U.S. patent application number 17/521512 was filed with the patent office on 2022-09-15 for gene therapy for treating hemophilia b.
The applicant listed for this patent is The Trustees of the University of Pennsylvania. Invention is credited to Lili Wang, James M. Wilson.
Application Number | 20220288233 17/521512 |
Document ID | / |
Family ID | 1000006374415 |
Filed Date | 2022-09-15 |
United States Patent
Application |
20220288233 |
Kind Code |
A1 |
Wang; Lili ; et al. |
September 15, 2022 |
GENE THERAPY FOR TREATING HEMOPHILIA B
Abstract
Compositions and regimens useful in treating hemophilia B are
provided. The method involves administering to the human subject
via a peripheral vein by infusion of a suspension of replication
deficient recombinant adeno-associated virus (rAAV).
Inventors: |
Wang; Lili; (Phoenixville,
PA) ; Wilson; James M.; (Philadelphia, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Trustees of the University of Pennsylvania |
Philadelphia |
PA |
US |
|
|
Family ID: |
1000006374415 |
Appl. No.: |
17/521512 |
Filed: |
November 8, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16093796 |
Oct 15, 2018 |
11191847 |
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PCT/US2017/027400 |
Apr 13, 2017 |
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17521512 |
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62323375 |
Apr 15, 2016 |
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62331064 |
May 3, 2016 |
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62428804 |
Dec 1, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2750/14143
20130101; C12N 2750/14132 20130101; A61K 48/0083 20130101; A61K
48/0058 20130101; A61K 48/0075 20130101; A61K 48/0008 20130101;
A61P 7/04 20180101; C12N 15/86 20130101; A61K 48/005 20130101; C07H
21/04 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/86 20060101 C12N015/86 |
Claims
1. A recombinant adeno-associated virus (rAAV) useful as a
liver-directed therapeutic for hemophilia B, said rAAV comprising
an AAVrh10 capsid and a vector genome packaged therein, said vector
genome comprising: (a) an AAV 5' inverted terminal repeat (ITR);
(b) a coding sequence for a human Factor IX (F9) having coagulation
function operably linked to regulatory elements which direct
expression of the human Factor IX in liver cells, wherein the
coding sequence is SEQ ID NO: 2, and wherein the regulatory
elements comprise (i) two copies of an alpha-1
microglobulin/bikunin enhancer and (ii) a thyroid hormone binding
globulin (TBG) promoter; and (c) an AAV 3' ITR.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Nonprovisional
patent application Ser. No. 16/093,796, filed Oct. 15, 2018, which
is a national stage entry of International Patent Application No.
PCT/US2017/027400, and which claims the benefit of the priority of
US Provisional Patent Application Nos. 62/323,375, filed Apr. 15,
2016, 62/331,064, filed May 3, 2016, and 62/428,804, filed Dec. 1,
2016, which are incorporated herein by reference.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC
FORM
[0002] Applicant hereby incorporates by reference the Sequence
Listing material filed in electronic form herewith. This file is
labeled "UPN-16-7797USC1_ST25".
1. INTRODUCTION
[0003] The application describes embodiments useful for gene
therapy for treating hemophilia B.
2. BACKGROUND
[0004] Hemophilia B is an X-linked bleeding disorder caused by
abnormalities in the function or expression of blood coagulation
Factor IX (FIX). Females who have one normal allele and one mutant
allele have sufficient FIX levels to be asymptomatic. Thus there is
not a strong requirement for a gene therapy product for hemophilia
B to deliver more than 50% of normal FIX blood levels. Because
males have a single X chromosome, presence of one abnormal allele
leads to a clinical presentation of hemophilia. Milder cases
demonstrate excessive bleeding in response to surgery or trauma;
with more severe cases, spontaneous internal bleeding may happen in
any part of the body, with bleeding into joints being most common.
Chronic joint deformities may occur from bleeding, and
intracerebral hemorrhage can occur, the latter with potentially
life-threatening consequences.
[0005] The molecular basis of hemophilia B lies in the gene that
encodes blood coagulation FIX. The incidence of hemophilia B in the
United States is about 1:25,000 live male births. Characterization
of mutant alleles has revealed a variety of mutations including
deletions, insertions, missense mutations, and nonsense mutations.
This genotypic heterogeneity leads to variable consequences in the
biochemical function. Disease severity ranges from mild to severe,
depending on the residual FIX activity, with the majority of
patients falling into the moderate-to-severe categories. Patients
with 5% to less than 50% of normal activity have mild disease, and
they may not show symptoms except in cases of trauma or surgery.
Patients with 1-5% of normal activity have moderate disease, have
excessive bleeding with trauma and may experience spontaneous
bleeding. Patients with less than 1% of normal activity have severe
disease, with frequent spontaneous bleeding, especially into joints
and muscle. Patients with severe hemophilia are typically treated
with regular injections of purified or recombinant FIX
(prophylaxis) to prevent spontaneous bleeds. Despite the
effectiveness of these regimens, they all do require frequent
burdensome intravenous infusions. Furthermore, the nature of these
treatment regimens leads to the risk of trough levels of Factor IX,
which is related to the risk of breakthrough bleeding episodes. The
patient with severe hemophilia B (Factor IX levels <1% of
normal) is still dependent on frequent intravenous infusions and is
still at risk for breakthrough bleeding complications.
[0006] Hemophilia may be first diagnosed in an infant boy when
prolonged bleeding is observed such as after a heel stick, a blood
draw or circumcision but also may be diagnosed when the child
starts crawling and walking, when large bruises may result from
even small falls. Patients with mild disease, having 5-50% of
normal FIX activity, typically only show symptoms of abnormal
bleeding in response to injury, including surgery or tooth
extraction. In patients with moderately severe disease, having 1-5%
of normal FIX activity, spontaneous hemorrhages may also occur, but
are infrequent. In the most severe form of the disease (<1% of
FIX activity; about 60% of all hemophilia B), frequent spontaneous
hemorrhages are the distinguishing characteristic. These result in
muscle hematomas, hemorrhages in the central nervous system, and
hemophilic arthropathy--damage caused by repetitive bleeding
episodes in the joints. Without appropriate treatment with FIX
replacement therapy, the disease can have disabling or even fatal
results. This genetic information strongly supports the idea that
raising the levels of FIX to >1% of normal activity has a large
beneficial effect on disease course in severe hemophiliacs, with
potentially even better results at 5% of normal activity.
Prevention of bleeding, rather than just treating bleeding
episodes, can have a significantly improved outcome, particularly
in preventing disabling joint damage.
[0007] In the developed world, where the Factor IX protein
replacement products are readily available, patients can be treated
with the products in response to injury, or in the case of severe
hemophilia, patients can be treated prophylactically. Patients with
hemophilia B with access to protein replacement now have a normal
life expectancy. However, there is still morbidity from spontaneous
bleeding in patients with insufficient prophylaxis. This can result
from several factors. Patients may develop neutralizing antibodies
(inhibitors) to Factor IX, reducing its ability to participate in
the clotting cascade. In the moderate to mildly affected groups,
spontaneous bleeds can occur, but because they are rare, these
patients are not usually given prophylactic therapy to prevent
those bleeds. Interestingly, joint disease may have become more
common in the less severe patients than in severe patients due to
the latter group's use of prophylaxis. Prophylaxis requires
frequent venipuncture, which in children may result in the need for
a venous access device, and also takes time to deliver, both to
prepare the therapeutic and for the infusion itself. Finally, in
many parts of the world, these clotting factors are not readily
available to patients, nor does every patient adhere to the
prescribed regimen.
[0008] The limitations of Factor IX (FIX) protein replacement have
been described above. Although FIX protein replacement therapy is
available to patients in the developed world, it requires a
lifetime of intravenous infusions every few days for optimal
prophylaxis, due to the relatively short half-life of FIX. Although
moderately affected patients could benefit from prophylaxis,
especially to prevent joint bleeding, they typically do not use a
prophylactic regimen. Gene replacement therapy is expected to be
effective, since hemophilia is caused by the lack of the single
gene product, FIX. Continuous synthesis of FIX by the liver,
recapitulating to an extent the normal state, is expected to be
even more effective at preventing bleeds than bolus infusions of
recombinant FIX. Patients would also be able to avoid the risks and
inconvenience of regular FIX infusion. It also has a greater
potential to treat hemophilia patients in the developing world, as
a single treatment is anticipated to provide many years of therapy.
Tight regulation of levels of FIX expression using gene therapy is
not expected to be required, due to the known efficacy of a wide
range of FIX levels in hemophilia B animal models as well as human
experience with FIX protein replacement.
