VHL), adenomatous polyposis coli (APC)), a
peptide with immunomodulatory properties, a tolerogenic or
immunogenic peptide or protein Tregitopes [de Groot et al., Blood
2008 Oct. 15; 112(8):3303], or hCDR1 [Sharabi et al., Proc Natl
Acad Sci USA. 2006 Jun. 6; 103(23):8810-5], insulin, glucokinase,
guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1
(Choroideremia), LCA 5 (LCA-Lebercilin), ornithine ketoacid
aminotransferase (Gyrate Atrophy), Retinoschisin 1 (X-linked
Retinoschisis), USH1C (Usher's Syndrome 1C), X-linked retinitis
pigmentosa GTPase (XLRP), MERTK (AR forms of RP: retinitis
pigmentosa), DFNB1 (Connexin 26 deafness), ACHM 2, 3 and 4
(Achromatopsia), PKD-1 or PKD-2 (Polycystic kidney disease), TPP1,
CLN2, gene deficiencies causative of lysosomal storage diseases
(e.g., sulfatases, N-acetylglucosamine-1-phosphate transferase,
cathepsin A, GM2-AP, NPC1, VPC2, Sphingolipid activator proteins,
etc.), one or more zinc finger nucleases for genome editing, or
donor sequences used as repair templates for genome editing.
[0026] All mammalian and non-mammalian forms of polynucleotides
encoding gene products, including the non-limiting genes and
proteins disclosed herein are expressly included, either known or
unknown. Thus, the invention includes genes and proteins from
non-mammals, mammals other than humans, and humans, which genes and
proteins function in a substantially similar manner to the human
genes and proteins described herein. A non-limiting example of
non-mammalian gene is a Fok nuclease domain, which is bacterial in
origin. Non-limiting examples of mammalian non-human FIX sequences
are described in Yoshitake et al., 1985, supra; Kurachi et al.,
1995, supra; Jallat et al., 1990, supra; Kurachi et al., 1982,
Proc. Natl. Acad. Sci. USA 79:6461-6464; Jaye et al., 1983, Nucl.
Acids Res. 11:2325-2335; Anson et al., 1984, EMBO J. 3: 1053-1060;
Wu et al., 1990, Gene 86:275-278; Evans et al., Proc Natl Acad Sci
USA 86:10095 (1989), Blood 74:207-212; Pendurthi et al., 1992,
Thromb. Res. 65:177-186; Sakar et al., 1990, Genomics 1990,
6:133-143; and, Katayama et al., 1979, Proc. Natl. Acad. Sci. USA
76:4990-4994.
[0027] Polynucleotides, polypeptides and subsequences thereof
include modified and variant forms. As used herein, the terms
"modify" or "variant" and grammatical variations thereof, mean that
a polynucleotide, polypeptide or subsequence thereof deviates from
a reference sequence. Modified and variant sequences may therefore
have substantially the same, greater or less activity or function
than a reference sequence, but at least retain partial activity or
function of the reference sequence.
[0028] Accordingly, the invention also includes naturally and
non-naturally occurring variants. Such variants include gain and
loss of function variants. For example, wild type human FIX DNA
sequences, which protein variants or mutants retain activity, or
are therapeutically effective, or are comparably or even more
therapeutically active than invariant human FIX in the methods and
uses of the invention. In a particular example, collagen IV serves
to trap FIX, meaning that when introduced into the muscle tissue of
a mammal some of the FIX is not available for participation in
blood coagulation because it is retained in the interstitial spaces
in the muscle tissue. A mutation in the sequence of FIX that
results in a protein with reduced binding to collagen IV (e.g.,
loss of function) is a mutant useful in the methods of the
invention, for example, for treatment of hemophilia. An example of
such a mutant human FIX gene encodes a human FIX protein with the
amino acid alanine in place of lysine in the fifth amino acid
position from the beginning of the mature protein.
[0029] Non-limiting examples of modifications include one or more
amino acid substitutions (e.g., 1-3, 3-5, 5-10, 10-15, 15-20,
20-25, 25-30, 30-40, 40-50, or more residues), additions (e.g.,
insertions or 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40,
40-50, or more residues) and deletions (e.g., subsequences or
fragments) of a reference sequence. In particular embodiments, a
modified or variant sequence retains at least part of a function or
an activity of unmodified sequence. Such modified forms and
variants can have less than, the same, or greater, but at least a
part of, a function or activity of a reference sequence, for
example, as described herein.
[0030] A variant can have one or more non-conservative or a
conservative amino acid sequence differences or modifications, or
both. A "conservative substitution" is the replacement of one amino
acid by a biologically, chemically or structurally similar residue.
Biologically similar means that the substitution does not destroy a
biological activity. Structurally similar means that the amino
acids have side chains with similar length, such as alanine,
glycine and serine, or a similar size. Chemical similarity means
that the residues have the same charge or are both hydrophilic or
hydrophobic. Particular examples include the substitution of one
hydrophobic residue, such as isoleucine, valine, leucine or
methionine for another, or the substitution of one polar residue
for another, such as the substitution of arginine for lysine,
glutamic for aspartic acids, or glutamine for asparagine, serine
for threonine, and the like. Particular examples of conservative
substitutions include the substitution of a hydrophobic residue
such as isoleucine, valine, leucine or methionine for another, the
substitution of a polar residue for another, such as the
substitution of arginine for lysine, glutamic for aspartic acids,
or glutamine for asparagine, and the like. For example,
conservative amino acid substitutions typically include
substitutions within the following groups: glycine, alanine;
valine, isoleucine, leucine; aspartic acid, glutamic acid;
asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine. A "conservative substitution" also
includes the use of a substituted amino acid in place of an
unsubstituted parent amino acid.
[0031] Accordingly, the invention includes gene and protein
variants (e.g., of polynucleotides encoding proteins described
herein) which retain one or more biological activities (e.g.,
function in blood clotting, etc.). Such variants of proteins or
polypeptides include proteins or polypeptides which have been or
may be modified using recombinant DNA technology such that the
protein or polypeptide possesses altered or additional properties,
for example, variants conferring enhanced protein stability in
plasma or enhanced activity of the protein. Variants can differ
from a reference sequence, such as naturally occurring
polynucleotides, proteins or peptides.
[0032] At the nucleotide sequence level, a naturally and
non-naturally occurring variant gene will typically be at least
about 50% identical, more typically about 70% identical, even more
typically about 80% identical (90% or more identity) to the
reference gene. At the amino acid sequence level, a naturally and
non-naturally occurring variant protein will typically be at least
about 70% identical, more typically about 80% identical, even more
typically about 90% or more identity to the reference protein,
although substantial regions of non-identity are permitted in
non-conserved regions (e.g., less, than 70% identical, such as less
than 60%, 50% or even 40%). In other embodiments, the sequences
have at least 60%, 70%, 75% or more identity (e.g., 80%, 85% 90%,
95%, 96%, 97%, 98%, 99% or more identity) to a reference sequence.
Procedures for the introduction of nucleotide and amino acid
changes in a polynucleotide, protein or polypeptide are known to
the skilled artisan (see, e.g., Sambrook et al. (1989)).
[0033] The term "identity," "homology" and grammatical variations
thereof, mean that two or more referenced entities are the same,
when they are "aligned" sequences. Thus, by way of example, when
two polypeptide sequences are identical, they have the same amino
acid sequence, at least within the referenced region or portion.
Where two polynucleotide sequences are identical, they have the
same polynucleotide sequence, at least within the referenced region
or portion. The identity can be over a defined area (region or
domain) of the sequence. An "area" or "region" of identity refers
to a portion of two or more referenced entities that are the same.
Thus, where two protein or nucleic acid sequences are identical
over one or more sequence areas or regions they share identity
within that region. An "aligned" sequence refers to multiple
polynucleotide or protein (amino acid) sequences, often containing
corrections for missing or additional bases or amino acids (gaps)
as compared to a reference sequence
[0034] The identity can extend over the entire sequence length or a
portion of the sequence. In particular aspects, the length of the
sequence sharing the percent identity is 2, 3, 4, 5 or more
contiguous polynucleotide or amino acids, e.g., 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, etc. contiguous amino acids. In
additional particular aspects, the length of the sequence sharing
identity is 20 or more contiguous polynucleotide or amino acids,
e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, etc. contiguous amino acids. In further particular aspects, the
length of the sequence sharing identity is 35 or more contiguous
polynucleotide or amino acids, e.g., 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 45, 47, 48, 49, 50, etc., contiguous amino acids.
In yet further particular aspects, the length of the sequence
sharing identity is 50 or more contiguous polynucleotide or amino
acids, e.g., 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85,
85-90, 90-95, 95-100, 100-110, etc. contiguous polynucleotide or
amino acids.
[0035] The terms "homologous" or "homology" mean that two or more
referenced entities share at least partial identity over a given
region or portion. "Areas, regions or domains" of homology or
identity mean that a portion of two or more referenced entities
share homology or are the same. Thus, where two sequences are
identical over one or more sequence regions they share identity in
these regions. "Substantial homology" means that a molecule is
structurally or functionally conserved such that it has or is
predicted to have at least partial structure or function of one or
more of the structures or functions (e.g., a biological function or
activity) of the reference molecule, or relevant/corresponding
region or portion of the reference molecule to which it shares
homology.