[0009] Liver-targeted recombinant adeno-associated virus (rAAV)
expressing canine factor IX cDNA in animals for Hemophilia B have
been described. See, e.g., Wang, Lili, et al. "Sustained correction
of bleeding disorder in hemophilia B mice by gene therapy."
Proceedings of the National Academy of Sciences 96.7 (1999):
3906-3910; and Wang, Lili, et al. "Sustained expression of
therapeutic level of factor IX in hemophilia B dogs by AAV-mediated
gene therapy in liver." Molecular therapy 1.2 (2000): 154-158, each
of which is incorporated herein by reference. A human factor IX in
AAV vector was generated by John T. Gray and a clinical trial using
such vectors has been described by Nathwani et al. Please see, U.S.
Pat. Nos. 8,030,065; 8,168,425; Nathwani, Amit C., et al.
"Adenovirus-associated virus vector-mediated gene transfer in
hemophilia B." N Engl J Med 2011.365 (2011): 2357-2365; and
Nathwani, Amit C., et al. "Long-term safety and efficacy of factor
IX gene therapy in hemophilia B." New England Journal of Medicine
371.21 (2014): 1994-2004, each of which is incorporated herein by
reference. However, adverse events were observed, such as an
asymptomatic elevation in the alanine aminotransferase (ALT)
level.
[0010] What are needed are safe and effective treatments for
Hemophilia B.
3. SUMMARY
[0011] Described herein are AAV gene therapy vectors for delivering
normal human FIX to a subject in need thereof, following
intravenous administration of the vector resulting in long-term,
perhaps 10 years or more, of clinically meaningful correction of
the bleeding defect. The subject patient population is patients
with severe hemophilia B. The intended vector dose described herein
is expected to deliver FIX blood levels of approximately 5% or
greater. The goal for the AAV vector treatment is conversion of
severe hemophilia B patients to either moderate or mild hemophilia
B thus relieving such patients of the need to be on a prophylaxis
regimen.
[0012] The gene therapy product described herein provides multiple
important advantages to currently available prophylactic approaches
to the management of severe Hemophilia B. First, preclinical
results with the investigational product are consistent with its
potential to achieve circulating levels of Factor IX of 5% or more
of normal, levels which would be transformative in the target
patient population. Second, the product should lead to effectively
constant Factor IX blood levels, avoiding the trough levels
currently seen with administration of exogenous factor. Third, by
only requiring a single administration, the requirement for
frequent intravenous administrations could be reduced for an
extended period of time, perhaps for a decade or more.
[0013] This application provides the use of a replication deficient
adeno-associated virus (AAV) to deliver a human Factor I (hFIX)
gene to liver cells of patients (human subjects) diagnosed with
hemophilia B. The recombinant AAV vector (rAAV) used for delivering
the hFIX gene ("rAAV.hFIX") should have a tropism for the liver
(e.g., an rAAV bearing an AAVrh.10 capsid), and the hFIX transgene
should be controlled by liver-specific expression control elements.
In one embodiment, the expression control elements include one or
more of the following: an alpha-1 microglobulin/bikunin enhancer; a
thyroid hormone binding globulin promoter (TBG); a human beta
globin IVS2 intron; a WPRE; and a polyA signal. Such elements are
further described herein.
[0014] The coding sequence for hFIX is, in one embodiment, codon
optimized for expression in humans. Such sequence may share less
than 80% identity to the native hFIX coding sequence (SEQ ID NO:
1). In one embodiment, the hFIX coding sequence is that shown in
SEQ ID NO: 2. In one embodiment, the hFIX coding sequence is that
shown in SEQ ID NO: 13.
[0015] In another aspect, provided herein is an aqueous suspension
suitable for administration to a hemophilia B patient which
includes the rAAV described herein. In some embodiments, the
suspension includes an aqueous suspending liquid and about
1.times.10.sup.12 to about 5.times.10.sup.13 genome copies (GC) of
the rAAV/mL. The suspension is, in one embodiment, suitable for
intravenous injection. In other embodiment, the suspension further
includes a surfactant, preservative, and/or buffer dissolved in the
aqueous suspending liquid.
[0016] In another embodiment, provided herein is a method of
treating a patient having hemophilia B with an rAAV as described
herein. In one embodiment, about 1.times.10.sup.11 to about
1.times.10.sup.13 genome copies (GC) of the rAAV/kg patient body
weight are delivered the patient in an aqueous suspension. All
ranges described herein are inclusive of the endpoints.
[0017] The goal of the treatment is to functionally replace the
patient's defective hFIX via rAAV-based liver-directed gene therapy
as a viable approach to treat this disease and improve response to
current therapies. The embodiments described in the application are
based, in part, on the development of therapeutic compositions and
methods that allow for the safe delivery of efficacious doses; and
improved manufacturing methods to meet the purification production
requirement for efficacious dosing in human subjects.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1. Schematic representation of
AAV.LSP.IVS2.hFIXco3T.WPRE.bGH cassette.
[0019] FIG. 2. Schematic representation of
pENN.AAV.LSP.hFIXco3T.WPRE.bGH.KanR cis plasmid.
[0020] FIG. 3. Alignment of human FIX (NM_000133) coding sequence
(nucleotides 29 to 1412 of SEQ ID NO: 1) with codon-optimized hFIX
(hFIXco) sequence (SEQ ID NO: 2).
[0021] FIG. 4. Alignment of human ABP enhancer (SEQ ID NO: 18;
bottom sequence) with APB enhancer described herein (nucleotides 2
to 99 of SEQ ID NO: 4; top sequence).
[0022] FIG. 5. Alignment of human TBG promoter (SEQ ID NO: 19;
bottom sequence) with TBG promoter described herein (nucleotides 26
to 496 of SEQ ID NO: 5, top sequence).
[0023] FIG. 6. Graph showing hFIX expression levels in 4 FIX-KO
mice in a dose response experiment, as determined by ELISA.
Experiment as described in Section 8.4.1.
[0024] FIG. 7. Graph showing hFIX expression levels in 4 FIX-KO
mice in a dose response experiment, as determined by APTT assay.
Experiment as described in Section 8.4.1.
[0025] FIG. 8. Graphs showing absence of anti-hFIX IgG in 7 FIX-KO
mice in a dose response experiment, as determined by solid-phase
ELISA. Experiment as described in Section 8.4.1.
[0026] FIG. 9 are 6 graphs showing PT (prothrombin time) in FIX-KO
male mouse plasma, following AAV vector administration. PT was
determined using a Stago ST Art Start Hemostasis Coagulation
Analyzer set to PT mode and Dade Innovin Reagent (Reference
#B4212-40). Normal PT values for male FIX-KO mice range from
6.8-8.2 seconds with mean and standard deviation of 7.3.+-.0.3
seconds, respectively. Experiments as described in Section
8.4.1.
[0027] FIG. 10. Graph showing Vector genome copies in liver
determined by QPCR. *P<0.05; **P<0.001, Mann Whitney test
(n=7). Experiments as described in Section 8.4.1.
[0028] FIG. 11. Graph showing relative expression of hFIXco mRNA in
liver determined by RT-QPCR. *P<0.05; **P<0.001, Mann Whitney
test (n=7). Experiments as described in Section 8.4.1.
[0029] FIG. 12. Manufacturing Process Flow Diagram
[0030] FIG. 13. rAAVrh.10.LSP.hFIXco vector was assessed at six
doses after intravenous administration: 8.times.10.sup.7,
2.7.times.10.sup.8, 2.7.times.10.sup.9, 2.7.times.10.sup.10,
2.7.times.10.sup.11 and 8.times.10.sup.11 GC/mouse. Mice were bled
at 2 and 4 weeks following vector administration and Factor IX
antigen and activity levels were determined by hFIX ELISA and aPTT,
respectively. Experiments as described in Section 8.2.
[0031] FIG. 14. A dose-response study was conducted in C57Bl/6 male
mice. rAAVrh.10.LSP.hFIXco was assessed at four doses after
intravenous administration: 7.times.10.sup.8, 2.3.times.10.sup.9,
7.times.10.sup.9, and 2.3.times.10.sup.10 GC/mouse. Factor IX
levels were observed above therapeutic levels (5% of normal; 100%=5
ug/ml) at a dose of 2.3.times.10.sup.9 GC/mouse
(1.1.times.10.sup.11 GC/kg), and above normal levels at
2.3.times.10.sup.10 GC/mouse (1.1.times.10.sup.12 GC/kg).
[0032] FIG. 15. Schematic representation of pAAV2.rh10.KanR.
[0033] FIG. 16 shows a comparison of rhCG expression levels by
AAVrh10, AAV8, AAV3B and AAV5 vectors (first vector injection).
[0034] FIG. 17 shows expression of rhCG in the liver at different
time points.
[0035] FIGS. 18A-18D shows rhCG vector DNA copies in liver at
different time points.
[0036] FIGS. 19A-19B shows rhAFP levels after readministration
(second vector injection) with AAV3B or AAV5 vectors expressing
rhAFP.
[0037] FIGS. 20A and 20B show rhAFP vector genome copies in
liver.
[0038] FIG. 21 shows differential AAV Nab response in macaques.
[0039] FIG. 22 shows FIX antigen and activity levels in animals
injected with vectors carrying the hFIXco and hFIXco3T-Padua at 2
weeks post injection.
[0040] FIG. 23 shows FIX antigen and activity levels in animals
injected with vectors carrying the hFIXco and hFIXco3T-Padua at 4
weeks post injection.
[0041] FIG. 24 shows FIX antigen and activity levels in animals
injected with vectors carrying the hFIXco and hFIXco3T-Padua at 6
weeks post injection.
[0042] FIG. 25 shows vector genome copies (GC) in liver 6 weeks
post injection for various dosages of vector.
[0043] FIG. 26 shows a time course of prothrombin time (PT) in
animals treated with various dosages of vector at 2 weeks, 4 weeks
and 6 weeks post injection.
[0044] FIGS. 27A-27d are graphs showing intracellular cytokine
staining (ICS) of CD4+(FIGS. 27A and 27B) and CD8+(FIGS. 27C and
27D) peripheral blood mononuclear cells (PBMCs) in patients treated
with low-dose (1.6.times.1012 GC/kg) or mid-dose (5.0.times.1012
GC/kg) of AAVrh10.hFIXco3T.
[0045] FIG. 28A-28C are graphs showing neutralizing antibodies
(NAbs) and Immunoglobulin-G (IgG) responses to the AAV capsid of
interest (AAVrh.10) from isolated serum of six patients receiving
the low- or mid-dose of the vector discussed for FIGS. 27A and B.
All results are reported as the reciprocal of serum dilution.
[0046] FIG. 29 is a heatmap showing the multianalye profile in the
serum of six patients receiving the low- or mid-dose of the vector
discussed for FIGS. 27A and B. Any analyte showing an increase in
activity was coded in red while decreases were coded in blue.
[0047] FIG. 30A shows the human FIX amino acid sequence (SEQ ID NO:
10) with the mutations of five patients receiving the low- or
mid-dose of the vector discussed for FIGS. 27A and B. The mutation
of one patient is not shown as it is not a coding mutation. Using
prediction software, the MHC Class I binding affinity to various
alleles was predicted as shown in FIG. 30B.
5. DETAILED DESCRIPTION OF THE INVENTION
[0048] This invention relates to the use of a replication deficient
adeno-associated virus (AAV) to deliver a human Factor IX (hFIX)
gene to liver cells of patients (human subjects) diagnosed with
hemophilia B. The recombinant AAV vector (rAAV) used for delivering
the hFIX gene ("rAAV.hFIX") should have a tropism for the liver
(e.g., an rAAV bearing an AAVrh.10 capsid), and the hFIX transgene
should be controlled by liver-specific expression control elements.
In one embodiment, the expression control elements include one or
more of the following: an alpha-1 microglobulin/bikunin enhancer; a
thyroid hormone binding globulin promoter (TBG); a human beta
globin IVS2 intron; a WPRE; and a polyA signal. Such elements are
further described herein.
[0049] As used herein, "AAVrh10 capsid" refers to the AAVrh.10
capsid having the amino acid sequence of GenBank, accession:
AAO88201, SEQ ID NO: 14, which is incorporated by reference herein.
Some variation from this encoded sequence is envisioned, including
sequences having about 99% identity to the referenced amino acid
sequence in AAO88201 and US 2013/0045186A1 (i.e., less than about
1% variation from the referenced sequence), provided that the
integrity of the ligand-binding site for the affinity capture
purification is maintained and the change in sequences does not
substantially alter the pH range for the capsid for the ion
exchange resin purification. For example, studies indicate that
rh.39, rh.20, rh.25, AAV10, bb.1, bb.2 and pi.2 serotypes should
bind to the illustrated affinity resin column because their
sequence in the antibody-binding region is identical or very
similar to rh10. Methods of generating the capsid, coding sequences
therefore, and methods for production of rAAV viral vectors have
been described. See, e.g., Gao, et al, Proc. Natl. Acad. Sci.
U.S.A. 100 (10), 6081-6086 (2003) and US 2013/0045186A1.
[0050] As used herein, the term "NAb titer" a measurement of how
much neutralizing antibody (e.g., anti-AAV NAb) is produced which
neutralizes the physiologic effect of its targeted epitope (e.g.,
an AAV). Anti-AAV NAb titers may be measured as described in, e.g.,
Calcedo, R., et al., Worldwide Epidemiology of Neutralizing
Antibodies to Adeno-Associated Viruses. Journal of Infectious
Diseases, 2009. 199(3): p. 381-390, which is incorporated by
reference herein.
[0051] The terms "percent (%) identity", "sequence identity",
"percent sequence identity", or "percent identical" in the context
of amino acid sequences refers to the residues in the two sequences
which are the same when aligned for correspondence. Percent
identity may be readily determined for amino acid sequences over
the full-length of a protein, polypeptide, about 32 amino acids,
about 330 amino acids, or a peptide fragment thereof or the
corresponding nucleic acid sequence coding sequencers. A suitable
amino acid fragment may be at least about 8 amino acids in length,
and may be up to about 700 amino acids. Generally, when referring
to "identity", "homology", or "similarity" between two different
sequences, "identity", "homology" or "similarity" is determined in
reference to "aligned" sequences. "Aligned" sequences or
"alignments" refer to multiple nucleic acid sequences or protein
(amino acids) sequences, often containing corrections for missing
or additional bases or amino acids as compared to a reference
sequence. Alignments are performed using any of a variety of
publicly or commercially available Multiple Sequence Alignment
Programs. Sequence alignment programs are available for amino acid
sequences, e.g., the "Clustal Omega", "Clustal X", "MAP", "PIMA",
"MSA", "BLOCKMAKER", "MEME", and "Match-Box" programs. Generally,
any of these programs are used at default settings, although one of
skill in the art can alter these settings as needed. Alternatively,
one of skill in the art can utilize another algorithm or computer
program which provides at least the level of identity or alignment
as that provided by the referenced algorithms and programs. See,
e.g., J. D. Thomson et al, Nucl. Acids. Res., "A comprehensive
comparison of multiple sequence alignments", 27(13):2682-2690
(1999).
[0052] As used herein, the term "operably linked" refers to both
expression control sequences that are contiguous with the gene of
interest and expression control sequences that act in trans or at a
distance to control the gene of interest.
[0053] A "replication-defective virus" or "viral vector" refers to
a synthetic or artificial viral particle in which an expression
cassette containing a gene of interest is packaged in a viral
capsid or envelope, where any viral genomic sequences also packaged
within the viral capsid or envelope are replication-deficient;
i.e., they cannot generate progeny virions but retain the ability
to infect target cells. In one embodiment, the genome of the viral
vector does not include genes encoding the enzymes required to
replicate (the genome can be engineered to be "gutless"--containing
only the transgene of interest flanked by the signals required for
amplification and packaging of the artificial genome), but these
genes may be supplied during production. Therefore, it is deemed
safe for use in gene therapy since replication and infection by
progeny virions cannot occur except in the presence of the viral
enzyme required for replication.
[0054] It is to be noted that the term "a" or "an" refers to one or
more. As such, the terms "a" (or "an"), "one or more," and "at
least one" are used interchangeably herein.
[0055] The words "comprise", "comprises", and "comprising" are to
be interpreted inclusively rather than exclusively. The words
"consist", "consisting", and its variants, are to be interpreted
exclusively, rather than inclusively. While various embodiments in
the specification are presented using "comprising" language, under
other circumstances, a related embodiment is also intended to be
interpreted and described using "consisting of" or "consisting
essentially of" language.
[0056] As used herein, the term "about" means a variability of 10%
from the reference given, unless otherwise specified.
[0057] Unless defined otherwise in this specification, technical
and scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art and by reference to
published texts, which provide one skilled in the art with a
general guide to many of the terms used in the present
application.