[0036] The extent of identity (homology) between two sequences can
be ascertained using a computer program and mathematical algorithm.
Such algorithms that calculate percent sequence identity (homology)
generally account for sequence gaps and mismatches over the
comparison region or area. For example, a BLAST (e.g., BLAST 2.0)
search algorithm (see, e.g., Altschul et al., J. Mol. Biol. 215:403
(1990), publicly available through NCBI) has exemplary search
parameters as follows: Mismatch -2; gap open 5; gap extension 2.
For polypeptide sequence comparisons, a BLASTP algorithm is
typically used in combination with a scoring matrix, such as
PAM100, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and
FASTA3) and SSEARCH sequence comparison programs are also used to
quantitate extent of identity (Pearson et al., Proc. Natl. Acad.
Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132:185 (2000);
and Smith et al., J. Mol. Biol. 147:195 (1981)). Programs for
quantitating protein structural similarity using Delaunay-based
topological mapping have also been developed (Bostick et al.,
Biochem Biophys Res Commun. 304:320 (2003)).
[0037] Polynucleotides include additions and insertions, for
example, heterologous domains. An addition (e.g., heterologous
domain) can be a covalent or non-covalent attachment of any type of
molecule to a composition. Typically additions and insertions
(e.g., a heterologous domain) confer a complementary or a distinct
function or activity.
[0038] Additions and insertions include chimeric and fusion
sequences, which is a polynucleotide or protein sequence having one
or more molecules not normally present in a reference native (wild
type) sequence covalently attached to the sequence. The terms
"fusion" or "chimeric" and grammatical variations thereof, when
used in reference to a molecule means that a portions or part of
the molecule contains a different entity distinct (heterologous)
from the molecule as they do not typically exist together in
nature. That is, for example, one portion of the fusion or chimera,
includes or consists of a portion that does not exist together in
nature, and is structurally distinct.
[0039] As set forth herein, polynucleotide sequences include
inhibitory and antisense nucleic acid sequences Inhibitory,
antisense, miRNA, shRNA, and RNAi nucleic acids can modulate
expression of a target gene. Antisense includes single, double or
triple stranded polynucleotides and peptide nucleic acids (PNAs)
that bind RNA transcript or DNA (e.g., genomic DNA).
Oligonucleotides derived from the transcription initiation site of
a target gene, e.g., between positions -10 and +10 from the start
site, are another particular example. Triplex forming antisense can
bind to double strand DNA thereby inhibiting transcription of the
gene. "RNAi" is the use of single or double stranded RNA sequences
for inhibiting gene expression (see, e.g., Kennerdell et al., Cell
95:1017 (1998); and Fire et al., Nature, 391:806 (1998)). Double
stranded RNA sequences from a target gene coding region may
therefore be used to inhibit or prevent gene
expression/transcription in accordance with the methods and uses of
the invention. Antisense and RNAi can be produced based upon
nucleic acids encoding target gene sequences (e.g., HTT), such as
nucleic acid encoding mammalian and human HTT. For example, a
single or double stranded nucleic acid (e.g., RNA) can target HTT
transcript (e.g., mRNA).
[0040] Particular non-limiting examples of genes (e.g., genomic
DNA) or transcript of a pathogenic gene (e.g, RNA or mRNA) that may
be targeted with inhibitory nucleic acid sequences in accordance
with the invention include, but are not limited to: pathogenic
genes associated with polynucleotide repeat diseases such as
huntingtin (HTT) gene, a gene associated with
dentatorubropallidolusyan atropy (e.g., atrophin 1, ATN1); androgen
receptor on the X chromosome in spinobulbar muscular atrophy, human
Ataxin-1, -2, -3, and -7, Ca.sub.v2.1 P/Q voltage-dependent calcium
channel is encoded by the (CACNA1A), TATA-binding protein, Ataxin 8
opposite strand, also known as ATXN8OS, Serine/threonine-protein
phosphatase 2A 55 kDa regulatory subunit B beta isoform in
spinocerebellar ataxia (type 1, 2, 3, 6, 7, 8, 12 17), FMR1
(fragile X mental retardation 1) in fragile X syndrome, FMR1
(fragile X mental retardation 1) in fragile X-associated
tremor/ataxia syndrome, FMR1 (fragile X mental retardation 2) or
AF4/FMR2 family member 2 in fragile XE mental retardation;
Myotonin-protein kinase (MT-PK) in myotonic dystrophy; Frataxin in
Friedreich's ataxia; a mutant of superoxide dismutase 1 (SOD1) gene
in amyotrophic lateral sclerosis; a gene involved in pathogenesis
of Parkinson's disease and/or Alzheimer's disease; apolipoprotein B
(APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9),
hypercoloesterolemia; HIV Tat, human immunodeficiency virus
transactivator of transcription gene, in HIV infection; HIV TAR,
HIV TAR, human immunodeficiency virus transactivator response
element gene, in HIV infection; C--C chemokine receptor (CCR5) in
HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV
infection, liver-specific microRNA (miR-122) in hepatitis C virus
infection; p53, acute kidney injury or delayed graft function
kidney transplant or kidney injury acute renal failure; protein
kinase N3 (PKN3) in advance recurrent or metastatic solid
malignancies; LMP2, LMP2 also known as proteasome subunit beta-type
9 (PSMB 9), metastatic melanoma; LMP7, also known as proteasome
subunit beta-type 8 (PSMB 8), metastatic melanoma; MECL1 also known
as proteasome subunit beta-type 10 (PSMB 10), metastatic melanoma;
vascular endothelial growth factor (VEGF) in solid tumors; kinesin
spindle protein in solid tumors, apoptosis suppressor B-cell
CLL/lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide
reductase M2 (RRM2) in solid tumors; Furin in solid tumors;
polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol
acyltransferase 1 (DGAT1) in hepatitis C infection, beta-catenin in
familial adenomatous polyposis; beta2 adrenergic receptor,
glaucoma; RTP801/Redd1 also known as DAN damage-inducible
transcript 4 protein, in diabetic macular oedma (DME) or
age-related macular degeneration; vascular endothelial growth
factor receptor I (VEGFR1) in age-related macular degeneration or
choroidal neivascularization, caspase 2 in non-arteritic ischaemic
optic neuropathy; Keratin 6A N17K mutant protein in pachyonychia
congenital; influenza A virus genome/gene sequences in influenza
infection; severe acute respiratory syndrome (SARS) coronavirus
genome/gene sequences in SARS infection; respiratory syncytial
virus genome/gene sequences in respiratory syncytial virus
infection; Ebola filovirus genome/gene sequence in Ebola infection;
hepatitis B and C virus genome/gene sequences in hepatitis B and C
infection; herpes simplex virus (HSV) genome/gene sequences in HSV
infection, coxsackievirus B3 genome/gene sequences in
coxsackievirus B3 infection; silencing of a pathogenic allele of a
gene (allele-specific silencing) like torsin A (TOR1A) in primary
dystonia, pan-class I and HLA-allele specific in transplant; or
mutant rhodopsin gene (RHO) in autosomal dominantly inherited
retinitis pigmentosa (adRP).
[0041] As used herein, the term "recombinant," as a modifier of
AAV, such as recombinant AAV-Rh74 and related AAV vectors, as well
as a modifier of sequences such as recombinant polynucleotides and
polypeptides, means that the compositions have been manipulated
(i.e., engineered) in a fashion that generally does not occur in
nature. A particular example of a recombinant AAV would be where a
polynucleotide that is not normally present in the wild-type AAV is
within the AAV particle and/or genome. For example, a particular
example of a recombinant polynucleotide would be where a
polynucleotide (e.g., gene) encoding a protein is cloned into a
vector, with or without 5', 3' and/or intron regions that the gene
is normally associated within the AAV genome. Although the term
"recombinant" is not always used herein in reference to AAV, such
as AAV-Rh74 and related AAV vectors, as well as sequences such as
polynucleotides and polypeptides, recombinant forms of AAV,
AAV-Rh74 and related AAV vectors, and sequences including
polynucleotides and polypeptides, are expressly included in spite
of any such omission.
[0042] Polynucleotide sequences in accordance with the invention
can be inserted into a vector. The term "vector" refers to a
plasmid, virus (e.g., AAV) or other vehicle that can be manipulated
by insertion or incorporation of a polynucleotide. Such vectors can
be used for genetic manipulation (i.e., "cloning vectors"), to
introduce/transfer polynucleotides into cells, and to transcribe or
translate the inserted polynucleotide in cells. A vector generally
contains at least an origin of replication for propagation in a
cell and expression control element (e.g., a promoter). Control
elements, including expression control elements as set forth
herein, present within a vector are included to facilitate proper
transcription and if appropriate translation (e.g., splicing signal
for introns, maintenance of the correct reading frame of the gene
to permit in-frame translation of mRNA and, stop codons etc.).
[0043] Vectors including AAV-Rh74 and related AAV vectors of the
invention can include one or more "expression control elements."
Typically, expression control elements are nucleic acid
sequence(s), such as promoters and enhancers, that influence
expression of an operably linked polynucleotide. Such elements
typically act in cis but may also act in trans.