[0058] 5.1 Gene Therapy Vectors
[0059] In one aspect, a recombinant adeno-associated virus (rAAV)
vector carrying the human clotting factor 9 (hFIX or hF9) gene is
provided for use in gene therapy. The rAAV.hFIX vector should have
a tropism for the liver (e.g., an rAAV bearing an AAVrh.10 capsid)
and the hFIX transgene should be controlled by liver-specific
expression control elements. The vector is formulated in a
buffer/carrier suitable for infusion in human subjects. The
buffer/carrier should include a component that prevents the rAAV
from sticking to the infusion tubing but does not interfere with
the rAAV binding activity in vivo.
[0060] 5.1.1. The rAAV.hFIX Vector
[0061] 5.1.1.1. The hFIX Sequence
[0062] Human coagulation factor IX FIX is a vitamin K-dependent
single-chain glycoprotein, which is synthesized as a precursor
protein. The precursor undergoes extensive posttranslational
modification to become the fully gamma-carboxylated mature zymogen
that is secreted into the blood. The precursor protein has a signal
peptide at the amino (NH.sub.2) terminal end, which directs the
protein to the endoplasmic reticulum in the liver, and a prepro
leader sequence recognized by the gamma-glutamylcarboxylase, which
is responsible for the posttranslational modification
(carboxylation) of the glutamic acid residues (Gla) in the
NH.sub.2-terminal portion of the molecule. These 2 parts of the
molecule are removed before the protein is secreted into the
circulation. The full length protein (before cleavage) is 461 amino
acids as shown in SEQ ID NO: 10 (Genbank Accession #P00740). The
mature protein is about 415 amino acids.
[0063] In one embodiment, the hFIX gene encodes the hFIX protein
shown in SEQ ID NO: 10, i.e., the full length protein. In another
embodiment the hFIX gene encodes an hFIX protein which has a
polymorphism at aa194. In on embodiment, the polymorphis is
T194A.
[0064] Thus, in one embodiment, the hFIX transgene can include, but
is not limited to, one or more of the sequences provided by SEQ ID
NO:1, SEQ ID NO: 2, or SEQ ID NO: 13, which are provided in the
attached Sequence Listing, which is incorporated by reference
herein. SEQ ID NO: 1 provides the cDNA for native human FIX. SEQ ID
NO: 2 provides an engineered cDNA for human FIX, which has been
codon optimized for expression in humans (also called hFIXco or
hFIXco3T). SEQ ID NO: 13 provides an engineered cDNA for human FIX,
which has been codon optimized for expression in humans.
Alternatively or additionally, web-based or commercially available
computer programs, as well as service based companies may be used
to back translate the amino acids sequences to nucleic acid coding
sequences, including both RNA and/or cDNA. See, e.g., backtranseq
by EMBOSS, www.ebi.ac.uk/Tools/st/; Gene Infinity
(www.geneinfinity.org/sms-/sms_backtranslation.html); ExPasy
(www.expasy.org/tools/). It is intended that all nucleic acids
encoding the described hFIX polypeptide sequences are encompassed,
including nucleic acid sequences which have been optimized for
expression in the desired target subject (e.g., by codon
optimization). In one embodiment, the nucleic acid sequence
encoding hFIX shares at least 95% identity with the native hFIX
coding sequence of SEQ ID NO: 1. In another embodiment, the nucleic
acid sequence encoding hFIX shares at least 99%, 97%, 95%, 90%,
85%, 80%, 75%, 70%, or 65% identity with the native hFIX coding
sequence of SEQ ID NO: 1. In yet another embodiment, the nucleic
acid sequence encoding hFIX shares at least 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
97, 98, or 99% identity with the hFIX coding sequence of SEQ ID NO:
1 or SEQ ID NO: 2. In one embodiment, the nucleic acid sequence
encoding hFIX shares about 75% identity with the native hFIX coding
sequence of SEQ ID NO: 1. In one embodiment, the nucleic acid
sequence encoding hFIX is SEQ ID NO: 2. In one embodiment, the
nucleic acid sequence encoding hFIX is SEQ ID NO: 13.
[0065] Codon-optimized coding regions can be designed by various
different methods. This optimization may be performed using methods
which are available on-line (e.g., GeneArt), published methods, or
a company which provides codon optimizing services, e.g., as DNA2.0
(Menlo Park, Calif.). One codon optimizing approach is described,
e.g., in International Patent Publication No. WO 2015/012924, which
is incorporated by reference herein. See also, e.g., US Patent
Publication No. 2014/0032186 and US Patent Publication No.
2006/0136184. Suitably, the entire length of the open reading frame
(ORF) for the product is modified. However, in some embodiments,
only a fragment of the ORF may be altered. By using one of these
methods, one can apply the frequencies to any given polypeptide
sequence, and produce a nucleic acid fragment of a codon-optimized
coding region which encodes the polypeptide.
[0066] A number of options are available for performing the actual
changes to the codons or for synthesizing the codon-optimized
coding regions designed as described herein. Such modifications or
synthesis can be performed using standard and routine molecular
biological manipulations well known to those of ordinary skill in
the art. In one approach, a series of complementary oligonucleotide
pairs of 80-90 nucleotides each in length and spanning the length
of the desired sequence are synthesized by standard methods. These
oligonucleotide pairs are synthesized such that upon annealing,
they form double stranded fragments of 80-90 base pairs, containing
cohesive ends, e.g., each oligonucleotide in the pair is
synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond
the region that is complementary to the other oligonucleotide in
the pair. The single-stranded ends of each pair of oligonucleotides
are designed to anneal with the single-stranded end of another pair
of oligonucleotides. The oligonucleotide pairs are allowed to
anneal, and approximately five to six of these double-stranded
fragments are then allowed to anneal together via the cohesive
single stranded ends, and then they ligated together and cloned
into a standard bacterial cloning vector, for example, a TOPO.RTM.
vector available from Invitrogen Corporation, Carlsbad, Calif. The
construct is then sequenced by standard methods. Several of these
constructs consisting of 5 to 6 fragments of 80 to 90 base pair
fragments ligated together, i.e., fragments of about 500 base
pairs, are prepared, such that the entire desired sequence is
represented in a series of plasmid constructs. The inserts of these
plasmids are then cut with appropriate restriction enzymes and
ligated together to form the final construct. The final construct
is then cloned into a standard bacterial cloning vector, and
sequenced. Additional methods would be immediately apparent to the
skilled artisan. In addition, gene synthesis is readily available
commercially.
[0067] 5.1.1.2. The rAAV Vector
[0068] Because hFIX is natively expressed in the hepatocytes, it is
desirable to use an AAV which shows tropism for liver. In one
embodiment, the AAV supplying the capsid is AAVrh.10. However, any
of a number of rAAV vectors with liver tropism can be used.
[0069] In a specific embodiment described in the Examples, infra,
the gene therapy vector is an AA Vrh.10 vector expressing an hFIX
transgene under control of a liver-specific promoter
(thyroxine-binding globulin, TBG) referred to as rAAVrh.10.TBG.hFIX
or rAAVrh.10.LSP.hFIXco. The external AAV vector component is a
serotype rh.10, T=1 icosahedral capsid consisting of 60 copies of
three AAV viral proteins, VP1, VP2, and VP3, at a ratio of 1:1:10.
The capsid contains a single-stranded DNA rAAV vector genome.
[0070] The rAAVrh. I0.TBG.hFIX genome contains an hFIX expression
cassette flanked by two AAV inverted terminal repeats (ITRs). The
hFIX expression cassette includes an enhancer, promoter, intron, an
hFIX coding sequence, a WPRE and polyadenylation (polyA) signal.
These control sequences are "operably linked" to the hFIX gene
sequences. The expression cassette and flanking ITRs may be
engineered onto a plasmid which is used for production of a viral
vector.
[0071] The ITRs are the genetic elements responsible for the
replication and packaging of the genome during vector production
and are the only viral cis elements required to generate rAAV. The
minimal sequences required to package the expression cassette into
an AAV viral particle are the AAV 5' and 3' ITRs, which may be of
the same AAV origin as the capsid, or which of a different AAV
origin (to produce an AAV pseudotype). In one embodiment, the ITR
sequences from AAV2, or the deleted version thereof (.DELTA.ITR),
are used for convenience and to accelerate regulatory approval.
However, ITRs from other AAV sources may be selected. Where the
source of the ITRs is from AAV2 and the AAV capsid is from another
AAV source, the resulting vector may be termed pseudotyped.
Typically, an expression cassette for an AAV vector comprises an
AAV 5' ITR, the hFIX coding sequences and any regulatory sequences,
and an AAV 3' ITR. However, other configurations of these elements
may be suitable. A shortened version of the 5' ITR, termed
.DELTA.ITR, has been described in which the D-sequence and terminal
resolution site (trs) are deleted. In other embodiments, the
full-length AAV 5' and 3' ITRs are used.
[0072] Expression of the hFIX coding sequence is driven from a
liver-specific promoter. An illustrative plasmid and vector
described herein uses the hepatocyte-specific promoter thyroxin
binding globulin (TBG). Alternatively, other liver-specific
promoters may be used including the alpha 1 anti-trypsin (A1AT);
human albumin (Miyatake et al., J. Virol., 71:5124 32 (1997)),
humAlb; and hepatitis B virus core promoter, (Sandig et al., Gene
Ther., 3:1002 9 (1996)), TTR minimal enhancer/promoter, or
alpha-antitrypsin promoter. See, e.g., The Liver Specific Gene
Promoter Database, Cold Spring Harbor, rulai.schl.edu/LSPD,
incorporated by reference herein. Although less desired, other
promoters, such as viral promoters, constitutive promoters,
regulatable promoters [see, e.g., WO 2011/126808 and WO
2013/04943], or a promoter responsive to physiologic cues may be
used in the vectors described herein.
[0073] In one embodiment, the expression control sequences include
an enhancer. In one embodiment the alpha 1 microglobulin/bikunin
enhancer element is included. In another embodiment, two copies of
the alpha 1 microglobulin/bikunin enhancer element precede the TBG
promoter to stimulate promoter activity. Together these elements
(two copies of the APB enhancer and TBG promoter) are termed "LSP"
as shown in nt 239 to nt 965 of SEQ ID NO: 11 or SEQ ID NO: 15 and
FIG. 1. See, Wang et al, Sustained correction of bleeding disorder
in hemophilia B mice by gene therapy, PNAS, 96:3906-10 (March
1999), which is incorporated herein by reference.
[0074] In addition to a promoter, an expression cassette and/or a
vector may contain other appropriate transcription initiation,
termination, enhancer sequences, and efficient RNA processing
signals. Such sequences include splicing and polyadenylation
(polyA) signals; sequences that stabilize cytoplasmic mRNA;
sequences that enhance translation efficiency (i.e., Kozak
consensus sequence); sequences that enhance protein stability; and
when desired, sequences that enhance secretion of the encoded
product. In one embodiment, a human beta globin IVS2 intron is
present to further enhance expression and a bovine growth hormone
(bGH) polyadenylation (polyA) signal is included to mediate
termination of hFIX mRNA transcripts. Examples of other suitable
polyA sequences include, e.g., SV40, rabbit beta globin, and TK
polyA. Examples of other suitable enhancers include, e.g., the
alpha fetoprotein enhancer, the TTR minimal promoter/enhancer,
human apolipoprotein hepatic control region, amongst others.
[0075] In other embodiments, spacers are inserted in the expression
cassette and/or vector. Such spacers may be included to adjust the
size of the total vector genome. In one embodiment, spacers are
included such that the vector genome is approximately the same size
as the native AAV vector genome (e.g., between 4.1 and 4.7 kb). In
one embodiment, spacers are included such that the vector genome is
approximately 4.7 kb. See, Wu et al, Effect of Genome Size on AAV
Vector Packaging, Mol Ther. 2010 January; 18(1): 80-86, which is
incorporated herein by reference. Spacer DNA may be non-coding DNA,
for example, an intron sequence.
[0076] In one embodiment, the vector is a self-complementary
vector. The abbreviation "sc" refers to self-complementary.
"Self-complementary AAV" refers a plasmid or vector having an
expression cassette in which a coding region carried by a
recombinant AAV nucleic acid sequence has been designed to form an
intra-molecular double-stranded DNA template. Upon infection,
rather than waiting for cell mediated synthesis of the second
strand, the two complementary halves of scAAV associate to form one
double stranded DNA (dsDNA) unit that is ready for immediate
replication and transcription. See, e.g., D M McCarty et al,
"Self-complementary recombinant adeno-associated virus (scAAV)
vectors promote efficient transduction independently of DNA
synthesis", Gene Therapy, (August 2001), Vol 8, Number 16, Pages
1248-1254. Self-complementary AAVs are described in, e.g., U.S.
Pat. Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is
incorporated herein by reference in its entirety.
[0077] In one embodiment the rAAV vector genome is nt 1 to nt 3951
of SEQ ID NO: 11. In another embodiment, the rAAV vector genome is
nt 7 to nt 4115 of SEQ ID NO: 12. In yet another embodiment, the
rAAV vector genome is nt 1 to nt 3951 of SEQ ID NO: 16.
[0078] 5.1.2. rAAV.hFIX Formulation
[0079] In one embodiment, the rAAV.hFIX vector is provided in a
pharmaceutical composition which comprises an aqueous carrier,
excipient, diluent or buffer. In a specific embodiment exemplified
herein, the rAAV.hFIX formulation is a suspension containing an
effective amount of rAAV.hFIX vector suspended in an aqueous
solution containing composed of 0.001% Pluronic F-68 in TMN200 (200
mM sodium chloride, 1 mM magnesium chloride, 20 mM Tris, pH 8.0).
However, various suitable solutions are known including those which
include one or more of: buffering saline, a surfactant, and a
physiologically compatible salt or mixture of salts adjusted to an
ionic strength equivalent to about 100 mM sodium chloride (NaCl) to
about 250 mM sodium chloride, or a physiologically compatible salt
adjusted to an equivalent ionic concentration. In another
embodiment, the buffer is PBS. Other suitable buffers include
Ringer's solution, Elliot's solution, and others known in the
art.
[0080] For example, a suspension as provided herein may contain
both NaCl and KCl. The pH may be in the range of 6.5 to 8.5, or 7
to 8.5, or 7.5 to 8. A suitable surfactant, or combination of
surfactants, may be selected from among a Poloxamers, i.e.,
nonionic triblock copolymers composed of a central hydrophobic
chain of polyoxypropylene (poly(propylene oxide)) flanked by two
hydrophilic chains of polyoxyethylene (poly(ethylene oxide)),
SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy
capryllic glyceride), polyoxy 10 oleyl ether, TWEEN
(polyoxyethylene sorbitan fatty acid esters), ethanol and
polyethylene glycol. In one embodiment, the formulation contains a
poloxamer. These copolymers are commonly named with the letter "P"
(for poloxamer) followed by three digits: the first two
digits.times.100 give the approximate molecular mass of the
polyoxypropylene core, and the last digit.times.10 gives the
percentage polyoxyethylene content. In one embodiment Poloxamer 188
is selected. The surfactant may be present in an amount up to about
0.0005% to about 0.001% of the suspension. In another embodiment,
the vector is suspended in an aqueous solution containing 180 mM
sodium chloride, 10 mM sodium phosphate, 0.001% Poloxamer 188, pH
7.3.
[0081] In one embodiment, the formulation is suitable for use in
human subjects and is administered intravenously. In one
embodiment, the formulation is delivered via a peripheral vein by
bolus injection. In one embodiment, the formulation is delivered
via a peripheral vein by infusion over about 10 minutes (.+-.5
minutes). In one embodiment, the formulation is delivered via a
peripheral vein by infusion over about 60 minutes (.+-.5 minutes).
However, these times may be adjusted as needed or desired. Any
suitable method or route can be used to administer an
AAV-containing composition as described herein, and optionally, to
co-administer other active drugs or therapies in conjunction with
the AAV-mediated antibodies described herein. Routes of
administration include, for example, systemic, oral, inhalation,
intranasal, intratracheal, intraarterial, intraocular, intravenous,
intramuscular, subcutaneous, intradermal, and other parental routes
of administration.
[0082] In one embodiment, the formulation may contain, e.g., about
1.5.times.10.sup.11 genome copies per kilogram of patient body
weight (GC/kg) to about 3.times.10.sup.13 GC/kg, about
1.6.times.10.sup.10 to about 5.times.10.sup.10 GC/kg, about
5.times.10.sup.11 genome copies per kilogram of patient body weight
(GC/kg) to about 2.times.10.sup.13 GC/kg, or about
1.times.10.sup.12 to about 1.25.times.10.sup.13 GC/kg, as measured
by oqPCR or digital droplet PCR (ddPCR) as described in, e.g., M.
Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014
April; 25(2):115-25. doi: 10.1089/hgtb.2013.131. Epub 2014 Feb. 14,
which is incorporated herein by reference. In one embodiment, the
rAAV.hFIX formulation is a suspension containing at least
1.times.10.sup.13 genome copies (GC)/mL, or greater, as measured by
oqPCR or digital droplet PCR (ddPCR) as described in, e.g., M. Lock
et al, supra.