[0044] Expression control can be effected at the level of
transcription, translation, splicing, message stability, etc.
Typically, an expression control element that modulates
transcription is juxtaposed near the 5' end of the transcribed
polynucleotide (i.e., "upstream"). Expression control elements can
also be located at the 3' end of the transcribed sequence (i.e.,
"downstream") or within the transcript (e.g., in an intron).
Expression control elements can be located at a distance away from
the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to
5000, 5000 to 10,000 or more nucleotides from the polynucleotide),
even at considerable distances. Nevertheless, owing to the
polynucleotide length limitations, for AAV-Rh74 and related AAV
vectors, such expression control elements will typically be within
1 to 1000 nucleotides from the polynucleotide.
[0045] Functionally, expression of the operably linked
polynucleotide is at least in part controllable by the element
(e.g., promoter) such that the element modulates transcription of
the polynucleotide and, as appropriate, translation of the
transcript. A specific example of an expression control element is
a promoter, which is usually located 5' of the transcribed
sequence. Another example of an expression control element is an
enhancer, which can be located 5', 3' of the transcribed sequence,
or within the transcribed sequence.
[0046] Expression control elements and promoters include those
active in a particular tissue or cell type, referred to herein as a
"tissue-specific expression control elements/promoters."
Tissue-specific expression control elements are typically active in
specific cell or tissue (e.g., liver, brain, central nervous
system, spinal cord, eye, retina or lung). Expression control
elements are typically active in these cells, tissues or organs
because they are recognized by transcriptional activator proteins,
or other regulators of transcription, that are unique to a specific
cell, tissue or organ type.
[0047] Expression control elements also include ubiquitous or
promiscuous promoters/enhancers which are capable of driving
expression of a polynucleotide in many different cell types. Such
elements include, but are not limited to the cytomegalovirus (CMV)
immediate early promoter/enhancer sequences, the Rous sarcoma virus
(RSV) promoter/enhancer sequences and the other viral
promoters/enhancers active in a variety of mammalian cell types, or
synthetic elements that are not present in nature.
[0048] Expression control elements also can confer expression in a
manner that is regulatable, that is, a signal or stimuli increases
or decreases expression of the operably linked polynucleotide. A
regulatable element that increases expression of the operably
linked polynucleotide in response to a signal or stimuli is also
referred to as an "inducible element" (i.e., is induced by a
signal). Particular examples include, but are not limited to, a
hormone (e.g., steroid) inducible promoter. A regulatable element
that decreases expression of the operably linked polynucleotide in
response to a signal or stimuli is referred to as a "repressible
element" (i.e., the signal decreases expression such that when the
signal, is removed or absent, expression is increased). Typically,
the amount of increase or decrease conferred by such elements is
proportional to the amount of signal or stimuli present; the
greater the amount of signal or stimuli, the greater the increase
or decrease in expression.
[0049] As used herein, the term "operable linkage" or "operably
linked" refers to a physical or functional juxtaposition of the
components so described as to permit them to function in their
intended manner. In the example of an expression control element in
operable linkage with a polynucleotide, the relationship is such
that the control element modulates expression of the nucleic acid.
More specifically, for example, two DNA sequences operably linked
means that the two DNAs are arranged (cis or trans) in such a
relationship that at least one of the DNA sequences is able to
exert a physiological effect upon the other sequence.
[0050] Vectors including AAV-Rh74 and related AAV vectors of the
invention can include still additional nucleic acid elements. These
elements include, without limitation one or more copies of an AAV
ITR sequence, a promoter/enhancer element, a transcription
termination signal, 5' or 3' untranslated regions (e.g.,
polyadenylation sequences) which flank a polynucleotide sequence,
or all or a portion of intron I. Such elements also optionally
include a transcription termination signal. A particular
non-limiting example of a transcription termination signal is the
SV40 transcription termination signal.
[0051] Inclusion of an intron element may enhance expression
compared with expression in the absence of the intron element
(Kurachi et al., 1995, supra). AAV vectors typically accept inserts
of DNA having a defined size range which is generally about 4 kb to
about 5.2 kb, or slightly more. Thus, for shorter sequences, it may
be necessary to include additional nucleic acid in the insert
fragment in order to achieve the required length which is
acceptable for the AAV vector. Introns and intron fragments (e.g.
portion of intron I of FIX) fulfill this requirement while also
enhancing expression. Thus, the invention is not limited to the
inclusion of intron I sequences in the AAV vector, and include
other introns or other DNA sequences in place of portions of intron
I. Accordingly, other 5' and 3' untranslated regions of nucleic
acid may be used in place of those recited for human FIX,
particularly when polynucleotides encoding proteins other than
human FIX are used in the AAV-Rh74 and related AAV vectors of the
invention.
[0052] A "portion of intron I" as used herein, is meant region of
intron I having a nucleotide length of from about 0.1 kb to about
1.7 kb, which region enhances expression of FIX, typically by about
1.5-fold or more on a plasmid or viral vector template when
compared with expression of FIX in the absence of a portion of
intron I. A more specific portion is a 1.3 kb portion of intron
1.
[0053] Polynucleotides and polypeptides including modified forms
can be made using various standard cloning, recombinant DNA
technology, via cell expression or in vitro translation and
chemical synthesis techniques. Purity of polynucleotides can be
determined through sequencing, gel electrophoresis and the like.
For example, nucleic acids can be isolated using hybridization or
computer-based database screening techniques. Such techniques
include, but are not limited to: (1) hybridization of genomic DNA
or cDNA libraries with probes to detect homologous nucleotide
sequences; (2) antibody screening to detect polypeptides having
shared structural features, for example, using an expression
library; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA
using primers capable of annealing to a nucleic acid sequence of
interest; (4) computer searches of sequence databases for related
sequences; and (5) differential screening of a subtracted nucleic
acid library.
[0054] Polynucleotides and polypeptides including modified forms
can also be produced by chemical synthesis using methods known in
the art, for example, an automated synthesis apparatus (see, e.g.,
Applied Biosystems, Foster City, Calif.). Peptides can be
synthesized, whole or in part, using chemical methods (see, e.g.,
Caruthers (1980). Nucleic Acids Res. Symp. Ser. 215; Horn (1980);
and Banga, A. K., Therapeutic Peptides and Proteins, Formulation,
Processing and Delivery Systems (1995) Technomic Publishing Co.,
Lancaster, Pa.). Peptide synthesis can be performed using various
solid phase techniques (see, e.g., Roberge Science 269:202 (1995);
Merrifield, Methods Enzymol. 289:3 (1997)) and automated synthesis
may be achieved, e.g., using the ABI 431A Peptide Synthesizer
(Perkin Elmer) in accordance with the manufacturer's
instructions.
[0055] The term "isolated," when used as a modifier of a
composition, means that the compositions are made by the hand of
man or are separated, completely or at least in part, from their
naturally occurring in vivo environment. Generally, isolated
compositions are substantially free of one or more materials with
which they normally associate with in nature, for example, one or
more protein, nucleic acid, lipid, carbohydrate, cell membrane. The
term "isolated" does not exclude alternative physical forms of the
composition, such as fusions/chimeras, multimers/oligomers,
modifications (e.g., phosphorylation, glycosylation, lipidation) or
derivatized forms, or forms expressed in host cells produced by the
hand of man.
[0056] In accordance with the invention, treatment methods and uses
are provided that include therapeutic methods and uses. Methods and
uses of the invention are broadly applicable to diseases amenable
to treatment by introducing a gene encoding a protein, or
increasing or stimulating gene expression or function, e.g., gene
addition or replacement. Methods and uses of the invention are also
broadly applicable to diseases amenable to treatment by reducing or
decreasing gene expression or function, e.g., gene knockout or
reduction of gene expression.
[0057] Non-limiting particular examples of diseases treatable in
accordance with the invention include those set forth herein as
well as a lung disease (e.g., cystic fibrosis), a blood coagulation
or bleeding disorder (e.g., hemophilia A or hemophilia B with or
without inhibitors), thalassemia, a blood disorder (e.g., anemia),
Alzheimer's disease, Parkinson's disease, Huntington's disease,
amyotrophic lateral sclerosis (ALS), epilepsy, lysosomal storage
diseases, a copper or iron accumulation disorders (e.g., Wilson's
or Menkes disease) lysosomal acid lipase deficiency, a neurological
or neurodegenerative disorder, cancer, type 1 or type 2 diabetes,
Gaucher's disease, Hurler's disease, adenosine deaminase
deficiency, a metabolic defect (e.g., glycogen storage diseases), a
retinal degenerative disease (such as RPE65 deficiency,
choroideremia, and other diseases of the eye), and a disease of a
solid organ (e.g., brain, liver, kidney, heart).
[0058] Thus, in one embodiment, a method of the invention includes:
(a) providing adeno-associated virus (AAV) vector, said vector
comprising a heterologous polynucleotide encoding a protein,
wherein the heterologous polynucleotide sequence is operably linked
to an expression control element conferring transcription of said
polynucleotide sequence; and (b) administering an amount of the AAV
vector to the mammal wherein said protein is expressed in the
mammal. In particular aspects, expression of the protein provides a
therapeutic benefit to the mammal
[0059] Methods of the invention include treatment methods, which
result in any therapeutic or beneficial effect. In various methods
embodiments, an methods of the invention further include
inhibiting, decreasing or reducing one or more adverse (e.g.,
physical) symptoms, disorders, illnesses, diseases or complications
caused by or associated with the disease, such as reduced blood
clotting time, reduced administration dosage of supplemental
clotting factor protein.