[0083] In order to ensure that empty capsids are removed from the
dose of AAV.hFIX that is administered to patients, empty capsids
are separated from vector particles during the vector purification
process, e.g., using the method discussed herein. In one
embodiment, the vector particles containing packaged genomes are
purified from empty capsids using the process described in U.S.
Patent Application No. 62/322,055, filed on Apr. 13, 2016, and
entitled "Scalable Purification Method for AAVrh.10", which is
incorporated by reference herein. Briefly, a two-step purification
scheme is described which selectively captures and isolates the
genome-containing rAAV vector particles from the clarified,
concentrated supernatant of a rAAV production cell culture. The
process utilizes an affinity capture method performed at a high
salt concentration followed by an anion exchange resin method
performed at high pH to provide rAAV vector particles which are
substantially free of rAAV intermediates. Other purification
methods are described, e.g., in US Patent Application Nos.
62/266,347, 62/266,357, 62/322,071, 62/266,351, 62/322,083,
62/266,341, and 62/322,098, each of which is incorporated herein by
reference.
[0084] While any conventional manufacturing process can be
utilized, the process described herein (and in U.S. Patent
Application No. 62/322,055) yields vector preparations wherein
between 50 and 70% of the particles have a vector genome, i.e., 50
to 70% full particles. Thus for an exemplary dose of
5.times.10.sup.11 GC/kg body weight, the total particle dose is
between 7.times.10.sup.11 GC and 1.times.10.sup.12 GC. On the basis
of peer-reviewed and published data, it can be estimated that the
total particle titer in starting dose in the clinical trial
described in Nathwani et al (Nathwani, Amit C., et al.
"Adenovirus-associated virus vector-mediated gene transfer in
hemophilia B." N Engl J Med 2011.365 (2011): and 2357-2365;
Nathwani, Amit C., et al. "Long-term safety and efficacy of factor
IX gene therapy in hemophilia B." New England Journal of Medicine
371.21 (2014): 1994-2004.) was approximately 2.times.10.sup.12
total particles. In another embodiment, the dosage is one half log
higher than the first dose, or 1.6.times.10.sup.12 GC/kg body
weight, and the total particle dose is between 2.3.times.10.sup.12
and 3.times.10.sup.12 particles. In another embodiment, the
proposed dose is one half log higher than the second dose, or
5.times.10.sup.12 GC/kg body weight, and the total particle dose is
between 7.6.times.10.sup.12 and 1.1.times.10.sup.13 particles. This
total particle dose is well below the estimated total particle dose
in the Nathwani trial that provoked a rise in ALT [Nathwani, Amit
C., et al. "Adenovirus-associated virus vector-mediated gene
transfer in hemophilia B." N Engl J Med 2011.365 (2011): 2357-2365;
and Nathwani, Amit C., et al. "Long-term safety and efficacy of
factor IX gene therapy in hemophilia B." New England Journal of
Medicine 371.21 (2014). 1994-2004.]. In one embodiment, the
formulation is characterized by an rAAV stock having a ratio of
"empty" to "full" of 1 or less, less than 0.75, less than 0.5, or
less than 0.3.
[0085] Briefly, in one embodiment, a method for separating AAVrh10
viral particles from AAVrh10 capsid intermediates is provided which
involves: subjecting a mixture comprising recombinant AAVrh10 viral
particles and AAV rh10 capsid intermediates to fast performance
liquid chromatography, wherein the AAVrh10 viral particles and
AAVrh10 intermediates are bound to an anion exchange resin
equilibrated at a pH of about 10.0 and subjected to a salt gradient
while monitoring eluate for ultraviolet absorbance at about 260 and
about 280, wherein the AAVrh10 full capsids are collected from a
fraction which is eluted when the ratio of A260/A280 reaches an
inflection point.
[0086] In one embodiment, the method further includes (a) mixing a
suspension comprising recombinant AAVrh10 viral particles and AAV
rh10 capsid intermediates and a Buffer A comprising 20 mM to 50 mM
Bis-Tris propane (BTP) and a pH of about 10.0; (b) loading the
suspension of (a) onto a strong anion exchange resin column; (c)
washing the loaded anion exchange resin with Buffer 1% B which
comprises a salt having the ionic strength of 10 mM to 40 mM NaCl
and BTP with a pH of about 10.0; (d) applying an increasing salt
concentration gradient to the loaded and washed anion exchange
resin, wherein the salt gradient is the equivalent of about 10 mM
to about 40 mM NaCl; and (e) collecting rAAVrh10 particles from
elute obtained at a salt concentration equivalent to at least 70 mM
NaCl, where the rAAVrh10 particles are at least about 90% purified
from AAVrh10 intermediates. In one embodiment, this is determined
by genome copies.
[0087] In one embodiment, the intermediates are eluted from the
anion exchange resin when the salt concentration is the equivalent
of greater than about 50 mM NaCl. In still a further embodiment,
Buffer A is further admixed with NaCl to a final concentration of
1M in order to form or prepare Buffer B. In yet another embodiment,
the salt gradient has an ionic strength equivalent to 10 mM to
about 190 mM NaCl. The elution gradient may be from 1% buffer B to
about 19% Buffer B. Optionally, the vessel containing the anion
exchange resin is a monolith column and where Buffer A, Buffer B,
and the salt gradient are in about 60 column volumes.
[0088] A stock or preparation of rAAVrh.10 particles (packaged
genomes) is "substantially free" of AAV empty capsids (and other
intermediates) when the rAAVrh.10 particles in the stock are at
least about 75% to about 100%, at least about 80%, at least about
85%, at least about 90%, at least about 95%, or at least 99% of the
rAAVrh.10 in the stock and "empty capsids" are less than about 1%,
less than about 5%, less than about 10%, less than about 15% of the
rAAVrh.10 in the stock or preparation.
[0089] In a further embodiment, the average yield of rAAV particles
is at least about 70%. This may be calculated by determining titer
(genome copies) in the mixture loaded onto the column and the
amount presence in the final elutions. Further, these may be
determined based on q-PCR analysis and/or SDS-PAGE techniques such
as those described herein or those which have been described in the
art.
[0090] For example, to calculate empty and full particle content,
VP3 band volumes for a selected sample (e.g., an iodixanol
gradient-purified preparation where # of GC=# of particles) are
plotted against GC particles loaded. The resulting linear equation
(y=mx+c) is used to calculate the number of particles in the band
volumes of the test article peaks. The number of particles (pt) per
20 .mu.L loaded is then multiplied by 50 to give particles (pt)
/mL. Pt/mL divided by GC/mL gives the ratio of particles to genome
copies (pt/GC). Pt/mL-GC/mL gives empty pt/mL. Empty pt/mL divided
by pt/mL and x 100 gives the percentage of empty particles.
[0091] Generally, methods for assaying for empty capsids and AAV
vector particles with packaged genomes have been known in the art.
See, e.g., Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et
al., Molec. Ther. (2003) 7:122-128. To test for denatured capsid,
the methods include subjecting the treated AAV stock to
SDS-polyacrylamide gel electrophoresis, consisting of any gel
capable of separating the three capsid proteins, for example, a
gradient gel containing 3-8% Tris-acetate in the buffer, then
running the gel until sample material is separated, and blotting
the gel onto nylon or nitrocellulose membranes. Anti-AAV capsid
antibodies are then used as the primary antibodies that bind to
denatured capsid proteins, for example an anti-AAV capsid
monoclonal antibody, such as the B1 anti-AAV-2 monoclonal antibody
(Wobus et al., J Virol. (2000) 74:9281-9293). A secondary antibody
is then used, one that binds to the primary antibody and contains a
means for detecting binding with the primary antibody, for example
an anti-IgG antibody containing a detection molecule covalently
bound to it, such as a sheep anti-mouse IgG antibody covalently
linked to horseradish peroxidase. A method for detecting binding is
used to semi-quantitatively determine binding between the primary
and secondary antibodies, for example a detection method capable of
detecting radioactive isotope emissions, electromagnetic radiation,
or colorimetric changes, such as a chemiluminescence detection kit.
For example, for SDS-PAGE, samples from column fractions can be
taken and heated in SDS-PAGE loading buffer containing reducing
agent (e.g., DTT), and capsid proteins were resolved on pre-cast
gradient polyacylamide gels (e.g., Novex). Silver staining may be
performed using SilverXpress (Invitrogen, CA) according to the
manufacturer's instructions. In one embodiment, the concentration
of AAV vector genomes (vg) in column fractions can be measured by
quantitative real time PCR (Q-PCR). Samples are diluted and
digested with DNase I (or another suitable nuclease) to remove
exogenous DNA. After inactivation of the nuclease, the samples are
further diluted and amplified using primers and a TaqMan.TM.
fluorogenic probe specific for the DNA sequence between the
primers. The number of cycles required to reach a defined level of
fluorescence (threshold cycle, Ct) is measured for each sample on
an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid
DNA containing identical sequences to that contained in the AAV
vector is employed to generate a standard curve in the Q-PCR
reaction. The cycle threshold (Ct) values obtained from the samples
are used to determine vector genome titer by normalizing it to the
Ct value of the plasmid standard curve. End-point assays based on
the digital PCR can also be used.