[0060] A therapeutic or beneficial effect of treatment is therefore
any objective or subjective measurable or detectable improvement or
benefit provided to a particular subject. A therapeutic or
beneficial effect can but need not be complete ablation of all or
any particular adverse symptom, disorder, illness, or complication
of a disease. Thus, a satisfactory clinical endpoint is achieved
when there is an incremental improvement or a partial reduction in
an adverse symptom, disorder, illness, or complication caused by or
associated with a disease, or an inhibition, decrease, reduction,
suppression, prevention, limit or control of worsening or
progression of one or more adverse symptoms, disorders, illnesses,
or complications caused by or associated with the disease, over a
short or long duration (hours, days, weeks, months, etc.).
[0061] Compositions, methods and uses of the invention, can be
administered in a sufficient or effective amount to a subject in
need thereof. An "effective amount" or "sufficient amount" refers
to an amount that provides, in single or multiple doses, alone or
in combination, with one or more other compositions (therapeutic
agents such as a drug), treatments, protocols, or therapeutic
regimens agents, a detectable response of any duration of time
(long or short term), an expected or desired outcome in or a
benefit to a subject of any measurable or detectable degree or for
any duration of time (e.g., for minutes, hours, days, months,
years, or cured).
[0062] The AAV vector dose to achieve a therapeutic effect, e.g.,
the dose in vector genomes/per kilogram of body weight (vg/kg),
will vary based on several factors including, but not limited to:
route of administration, the level of heterologous polynucleotide
expression required to achieve a therapeutic effect, the specific
disease treated, any host immune response to the AAV vector, a host
immune response to the heterologous polynucleotide or expression
product (protein), and the stability of the protein expressed. One
skilled in the art can readily determine a AAV virion dose range to
treat a patient having a particular disease or disorder based on
the aforementioned factors, as well as other factors. Generally,
doses will range from at least 1.times.10.sup.8, or more, for
example, 1.times.10.sup.9, 1.times.10.sup.10, 1.times.10.sup.11,
1.times.10.sup.12, 1.times.10.sup.13 or 1.times.10.sup.14, or more,
vector genomes per kilogram (vg/kg) of the weight of the subject,
to achieve a therapeutic effect.
[0063] Using hemophilia as an example, generally speaking, it is
believed that, in order to achieve a therapeutic effect, a blood
coagulation factor concentration that is greater than 1% of factor
concentration found in a normal individual is needed to change a
severe disease phenotype to a moderate one. A severe phenotype is
characterized by joint damage and life-threatening bleeds. To
convert a moderate disease phenotype into a mild one, it is
believed that a blood coagulation factor concentration greater than
5% of normal is needed. With respect to treating such a hemophilic
subject, a typical dose is at least 1.times.10.sup.10 vector
genomes (vg) per kilogram (vg/kg) of the weight of the subject, or
between about 1.times.10.sup.10 to 1.times.10.sup.11 vg/kg of the
weight of the subject, or between about 1.times.10.sup.11 to
1.times.10.sup.12 vg/kg of the weight of the subject, or between
about 1.times.10.sup.12 to 1.times.10.sup.13 vg/kg of the weight of
the subject, to achieve a desired therapeutic effect.
[0064] The doses of an "effective amount" or "sufficient amount"
for treatment (e.g., to ameliorate or to provide a therapeutic
benefit or improvement) typically are effective to provide a
response to one, multiple or all adverse symptoms, consequences or
complications of the disease, one or more adverse symptoms,
disorders, illnesses, pathologies, or complications, for example,
caused by or associated with the disease, to a measurable extent,
although decreasing, reducing, inhibiting, suppressing, limiting or
controlling progression or worsening of the disease is a
satisfactory outcome.
[0065] An effective amount or a sufficient amount can but need not
be provided in a single administration, may require multiple
administrations, and, can but need not be, administered alone or in
combination with another composition (e.g., agent), treatment,
protocol or therapeutic regimen. For example, the amount may be
proportionally increased as indicated by the need of the subject,
type, status and severity of the disease treated or side effects
(if any) of treatment. In addition, an effective amount or a
sufficient amount need not be effective or sufficient if given in
single or multiple doses without a second composition (e.g.,
another drug or agent), treatment, protocol or therapeutic regimen,
since additional doses, amounts or duration above and beyond such
doses, or additional compositions (e.g., drugs or agents),
treatments, protocols or therapeutic regimens may be included in
order to be considered effective or sufficient in a given subject.
Amounts considered effective also include amounts that result in a
reduction of the use of another treatment, therapeutic regimen or
protocol, such as administration of recombinant clotting factor
protein for treatment of a clotting disorder (e.g., hemophilia A or
B).
[0066] An effective amount or a sufficient amount need not be
effective in each and every subject treated, or a majority of
treated subjects in a given group or population. An effective
amount or a sufficient amount means effectiveness or sufficiency in
a particular subject, not a group or the general population. As is
typical for such methods, some subjects will exhibit a greater
response, or less or no response to a given treatment method or
use. Thus, appropriate amounts will depend upon the condition
treated, the therapeutic effect desired, as well as the individual
subject (e.g., the bioavailability within the subject, gender, age,
etc.).
[0067] The term "ameliorate" means a detectable or measurable
improvement in a subject's disease or symptom thereof, or an
underlying cellular response. A detectable or measurable
improvement includes a subjective or objective decrease, reduction,
inhibition, suppression, limit or control in the occurrence,
frequency, severity, progression, or duration of the disease, or
complication caused by or associated with the disease, or an
improvement in a symptom or an underlying cause or a consequence of
the disease, or a reversal of the disease.
[0068] Thus, a successful treatment outcome can lead to a
"therapeutic effect," or "benefit" of decreasing, reducing,
inhibiting, suppressing, limiting, controlling or preventing the
occurrence, frequency, severity, progression, or duration of a
disease, or one or more adverse symptoms or underlying causes or
consequences of the disease in a subject. Treatment methods and
uses affecting one or more underlying causes of the disease or
adverse symptoms are therefore considered to be beneficial. A
decrease or reduction in worsening, such as stabilizing the
disease, or an adverse symptom thereof, is also a successful
treatment outcome.
[0069] A therapeutic benefit or improvement therefore need not be
complete ablation of the disease, or any one, most or all adverse
symptoms, complications, consequences or underlying causes
associated with the disease. Thus, a satisfactory endpoint is
achieved when there is an incremental improvement in a subject's
disease, or a partial decrease, reduction, inhibition, suppression,
limit, control or prevention in the occurrence, frequency,
severity, progression, or duration, or inhibition or reversal, of
the disease (e.g., stabilizing one or more symptoms or
complications), over a short or long duration of time (hours, days,
weeks, months, etc.). Effectiveness of a method or use, such as a
treatment that provides a potential therapeutic benefit or
improvement of a disease, can be ascertained by various
methods.
[0070] Invention methods and uses can be combined with any
compound, agent, drug, treatment or other therapeutic regimen or
protocol having a desired therapeutic, beneficial, additive,
synergistic or complementary activity or effect. Exemplary
combination compositions and treatments include second actives,
such as, biologics (proteins), agents and drugs. Such biologics
(proteins), agents, drugs, treatments and therapies can be
administered or performed prior to, substantially contemporaneously
with or following any other method or use of the invention, for
example, a therapeutic method of treating a subject for a blood
clotting disease.
[0071] The compound, agent, drug, treatment or other therapeutic
regimen or protocol can be administered as a combination
composition, or administered separately, such as concurrently or in
series or sequentially (prior to or following) an AAV vector of the
invention. The invention therefore provides combinations in which a
method or use of the invention is in a combination with any
compound, agent, drug, therapeutic regimen, treatment protocol,
process, remedy or composition, set forth herein or known to one of
skill in the art. The compound, agent, drug, therapeutic regimen,
treatment protocol, process, remedy or composition can be
administered or performed prior to, substantially contemporaneously
with or following administration of an AAV vector of the invention,
to a subject. Specific non-limiting examples of combination
embodiments therefore include the foregoing or other compound,
agent, drug, therapeutic regimen, treatment protocol, process,
remedy or composition.
[0072] Methods and uses of the invention also include, among other
things, methods and uses that result in a reduced need or use of
another compound, agent, drug, therapeutic regimen, treatment
protocol, process, or remedy. For example, for a blood clotting
disease, a method or use of the invention has a therapeutic benefit
if in a given subject a less frequent or reduced dose or
elimination of administration of a recombinant clotting factor
protein to supplement for the deficient or defective (abnormal or
mutant) endogenous clotting factor in the subject. Thus, in
accordance with the invention, methods and uses of reducing need or
use of another treatment or therapy are provided.
[0073] The term "subject" refers to an animal, typically a mammal,
such as humans, non-human primates (apes, gibbons, gorillas,
chimpanzees, orangutans, macaques), a domestic animal (dogs and
cats), a farm animal (poultry such as chickens and ducks, horses,
cows, goats, sheep, pigs), and experimental animals (mouse, rat,
rabbit, guinea pig). Subjects include animal disease models, for
example, mouse and other animal models of blood clotting diseases
and others known to those of skill in the art.
[0074] Subjects appropriate for treatment in accordance with the
invention include those having or at risk of producing an
insufficient amount or having a deficiency in a functional gene
product (protein), or produce an aberrant, partially functional or
non-functional gene product (protein), which can lead to disease.
Subjects appropriate for treatment in accordance with the invention
also include those having or at risk of producing an aberrant, or
defective (mutant) gene product (protein) that leads to a disease
such that reducing amounts, expression or function of the aberrant,
or defective (mutant) gene product (protein) would lead to
treatment of the disease, or reduce one or more symptoms or
ameliorate the disease. Target subjects therefore include subjects
that have such defects regardless of the disease type, timing or
degree of onset, progression, severity, frequency, or type or
duration of the symptoms.
[0075] Subjects appropriate for treatment in accordance with the
invention also include those having or at risk of producing
antibodies against AAV. AAV vectors can be administered or
delivered to such subjects using several techniques. For example,
empty capsid AAV (i.e., AAV lacking a heterologous polynucleotide)
can be delivered to bind to the AAV antibodies thereby allowing the
AAV vector bearing the heterologous polynucleotide to transform
cells of the subject. Amounts of empty capsid AAV to administer can
be calibrated based upon the amount of AAV antibodies produced in a
particular subject. Alternatively or in addition to, AAV vector can
be delivered by direct intramuscular injection (e.g., one or more
slow-twitch fibers of a muscle). In another alternative, a catheter
introduced into the femoral artery can be used to delivery AAV
vectors to liver via the hepatic artery. Non-surgical means can
also be employed, such as endoscopic retrograde
cholangiopancreatography (ERCP), to deliver AAV vectors directly to
the liver, thereby bypassing the bloodstream and AAV antibodies.
Other ductal systems, such as the ducts of the submandibular gland,
can also be used as portals for delivering AAV vectors into a
subject with preexisting anti-AAV antibodies.
[0076] "Prophylaxis" and grammatical variations thereof mean a
method in which contact, administration or in vivo delivery to a
subject is prior to disease. Administration or in vivo delivery to
a subject can be performed prior to development of an adverse
symptom, condition, complication, etc. caused by or associated with
the disease. For example, a screen (e.g., genetic) can be used to
identify such subjects as candidates for invention methods and
uses, but the subject may not manifest the disease. Such subjects
therefore include those screened positive for an insufficient
amount or a deficiency in a functional gene product (protein), or
produce an aberrant, partially functional or non-functional gene
product (protein), which can lead to disease; and subjects that
screen positive for an aberrant, or defective (mutant) gene product
(protein) that leads to disease, even though such subjects do not
manifest symptoms of the disease.
[0077] Methods and uses of the invention include delivery and
administration systemically, regionally or locally, or by any
route, for example, by injection, infusion, orally (e.g., ingestion
or inhalation), or topically (e.g., transdermally). Such delivery
and administration include intravenously, intramuscularly,
intraperitoneally, intradermally, subcutaneously, intracavity,
intracranially, transdermally (topical), parenterally, e.g.
transmucosally or rectally. Exemplary administration and delivery
routes include intravenous (i.v.), intraperitoneal (i.p.),
intrarterial, intramuscular, parenteral, subcutaneous,
intra-pleural, topical, dermal, intradermal, transdermal,
parenterally, e.g. transmucosal, intra-cranial, intra-spinal, oral
(alimentary), mucosal, respiration, intranasal, intubation,
intrapulmonary, intrapulmonary instillation, buccal, sublingual,
intravascular, intrathecal, intracavity, iontophoretic,
intraocular, ophthalmic, optical, intraglandular, intraorgan,
intralymphatic.
[0078] Doses for delivery and administration can be based upon
current existing protocols, empirically determined, using animal
disease models or optionally in human clinical trials. Initial
study doses can be based upon animal studies set forth herein, for
a mouse or dog, for example.
[0079] Doses can vary and depend upon whether the treatment is
prophylactic or therapeutic, the type, onset, progression,
severity, frequency, duration, or probability of the disease to
which treatment is directed, the clinical endpoint desired,
previous or simultaneous treatments, the general health, age,
gender, race or immunological competency of the subject and other
factors that will be appreciated by the skilled artisan. The dose
amount, number, frequency or duration may be proportionally
increased or reduced, as indicated by any adverse side effects,
complications or other risk factors of the treatment or therapy and
the status of the subject. The skilled artisan will appreciate the
factors that may influence the dosage and timing required to
provide an amount sufficient for providing a therapeutic or
prophylactic benefit.
[0080] Methods and uses of the invention as disclosed herein can be
practiced within 1-2, 2-4, 4-12, 12-24 or 24-72 hours after a
subject has been identified as having the disease targeted for
treatment, has one or more symptoms of the disease, or has been
screened and is identified as positive as set forth herein even
though the subject does not have one or more symptoms of the
disease. Of course, methods and uses of the invention can be
practiced 1-7, 7-14, 14-21, 21-48 or more days, months or years
after a subject has been identified as having the disease targeted
for treatment, has one or more symptoms of the disease, or has been
screened and is identified as positive as set forth herein.
[0081] AAV vectors and other compositions, agents, drugs, biologics
(proteins) can be incorporated into pharmaceutical compositions,
e.g., a pharmaceutically acceptable carrier or excipient. Such
pharmaceutical compositions are useful for, among other things,
administration and delivery to a subject in vivo or ex vivo.
[0082] As used herein the term "pharmaceutically acceptable" and
"physiologically acceptable" mean a biologically acceptable
formulation, gaseous, liquid or solid, or mixture thereof, which is
suitable for one or more routes of administration, in vivo delivery
or contact. Such formulations include solvents (aqueous or
non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g.,
oil-in-water or water-in-oil), suspensions, syrups, elixirs,
dispersion and suspension media, coatings, isotonic and absorption
promoting or delaying agents, compatible with pharmaceutical
administration or in vivo contact or delivery. Aqueous and
non-aqueous solvents, solutions and suspensions may include
suspending agents and thickening agents. Such pharmaceutically
acceptable carriers include tablets (coated or uncoated), capsules
(hard or soft), microbeads, powder, granules and crystals.
Supplementary active compounds (e.g., preservatives, antibacterial,
antiviral and antifungal agents) can also be incorporated into the
compositions.
[0083] Pharmaceutical compositions can be formulated to be
compatible with a particular route of administration or delivery,
as set forth herein or known to one of skill in the art. Thus,
pharmaceutical compositions include carriers, diluents, or
excipients suitable for administration by various routes.
[0084] Formulations suitable for parenteral administration comprise
aqueous and non-aqueous solutions, suspensions or emulsions of the
active compound, which preparations are typically sterile and can
be isotonic with the blood of the intended recipient. Non-limiting
illustrative examples include water, saline, dextrose, fructose,
ethanol, animal, vegetable or synthetic oils.
[0085] For transmucosal or transdermal administration (e.g.,
topical contact), penetrants can be included in the pharmaceutical
composition. Penetrants are known in the art, and include, for
example, for transmucosal administration, detergents, bile salts,
and fusidic acid derivatives. For transdermal administration, the
active ingredient can be formulated into aerosols, sprays,
ointments, salves, gels, or creams as generally known in the art.
For contact with skin, pharmaceutical compositions typically
include ointments, creams, lotions, pastes, gels, sprays, aerosols,
or oils. Carriers which may be used include Vaseline, lanolin,
polyethylene glycols, alcohols, transdermal enhancers, and
combinations thereof.
[0086] Cosolvents and adjuvants may be added to the formulation.
Non-limiting examples of cosolvents contain hydroxyl groups or
other polar groups, for example, alcohols, such as isopropyl
alcohol; glycols, such as propylene glycol, polyethyleneglycol,
polypropylene glycol, glycol ether; glycerol; polyoxyethylene
alcohols and polyoxyethylene fatty acid esters. Adjuvants include,
for example, surfactants such as, soya lecithin and oleic acid;
sorbitan esters such as sorbitan trioleate; and
polyvinylpyrrolidone.
[0087] Pharmaceutical formulations and delivery systems appropriate
for the compositions and methods of the invention are known in the
art (see, e.g., Remington: The Science and Practice of Pharmacy
(2003) 20.sup.th ed., Mack Publishing Co., Easton, Pa.; Remington's
Pharmaceutical Sciences (1990) 18.sup.th ed., Mack Publishing Co.,
Easton, Pa.; The Merck Index (1996) 12.sup.th ed., Merck Publishing
Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage
Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel
and Stoklosa, Pharmaceutical Calculations (2001) 11.sup.th ed.,
Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et
al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford,
N.Y., pp. 253-315).