[0092] In one aspect, an optimized q-PCR method is provided herein
which utilizes a broad spectrum serine protease, e.g., proteinase K
(such as is commercially available from Qiagen). More particularly,
the optimized qPCR genome titer assay is similar to a standard
assay, except that after the DNase I digestion, samples are diluted
with proteinase K buffer and treated with proteinase K followed by
heat inactivation. Suitably samples are diluted with proteinase K
buffer in an amount equal to the sample size. The proteinase K
buffer may be concentrated to 2 fold or higher. Typically,
proteinase K treatment is about 0.2 mg/mL, but may be varied from
0.1 mg/mL to about 1 mg/mL. The treatment step is generally
conducted at about 55.degree. C. for about 15 minutes, but may be
performed at a lower temperature (e.g., about 37.degree. C. to
about 50.degree. C.) over a longer time period (e.g, about 20
minutes to about 30 minutes), or a higher temperature (e.g., up to
about 60.degree. C.) for a shorter time period (e.g., about 5 to 10
minutes) Similarly, heat inactivation is generally at about
95.degree. C. for about 15 minutes, but the temperature may be
lowered (e.g., about 70 to about 90.degree. C.) and the time
extended (e.g, about 20 minutes to about 30 minutes). Samples are
then diluted (e.g., 1000 fold) and subjected to TaqMan analysis as
described in the standard assay Additionally, or alternatively,
droplet digital PCR (ddPCR) may be used. For example, methods for
determining single-stranded and self-complementary AAV vector
genome titers by ddPCR have been described. See, e.g., M. Lock et
al, Hu Gene Therapy Methods, Hum Gene Ther Methods 2014 April;
25(2):115-25. doi: 10.1089/hgtb.2013.131 Epub 2014 Feb. 14.
[0093] 5.1.3 Manufacturing
[0094] The rAAV.hFIX vector can be manufactured as shown in the
flow diagram shown in FIG. 12. Briefly, cells (e.g. HEK 293 cells
or HeLa cells) are propagated in a suitable cell culture system and
transfected for vector generation. The rAAV.hFIX vector can then be
harvested, concentrated and purified to prepare bulk vector which
is then filled and finished in a downstream process.
[0095] Methods for manufacturing the gene therapy vectors described
herein include methods well known in the art such as generation of
plasmid DNA used for production of the gene therapy vectors,
generation of the vectors, and purification of the vectors. In some
embodiments, the gene therapy vector is an AAV vector and the
plasmids generated are an AAV cis-plasmid encoding the AAV genome
and the gene of interest, an AAV trans-plasmid containing AAV rep
and cap genes, and an adenovirus helper plasmid. The vector
generation process can include method steps such as initiation of
cell culture, passage of cells, seeding of cells, transfection of
cells with the plasmid DNA, post-transfection medium exchange to
serum free medium, and the harvest of vector-containing cells and
culture media. The harvested vector-containing cells and culture
media are referred to herein as crude cell harvest.
[0096] In one embodiment, the production plasmid is that shown in
SEQ ID NO: 11. In another embodiment, the production plasmid is
that shown in SEQ ID NO: 12. In yet another embodiment, the
production plasmid is that shown in SEQ ID NO: 16.
[0097] The crude cell harvest may thereafter be subject method
steps such as concentration of the vector harvest, diafiltration of
the vector harvest, microfluidization of the vector harvest,
nuclease digestion of the vector harvest, filtration of
microfluidized intermediate, purification by chromatography,
purification by ultracentrifugation, buffer exchange by tangential
flow filtration, and formulation and filtration to prepare bulk
vector.
[0098] In a specific embodiment, the methods used for manufacturing
the gene therapy vectors are described in Example 3 at Section 7,
infra.
[0099] 5.2 Patient Population
[0100] Severe hemophilia B patients are the chosen study population
for several reasons. Severe hemophilia B patients are defined as
having less than 1% of normal Factor IX (FIX) activity thus
requiring frequent infusions of FIX to control their bleeding
diathesis. This represents a significant burden with respect to
carrying on a normal life and in addition, the blood levels of FIX
go through the well-known peaks and troughs pattern, which is not
optimal. The fact that FIX blood levels in severe patients is less
than 1% makes it possible to reliably measure even low to moderate
increases in FIX blood levels after AAVrh.10.hFIX has been
administered. Recent clinical trials have borne out the validity of
this approach.
[0101] Patients who are candidates for treatment include adult
males .ltoreq.18 years of age, diagnosed with moderate/severe or
severe hemophilia B. In one embodiment, the patient has a baseline
FIX activity .ltoreq.2% of normal or documented history of FIX
activity .ltoreq.2%. In some embodiments, a patient .ltoreq.18
years of age can be treated. Candidates for treatment include
subjects who have had at least 3 bleeding episodes per year that
require on-demand treatment with FIX. Other candidates for
treatment include subjects who are treated with a prophylactic
regimen of FIX. Other criteria demonstrating that the subject is
appropriate for treatment includes at least 100 days exposure
history to FIX; no documented history of inhibitors (neutralizing
antibodies) to exogenous FIX; no known allergic reaction to
exogenous FIX or any component of rAAVrh.10.LSP.hFIXco.
[0102] Prior to treatment, the hemophilia B patient should be
assessed for NAb to the AAV serotype used to deliver the hFIX gene
(e.g, AAVrh.10). Such NAbs can interfere with transduction
efficiency and reduce therapeutic efficacy. Hemophilia B patients
that have a baseline serum NAb titer .ltoreq.1:5 are good
candidates for treatment with the rAAV.hFIX gene therapy protocol.
Treatment of Hemophilia B patients with titers of serum NAb >1:5
may require a combination therapy, such as plasmapheresis.
Alternative, empty capsids may be added to the final vector
formulation. See, Mingozzi F et al, 2013. PMID 23863832, which is
incorporated herein by reference. Further, transient co-treatment
with an immunosuppressant may be required to combat T cell response
to the capsid or transgene product. Immunosuppressants for such
co-therapy include, but are not limited to, steroids,
antimetabolites, T-cell inhibitors, and alkylating agents. For
example, such transient treatment may include a steroid (e.g.,
prednisole) dosed once daily for 7 days at a decreasing dose, in an
amount starting at about 60 mg, and decreasing by 10 mg/day (day 7
no dose). Other doses and medications may be selected.
[0103] Subjects may be permitted to continue their standard of care
treatment(s) (e.g., recombinant FIX therapy) prior to and
concurrently with the gene therapy treatment at the discretion of
their caring physician. In the alternative, the physician may
choose to stop standard of care therapies prior to administering
the gene therapy treatment and, optionally, resume standard of care
treatments as a co-therapy after administration of the gene
therapy.
[0104] Desirable endpoints of the gene therapy regimen are an
increase in FIX activity to 5% of normal from baseline up to 1
year, 5 years, 10 years or longer after administration of the gene
therapy treatment. In one embodiment, patients achieve desired
circulating FIX levels (e.g., 5% or greater) after treatment with
AAVrh.10.hFIX, alone without the use of adjunctive treatments over
the duration of the study, or over a period of time during the
study. In another embodiment, patients achieve circulating FIX
levels of 10%, 15%, 20% or greater after treatment with
AAVrh.10.hFIX, alone without the use of adjunctive treatments over
the duration of the study or over a period of time during the
study.
[0105] Nevertheless, patients having one or more of the following
characteristics may be excluded from treatment at the discretion of
their caring physician:
[0106] 1. History of significant liver disease (ie; portal
hypertension).
[0107] 2. Significant hepatic inflammation or cirrhosis.
[0108] 3. Evidence of active hepatitis B virus (HBV) or hepatitis C
virus (HCV) infection.
[0109] 4. History of human immunodeficiency virus (HIV) infection
AND any of the following: CD4+ cell count <350 cells/mm.sup.3,
change in antiretroviral therapy regimen within 6 months prior to
Day 0, or plasma viral load >200 copies/ml, on 2 separate
occasions, as measured by PCR.
[0110] 5. Anti-AAVrh10 neutralizing antibody titer >1:5.
[0111] 6. Participation (current or previous) in another gene
therapy study.