[0088] A "unit dosage form" as used herein refers to physically
discrete units suited as unitary dosages for the subject to be
treated; each unit containing a predetermined quantity optionally
in association with a pharmaceutical carrier (excipient, diluent,
vehicle or filling agent) which, when administered in one or more
doses, is calculated to produce a desired effect (e.g.,
prophylactic or therapeutic effect). Unit dosage forms may be
within, for example, ampules and vials, which may include a liquid
composition, or a composition in a freeze-dried or lyophilized
state; a sterile liquid carrier, for example, can be added prior to
administration or delivery in vivo. Individual unit dosage forms
can be included in multi-dose kits or containers. Pharmaceutical
formulations can be packaged in single or multiple unit dosage form
for ease of administration and uniformity of dosage.
[0089] The invention provides kits with packaging material and one
or more components therein. A kit typically includes a label or
packaging insert including a description of the components or
instructions for use in vitro, in vivo, or ex vivo, of the
components therein. A kit can contain a collection of such
components, e.g., an AAV vector and optionally a second active,
such as another compound, agent, drug or composition.
[0090] The term "packaging material" refers to a physical structure
housing the components of the kit. The packaging material can
maintain the components sterilely, and can be made of material
commonly used for such purposes (e.g., paper, corrugated fiber,
glass, plastic, foil, ampules, vials, tubes, etc.).
[0091] Labels or inserts can include identifying information of one
or more components therein, dose amounts, clinical pharmacology of
the active ingredient(s) including mechanism of action,
pharmacokinetics and pharmacodynamics. Labels or inserts can
include information identifying manufacturer, lot numbers,
manufacture location and date, expiration dates. Labels or inserts
can include information identifying manufacturer information, lot
numbers, manufacturer location and date. Labels or inserts can
include information on a disease for which a kit component may be
used. Labels or inserts can include instructions for the clinician
or subject for using one or more of the kit components in a method,
use, or treatment protocol or therapeutic regimen. Instructions can
include dosage amounts, frequency or duration, and instructions for
practicing any of the methods, uses, treatment protocols or
prophylactic or therapeutic regimes described herein.
[0092] Labels or inserts can include information on any benefit
that a component may provide, such as a prophylactic or therapeutic
benefit. Labels or inserts can include information on potential
adverse side effects, complications or reactions, such as warnings
to the subject or clinician regarding situations where it would not
be appropriate to use a particular composition. Adverse side
effects or complications could also occur when the subject has,
will be or is currently taking one or more other medications that
may be incompatible with the composition, or the subject has, will
be or is currently undergoing another treatment protocol or
therapeutic regimen which would be incompatible with the
composition and, therefore, instructions could include information
regarding such incompatibilities.
[0093] Labels or inserts include "printed matter," e.g., paper or
cardboard, or separate or affixed to a component, a kit or packing
material (e.g., a box), or attached to an ampule, tube or vial
containing a kit component. Labels or inserts can additionally
include a computer readable medium, such as a bar-coded printed
label, a disk, optical disk such as CD- or DVD-ROM/RAM, DVD, MP3,
magnetic tape, or an electrical storage media such as RAM and ROM
or hybrids of these such as magnetic/optical storage media, FLASH
media or memory type cards.
[0094] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described herein.
[0095] All applications, publications, patents and other
references, GenBank citations and ATCC citations cited herein are
incorporated by reference in their entirety. In case of conflict,
the specification, including definitions, will control.
[0096] All of the features disclosed herein may be combined in any
combination. Each feature disclosed in the specification may be
replaced by an alternative feature serving a same, equivalent, or
similar purpose. Thus, unless expressly stated otherwise, disclosed
features (e.g., compound structures) are an example of a genus of
equivalent or similar features.
[0097] As used herein, the singular forms "a", "and," and "the"
include plural referents unless the context clearly indicates
otherwise. Thus, for example, reference to "a polynucleotide"
includes a plurality of such polynucleotides, such as a plurality
of genes.
[0098] As used herein, all numerical values or numerical ranges
include integers within such ranges and fractions of the values or
the integers within ranges unless the context clearly indicates
otherwise. Thus, to illustrate, reference to at least 90% identity,
includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, etc., as well
as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%,
92.4%, 92.5%, etc., and so forth.
[0099] Reference to a number with more (greater) or less than
includes any number greater or less than the reference number,
respectively. Thus, for example, a reference to less than 1,000,
includes 999, 998, 997, etc. all the way down to the number one
(1); and less than 100, includes 99, 98, 97, etc. all the way down
to the number one (1).
[0100] As used herein, all numerical values or ranges include
fractions of the values and integers within such ranges and
fractions of the integers within such ranges unless the context
clearly indicates otherwise. Thus, to illustrate, reference to a
numerical range, such as a percentage range, 90-100%, includes 91%,
92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%,
91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so
forth. Reference to a range of 1-50 therefore includes 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., as
well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5,
etc., and so forth.
[0101] Reference to a series of ranges includes ranges which
combine the values of the boundaries of different ranges within the
series. Thus, to illustrate reference to a series of ranges of 2-72
hours, 2-48 hours, 4-24 hours, 4-18 hours and 6-12 hours, includes
ranges of 2-6 hours, 2, 12 hours, 2-18 hours, 2-24 hours, etc., and
4-27 hours, 4-48 hours, 4-6 hours, etc.
[0102] The invention is generally disclosed herein using
affirmative language to describe the numerous embodiments and
aspects. The invention also specifically includes embodiments in
which particular subject matter is excluded, in full or in part,
such as substances or materials, method steps and conditions,
protocols, or procedures. For example, in certain embodiments or
aspects of the invention, materials and/or method steps are
excluded. Thus, even though the invention is generally not
expressed herein in terms of what the invention does not include
aspects that are not expressly excluded in the invention are
nevertheless disclosed herein.
[0103] A number of embodiments of the invention have been
described. Nevertheless, one skilled in the art, without departing
from the spirit and scope of the invention, can make various
changes and modifications of the invention to adapt it to various
usages and conditions. Accordingly, the following examples are
intended to illustrate but not limit the scope of the invention
claimed.
EXAMPLES
Example 1
[0104] This example includes a description of various materials and
methods.
[0105] Mice:
[0106] Male C57BL/6J (WT) mice 8-10 weeks of age, n=5 per
experimental group. The dog is a HB dog from the University of
North Carolina Chapel Hill colony carrying a missense mutation in
the FIX gene (Evans et al., Proc Natl Acad Sci USA 86:10095
(1989)).
[0107] AAV Vector Constructs:
[0108] The in vivo studies in mice were performed using a construct
expressing human FIX under the control of the ApoE-hAAT liver
specific promoter. The study in dogs used a nearly identical
promoter and the canine FIX transgene.
[0109] Gene Transfer Methodology:
[0110] All vectors were delivered intravenously. In mice via the
tail vein (a volume of 200 microliters per mouse was administered,
vector was diluted in PBS). In dogs the vector was delivered via
the saphenous vein.
[0111] FIX Expression Determination:
[0112] ELISA was used to measure FIX levels. In mice, the human FIX
ELISA antibody pair (capture and secondary) is from Affinity
Biologicals. In dogs, an antibody pair also from Affinity
Biologicals was used as described in Haurigot et al. (Mol Ther
18:1318 (2010)).
[0113] Statistical Analysis:
[0114] Statistical analysis was performed with unpaired, two tailed
t test. p values<0.05 were considered statistically
significant.
[0115] AAV Antibody Measurements:
[0116] An in vitro neutralization assay described in Manno et al.,
(Nat Med 12:342 (2006)) and Mingozzi et al. (Nat Med 13:419 (2007))
was used for antibody measurement. In brief, two AAV vector
constructs were used in the assay, a single-stranded vector
expressing .beta.-galactosidase under the control of the CMV
promoter (ssAAV-LacZ), or a self-complementary vector expressing
the Renilla reporter gene, AAV-Rh74-CBA-Renilla, under the control
of the chicken .beta.-actin promoter (CBA). To increase the
efficiency of transduction of AAV vectors in vitro, 2V6.11 cells
(ATCC) were used, which expressed the adenoviral gene E4 under the
control of an inducible promoter. Cells were seeded in a 96-well
plate at a density of 1.25.times.10.sup.4 cells/well and a 1:1000
dilution of ponasterone A (Invitrogen) was added to the medium to
induce E4 expression. At the day of assay, serial half-log
dilutions of heat-inactivated test serum were mixed with medium
containing virus. For the ssAAV-LacZ vector, virus concentration
used in the assay was .about.1.times.10.sup.10 vg/ml for AAV2 and
.about.5.5.times.10.sup.10 vg/ml for AAV5, 6, or 8. For the
scAAV-Luc vector, virus concentration in the assay was between
.about.50 and 150-fold lower. Residual activity of the reporter
transgene was measured using either a colorimetric assay
(ssAAV-LacZ) or a luminometer (scAAV-Luc).
[0117] Anti-AAV capsid total IgG or Ig subclasses were measured
with a capture assay; ELISA plates were coated with
5.times.10.sup.10 capsid particles/ml of AAV empty capsids. Plates
were blocked with 2% BSA, 0.05% Tween 20 in PBS for 2 hours at room
temperature and serial dilutions of samples were loaded onto the
wells and incubated overnight at 4.degree. C. Biotin-conjugated
anti-human IgG1, IgG2, IgG3, IgG4, or IgM antibody (Sigma) were
used as detecting antibodies; streptavidin-HRP was the added for
substrate detection. Ig concentration was determined against
standard curves made with serial dilution of human purified IgG1,
IgG2, IgG3, IgG4, or IgM (Sigma).
[0118] AAV Production:
[0119] The process for vector production is described in detail in
Ayuso et al. (Gene Ther 17:503 (2010)).
Example 2
[0120] This example includes a description of human FIX gene
transfer animal (Mice) studies and FIX expression after gene
transfer.
[0121] C57BL/6 mice (n=5 per group) were injected via the tail vein
with AAV vectors bearing the Factor IX (FIX) gene (2.5.sup.10
vector genomes per mouse) under the control of a liver-specific
promoter. Human FIX transgene product (protein) plasma levels in
the mice were determined by ELISA at week 1, 2, and 4 post gene
transfer, and are illustrated in FIG. 1. AAV-Rh74 showed the
highest level of transgene expression in the animals.
Example 3
[0122] This example includes a description of animal studies and
data demonstrating effective AAV-Rh74 mediated delivery of FIX at
therapeutic levels in hemophilia dogs.
[0123] In brief, hemophilia B dogs were infused intravenously (IV)
though the saphenous vein with 3.times.10.sup.12 vector genomes per
kg of weight. Expression of the therapeutic FIX transgene was
driven by a liver specific promoter. Vectors and FIX levels were
monitored by ELISA. Canine FIX plasma levels are shown in FIG. 2.
AAV-Rh74 and AAV8 performed roughly equally in hemophilia B dogs,
and both were superior to AAV6.
Example 4
[0124] This example includes a description of studies showing the
presence of anti-AAV neutralizing antibodies (NAb) in humans.
[0125] The data in Table 1 show anti-AAV neutralizing antibodies
(NAb) measured in humans with an in vitro assay. Subjects with a
NAb titer of 1:1 are defined as naive or low-titer for anti-AAV
antibodies, and are eligible for gene transfer for that AAV
serotype (highlighted in grey). AAV-Rh74 exhibited the lowest
prevalence of anti-AAV Nab compared to AAV-2 and AAV-8.
TABLE-US-00001 TABLE 1 ##STR00001##
Example 5
[0126] This example includes a description of data showing
production amounts of different AAV serotypes including
AAV-Rh74.
[0127] The data in Table 2 show production yield of different AAV
serotypes. Reported are the virus batch size in roller bottles, the
total vector yield, and the yield per bottle. All serotypes were
packaged with the same expression cassette. AAV-Rh74 has a yield
comparable to or greater than the other serotypes evaluated, namely
AAV-8, AAV-dj, and AAV-2.
TABLE-US-00002 TABLE 2 Total vector Yield per roller Batch size
yield bottle (number of roller (vector (vector Serotype bottles)
genomes) genomes) AAV-Rh74 80 1.21E+15 1.51E+13 AAV-Rh74 10
1.23E+14 1.23E+13 AAV-8 30 2.54E+14 8.47E+12 AAV-dj 20 1.79E+14
8.95E+12 AAV-2 30 1.38E+14 4.60E+12
Example 6
[0128] This example includes a description of data showing that
AAVrh74 vector expressing human Factor IX (FIX) under the control
of a liver-specific promoter administered to rhesus macaques led to
production amounts of FIX in animals, and at higher levels than
AAV8 vector administered at the same amount.
[0129] In brief, animals were administered either AAV8 or AAVrh74
at a dose of 2.times.10.sup.12 vector genomes (vg)/kg of weight.
Vectors were either formulated in saline or in a mixture of vector
and empty AAV capsid (denoted EC).
[0130] FIG. 4 is a histogram plot of the average (weeks 2 to 8) and
standard error or the mean of human FIX measured by an ELISA that
detects specifically human FIX in rhesus macaque plasma. Animals
receiving the AAVrh74-FIX vector are shown in the last two bars
towards the right margin. The data shows that animals receiving the
AAVrh74 vectors (last two bars towards right margin) expressed the
FIX transgene at higher levels compared to the other groups of
animals injected at the same dose (black and grey bars). Average
levels were compared using unpaired, two-tailed student t test.
Sequence CWU 1
1
41738PRTAdeno-associated virus 1Met Ala Ala Asp Gly Tyr Leu Pro Asp
Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp
Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln
Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys
Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val
Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70
75 80 Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His
Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser
Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys
Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Ser Pro Val Lys
Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Pro Ser Pro Gln
Arg Ser Pro Asp Ser Ser Thr Gly Ile 145 150 155 160 Gly Lys Lys Gly
Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln 165 170 175 Thr Gly
Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro 180 185 190
Pro Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly 195
200 205 Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly
Ser 210 215 220 Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly
Asp Arg Val 225 230 235 240 Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu
Pro Thr Tyr Asn Asn His 245 250 255 Leu Tyr Lys Gln Ile Ser Asn Gly
Thr Ser Gly Gly Ser Thr Asn Asp 260 265 270 Asn Thr Tyr Phe Gly Tyr
Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn 275 280 285 Arg Phe His Cys
His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn 290 295 300 Asn Asn
Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn 305 310 315
320 Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala
325 330 335 Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu
Tyr Gln 340 345 350 Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys
Leu Pro Pro Phe 355 360 365 Pro Ala Asp Val Phe Met Ile Pro Gln Tyr
Gly Tyr Leu Thr Leu Asn 370 375 380 Asn Gly Ser Gln Ala Val Gly Arg
Ser Ser Phe Tyr Cys Leu Glu Tyr 385 390 395 400 Phe Pro Ser Gln Met
Leu Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr 405 410 415 Asn Phe Glu
Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser 420 425 430 Leu
Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu 435 440
445 Ser Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Gln Gln Leu Leu
450 455 460 Phe Ser Gln Ala Gly Pro Asn Asn Met Ser Ala Gln Ala Lys
Asn Trp 465 470 475 480 Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val
Ser Thr Thr Leu Ser 485 490 495 Gln Asn Asn Asn Ser Asn Phe Ala Trp
Thr Gly Ala Thr Lys Tyr His 500 505 510 Leu Asn Gly Arg Asp Ser Leu
Val Asn Pro Gly Val Ala Met Ala Thr 515 520 525 His Lys Asp Asp Glu
Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met 530 535 540 Phe Gly Lys
Gln Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Ser Val 545 550 555 560
Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr 565
570 575 Glu Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gln Gln Asn Ala
Ala 580 585 590 Pro Ile Val Gly Ala Val Asn Ser Gln Gly Ala Leu Pro
Gly Met Val 595 600 605 Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro
Ile Trp Ala Lys Ile 610 615 620 Pro His Thr Asp Gly Asn Phe His Pro
Ser Pro Leu Met Gly Gly Phe 625 630 635 640 Gly Leu Lys His Pro Pro
Pro Gln Ile Leu Ile Lys Asn Thr Pro Val 645 650 655 Pro Ala Asp Pro
Pro Thr Thr Phe Asn Gln Ala Lys Leu Ala Ser Phe 660 665 670 Ile Thr
Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu 675 680 685
Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr 690
695 700 Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr
Glu 705 710 715 720 Gly Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg
Tyr Leu Thr Arg 725 730 735 Asn Leu 22217DNAAdeno-associated virus
2atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc
60gagtggtggg acctgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac
120aacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa
cggactcgac 180aagggggagc ccgtcaacgc ggcggacgca gcggccctcg
agcacgacaa ggcctacgac 240cagcagctcc aagcgggtga caatccgtac
ctgcggtata atcacgccga cgccgagttt 300caggagcgtc tgcaagaaga
tacgtctttt gggggcaacc tcgggcgcgc agtcttccag 360gccaaaaagc
gggttctcga acctctgggc ctggttgaat cgccggttaa gacggctcct
420ggaaagaaga gaccggtaga gccatcaccc cagcgctctc cagactcctc
tacgggcatc 480ggcaagaaag gccagcagcc cgcaaaaaag agactcaatt
ttgggcagac tggcgactca 540gagtcagtcc ccgaccctca accaatcgga
gaaccaccag caggcccctc tggtctggga 600tctggtacaa tggctgcagg
cggtggcgct ccaatggcag acaataacga aggcgccgac 660ggagtgggta
gttcctcagg aaattggcat tgcgattcca catggctggg cgacagagtc
720atcaccacca gcacccgcac ctgggccctg cccacctaca acaaccacct
ctacaagcaa 780atctccaacg ggacctcggg aggaagcacc aacgacaaca
cctacttcgg ctacagcacc 840ccctgggggt attttgactt caacagattc
cactgccact tttcaccacg tgactggcag 900cgactcatca acaacaactg
gggattccgg cccaagaggc tcaacttcaa gctcttcaac 960atccaagtca
aggaggtcac gcagaatgaa ggcaccaaga ccatcgccaa taaccttacc
1020agcacgattc aggtctttac ggactcggaa taccagctcc cgtacgtgct
cggctcggcg 1080caccagggct gcctgcctcc gttcccggcg gacgtcttca
tgattcctca gtacgggtac 1140ctgactctga acaatggcag tcaggctgtg
ggccggtcgt ccttctactg cctggagtac 1200tttccttctc aaatgctgag
aacgggcaac aactttgaat tcagctacaa cttcgaggac 1260gtgcccttcc
acagcagcta cgcgcacagc cagagcctgg accggctgat gaaccctctc
1320atcgaccagt acttgtacta cctgtcccgg actcaaagca cgggcggtac
tgcaggaact 1380cagcagttgc tattttctca ggccgggcct aacaacatgt
cggctcaggc caagaactgg 1440ctacccggtc cctgctaccg gcagcaacgc
gtctccacga cactgtcgca gaacaacaac 1500agcaactttg cctggacggg
tgccaccaag tatcatctga atggcagaga ctctctggtg 1560aatcctggcg
ttgccatggc tacccacaag gacgacgaag agcgattttt tccatccagc
1620ggagtcttaa tgtttgggaa acagggagct ggaaaagaca acgtggacta
tagcagcgtg 1680atgctaacca gcgaggaaga aataaagacc accaacccag
tggccacaga acagtacggc 1740gtggtggccg ataacctgca acagcaaaac
gccgctccta ttgtaggggc cgtcaatagt 1800caaggagcct tacctggcat
ggtgtggcag aaccgggacg tgtacctgca gggtcccatc 1860tgggccaaga
ttcctcatac ggacggcaac tttcatccct cgccgctgat gggaggcttt
1920ggactgaagc atccgcctcc tcagatcctg attaaaaaca cacctgttcc
cgcggatcct 1980ccgaccacct tcaatcaggc caagctggct tctttcatca
cgcagtacag taccggccag 2040gtcagcgtgg agatcgagtg ggagctgcag
aaggagaaca gcaaacgctg gaacccagag 2100attcagtaca cttccaacta
ctacaaatct acaaatgtgg actttgctgt caatactgag 2160ggtacttatt
ccgagcctcg ccccattggc acccgttacc tcacccgtaa tctgtaa
22173601PRTAdeno-associated virus 3Thr Ala Pro Gly Lys Lys Arg Pro
Val Glu Pro Ser Pro Gln Arg Ser 1 5 10 15 Pro Asp Ser Ser Thr Gly
Ile Gly Lys Lys Gly Gln Gln Pro Ala Lys 20 25 30 Lys Arg Leu Asn
Phe Gly Gln Thr Gly Asp Ser Glu Ser Val Pro Asp 35 40 45 Pro Gln
Pro Ile Gly Glu Pro Pro Ala Gly Pro Ser Gly Leu Gly Ser 50 55 60
Gly Thr Met Ala Ala Gly Gly Gly Ala Pro Met Ala Asp Asn Asn Glu 65
70 75 80 Gly Ala Asp Gly Val Gly Ser Ser Ser Gly Asn Trp His Cys
Asp Ser 85 90 95 Thr Trp Leu Gly Asp Arg Val Ile Thr Thr Ser Thr
Arg Thr Trp Ala 100 105 110 Leu Pro Thr Tyr Asn Asn His Leu Tyr Lys
Gln Ile Ser Asn Gly Thr 115 120 125 Ser Gly Gly Ser Thr Asn Asp Asn
Thr Tyr Phe Gly Tyr Ser Thr Pro 130 135 140 Trp Gly Tyr Phe Asp Phe
Asn Arg Phe His Cys His Phe Ser Pro Arg 145 150 155 160 Asp Trp Gln
Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg 165 170 175 Leu
Asn Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Gln Asn 180 185
190 Glu Gly Thr Lys Thr Ile Ala Asn Asn Leu Thr Ser Thr Ile Gln Val
195 200 205 Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr Val Leu Gly Ser
Ala His 210 215 220 Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp Val Phe
Met Ile Pro Gln 225 230 235 240 Tyr Gly Tyr Leu Thr Leu Asn Asn Gly
Ser Gln Ala Val Gly Arg Ser 245 250 255 Ser Phe Tyr Cys Leu Glu Tyr
Phe Pro Ser Gln Met Leu Arg Thr Gly 260 265 270 Asn Asn Phe Glu Phe
Ser Tyr Asn Phe Glu Asp Val Pro Phe His Ser 275 280 285 Ser Tyr Ala
His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile 290 295 300 Asp
Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr Gln Ser Thr Gly Gly Thr 305 310
315 320 Ala Gly Thr Gln Gln Leu Leu Phe Ser Gln Ala Gly Pro Asn Asn
Met 325 330 335 Ser Ala Gln Ala Lys Asn Trp Leu Pro Gly Pro Cys Tyr
Arg Gln Gln 340 345 350 Arg Val Ser Thr Thr Leu Ser Gln Asn Asn Asn
Ser Asn Phe Ala Trp 355 360 365 Thr Gly Ala Thr Lys Tyr His Leu Asn
Gly Arg Asp Ser Leu Val Asn 370 375 380 Pro Gly Val Ala Met Ala Thr
His Lys Asp Asp Glu Glu Arg Phe Phe 385 390 395 400 Pro Ser Ser Gly
Val Leu Met Phe Gly Lys Gln Gly Ala Gly Lys Asp 405 410 415 Asn Val
Asp Tyr Ser Ser Val Met Leu Thr Ser Glu Glu Glu Ile Lys 420 425 430
Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr Gly Val Val Ala Asp Asn 435
440 445 Leu Gln Gln Gln Asn Ala Ala Pro Ile Val Gly Ala Val Asn Ser
Gln 450 455 460 Gly Ala Leu Pro Gly Met Val Trp Gln Asn Arg Asp Val
Tyr Leu Gln 465 470 475 480 Gly Pro Ile Trp Ala Lys Ile Pro His Thr
Asp Gly Asn Phe His Pro 485 490 495 Ser Pro Leu Met Gly Gly Phe Gly
Leu Lys His Pro Pro Pro Gln Ile 500 505 510 Leu Ile Lys Asn Thr Pro
Val Pro Ala Asp Pro Pro Thr Thr Phe Asn 515 520 525 Gln Ala Lys Leu
Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val 530 535 540 Ser Val
Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp 545 550 555
560 Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Thr Asn Val
565 570 575 Asp Phe Ala Val Asn Thr Glu Gly Thr Tyr Ser Glu Pro Arg
Pro Ile 580 585 590 Gly Thr Arg Tyr Leu Thr Arg Asn Leu 595 600
4535PRTAdeno-associated virus 4Met Ala Ala Gly Gly Gly Ala Pro Met
Ala Asp Asn Asn Glu Gly Ala 1 5 10 15 Asp Gly Val Gly Ser Ser Ser
Gly Asn Trp His Cys Asp Ser Thr Trp 20 25 30 Leu Gly Asp Arg Val
Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro 35 40 45 Thr Tyr Asn
Asn His Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly 50 55 60 Gly
Ser Thr Asn Asp Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly 65 70
75 80 Tyr Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp
Trp 85 90 95 Gln Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys
Arg Leu Asn 100 105 110 Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val
Thr Gln Asn Glu Gly 115 120 125 Thr Lys Thr Ile Ala Asn Asn Leu Thr
Ser Thr Ile Gln Val Phe Thr 130 135 140 Asp Ser Glu Tyr Gln Leu Pro
Tyr Val Leu Gly Ser Ala His Gln Gly 145 150 155 160 Cys Leu Pro Pro
Phe Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly 165 170 175 Tyr Leu
Thr Leu Asn Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe 180 185 190
Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn 195
200 205 Phe Glu Phe Ser Tyr Asn Phe Glu Asp Val Pro Phe His Ser Ser
Tyr 210 215 220 Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu
Ile Asp Gln 225 230 235 240 Tyr Leu Tyr Tyr Leu Ser Arg Thr Gln Ser
Thr Gly Gly Thr Ala Gly 245 250 255 Thr Gln Gln Leu Leu Phe Ser Gln
Ala Gly Pro Asn Asn Met Ser Ala 260 265 270 Gln Ala Lys Asn Trp Leu
Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val 275 280 285 Ser Thr Thr Leu
Ser Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly 290 295 300 Ala Thr
Lys Tyr His Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly 305 310 315
320 Val Ala Met Ala Thr His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser
325 330 335 Ser Gly Val Leu Met Phe Gly Lys Gln Gly Ala Gly Lys Asp
Asn Val 340 345 350 Asp Tyr Ser Ser Val Met Leu Thr Ser Glu Glu Glu
Ile Lys Thr Thr 355 360 365 Asn Pro Val Ala Thr Glu Gln Tyr Gly Val
Val Ala Asp Asn Leu Gln 370 375 380 Gln Gln Asn Ala Ala Pro Ile Val
Gly Ala Val Asn Ser Gln Gly Ala 385 390 395 400 Leu Pro Gly Met Val
Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro 405 410 415 Ile Trp Ala
Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro 420 425 430 Leu
Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile 435 440
445 Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Thr Phe Asn Gln Ala
450 455 460 Lys Leu Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val
Ser Val 465 470 475 480 Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser
Lys Arg Trp Asn Pro 485 490 495 Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr
Lys Ser Thr Asn Val Asp Phe 500 505 510 Ala Val Asn Thr Glu Gly Thr
Tyr Ser Glu Pro Arg Pro Ile Gly Thr 515 520 525 Arg Tyr Leu Thr Arg
Asn Leu 530 535
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