[0112] 7. Participation in another investigational medicine study
within 3 months before screening.
[0113] In other embodiments, a caring physician may determine that
the presence of one or more of these physical characteristics
(medical history) should not preclude treatment as provided
herein.
[0114] 5.3. Dosing & Route of Administration
[0115] In another embodiment, provided herein is a method of
treating a patient having hemophilia B with an rAAV as described
herein. In one embodiment, about 1.times.10.sup.11 to about
1.times.10.sup.13 genome copies (GC) of the rAAV/kg patient body
weight are delivered the patient in an aqueous suspension. All
ranges described herein are inclusive of the endpoints.
[0116] In one embodiment, the rAAV.hFIX vector is delivered as a
single dose per patient. In one embodiment, the subject is
delivered the minimal effective dose (MED) (as determined by
preclinical study described in the Examples herein). As used
herein, MED refers to the rAAV.hFIX dose required to achieve 5% of
normal Factor IX activity.
[0117] As is conventional, the vector titer is determined on the
basis of the DNA content of the vector preparation. In one
embodiment, quantitative PCR or optimized quantitative PCR as
described in the Examples is used to determine the DNA content of
the rAAV.hFIX vector preparations. In one embodiment, digital
droplet PCR as described in the Examples is used to determine the
DNA content of the rAAV.hFIX vector preparations. In one
embodiment, the dosage is about 1.times.10.sup.11 genome copies
(GC)/kg body weight to about 1.times.10.sup.13 GC/kg, inclusive of
endpoints. In one embodiment, the dosage is 5.times.10.sup.11
GC/kg. In another embodiment, the dosage is 5.times.10.sup.12
GC/kg. In specific embodiments, the dose of rAAV.hFIX administered
to a patient is at least 5.times.10.sup.11 GC/kg, 1.times.10.sup.12
GC/kg, 1.5.times.10.sup.12 GC/kg, 2.0.times.10.sup.12 GC/kg,
2.5.times.10.sup.12 GC/kg, 3.0.times.10.sup.12 GC/kg,
3.5.times.10.sup.12 GC/kg, 4.0.times.10.sup.12 GC/kg,
4.5.times.10.sup.12 GC/kg, 5.0.times.10.sup.12 GC/kg,
5.5.times.10.sup.12 GC/kg, 6.0.times.10.sup.12 GC/kg,
6.5.times.10.sup.12 GC/kg, 7.0.times.10.sup.12 GC/kg, or
7.5.times.10.sup.12 GC/kg. Also, the replication-defective virus
compositions can be formulated in dosage units to contain an amount
of replication-defective virus that is in the range of about
1.0.times.10.sup.9 GC to about 1.0.times.10.sup.15 s GC. As used
herein, the term "dosage" can refer to the total dosage delivered
to the subject in the course of treatment, or the amount delivered
in a single (of multiple) administration.
[0118] In another embodiment, the composition is readministered at
a later date. Optionally, more than one readministration is
permitted. Such readministration may be with the same type of
vector or a different viral vector as described herein. In one
embodiment, the vector is readministered about 6 months after the
first administration. In another embodiment, the vector is
readministered about 1 year after the first administration. In
another embodiment, the vector is readministered about 2 years
after the first administration. In another embodiment, the vector
is readministered about 3 years after the first administration. In
another embodiment, the vector is readministered about 4 years
after the first administration. In another embodiment, the vector
is readministered about 5 years after the first administration. In
another embodiment, the vector is readministered about 6 years
after the first administration. In another embodiment, the vector
is readministered about 7 years after the first administration. In
another embodiment, the vector is readministered about 8 years
after the first administration. In another embodiment, the vector
is readministered about 9 years after the first administration. In
another embodiment, the vector is readministered about 10 years or
more after the first administration.
[0119] In one embodiment, the dosage is sufficient to increase the
Factor IX levels in the patient to 5% of normal. In another
embodiment, the dosage is sufficient to increase the Factor IX
levels in the patient to 10% of normal. In another embodiment, the
dosage is sufficient to increase the Factor IX levels in the
patient to 15% of normal. In another embodiment, the dosage is
sufficient to increase the Factor IX levels in the patient to 20%
or greater of normal. In another embodiment, the dosage is
sufficient to increase the Factor IX levels in the patient to 25%
or greater of normal. In another embodiment, the dosage is
sufficient to increase the Factor IX levels in the patient to 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or greater of
normal.
[0120] In some embodiments, rAAV.hFIX is administered in
combination with one or more therapies for the treatment of
hemophilia B, such as administration of recombinant FIX. In another
embodiment, rAAV.hFIX is administered as a combination product with
vectors having different AAV capsids. In one embodiment, the
combination product includes an rAAVrh.10.hFIX vector and an
rAAV3B.hFIX vector.
[0121] 5.4. Measuring Clinical Objectives
[0122] Measurements of efficacy of treatment can be measured by
transgene expression and activity as determined by plasma Factor IX
levels and Factor IX activity. Further assessment of efficacy can
be determined by clinical assessment of replacement Factor IX
requirements and frequency of spontaneous bleeding episodes. Such
assessments may be conducted twice a week for 4 weeks after the
administration of the product, weekly from week 6 to week 12,
monthly throughout the remainder of the first year and at 6 month
intervals for a total period of 5 years.
[0123] Safety of the gene therapy vector after administration can
be assessed by the number of adverse events, changes noted on
physical examination, and/or clinical laboratory parameters
assessed at multiple time points up to about 52 weeks post vector
administration. Although physiological effect may be observed
earlier, e.g., in about one week, in one embodiment, steady state
levels expression levels are reached by about 4 to about 8 weeks.
The following assessments may be conducted twice a week for 4 weeks
after the administration of the product, weekly from week 6 to week
12, monthly throughout the remainder of the first year and at 6
month intervals for a total period of 5 years. Such assessments
include: [0124] a. Physical examination [0125] b. ECG [0126] c.
Biochemical assessment: Serum electrolytes, BUN, creatinine,
calcium, phosphate, total protein, albumin, LDH, CPK, AST, ALT,
alkaline phosphatase, bilirubin [0127] d. Hematological assessment.
CBC and differential, coagulation profile [0128] e. Urinalysis
[0129] f. Immunological assessment: [0130] g. Serological response
to rh 10 capsid and to Factor IX [0131] h. T cell response to rh 10
capsid and Factor IX antigens [0132] i. Assessment of vector DNA;
qPCR measurements in plasma, urine and saliva.
[0133] hFIX increase achieved with rAAV.hFIX administration can be
assessed as a defined percent change in hFIX at about 4 to about 8
weeks, or at other desired timepoints, compared to hFIX levels of a
patient not having hemophilia B, i.e., so-called normal hFIX
levels, i.e., 100%. In another embodiment, the change is compared
to the patient's baseline hFIX levels. In one embodiment, the
desired efficacy is an increase in the Factor IX levels in the
patient to 5% of normal. In another embodiment, the dosage is
sufficient to increase the Factor IX levels in the patient to 10%
of normal. In another embodiment, the dosage is sufficient to
increase the Factor IX levels in the patient to 15% of normal. In
another embodiment, the dosage is sufficient to increase the Factor
IX levels in the patient to 20% or greater of normal.
[0134] As used herein, the rAAV.hFIX vector herein "functionally
replaces" or "functionally supplements" the patients defective FIX
with active FIX when the patient expresses a sufficient level of
FIX to achieve at least one of these clinical endpoints. Expression
levels of hFIX which achieve as low as about 5% to less than 100%
of normal wild-type clinical endpoint levels in a non-hemophilia
patient may provide functional replacement. Alternatively, levels
of hFIX which achieve as low as about 5% to less than 100% of
normal wild-type function patient may provide functional
replacement. In one embodiment, FIX activity is measured via
APTT.
[0135] In one embodiment, expression may be observed as early as
about 8 hours to about 24 hours post-dosing. One or more of the
desired clinical effects described above may be observed within
several days to several weeks post-dosing.
[0136] Long term (up to 260 weeks) safety and efficacy can be
assessed after rAAV.hFIX administration.
[0137] In one aspect, a regimen for delivery of a hFIX gene product
to a human patient is provided. The regimen comprises (a) delivery
of a first rAAV.hFIX vector comprising an expression cassette as
described herein; and (b) delivery of a second rAAV.hFIX vector
comprising an expression cassette as described herein, wherein the
first recombinant AAV vector or the second AAV vector has an AAV3B
capsid. In one embodiment, the other of the first or the second AAV
vector has an rh.10 capsid. Such regimens are described in
International Patent Application No. PCT/US16/42472, which is
incorporated herein by reference.
[0138] In one embodiment, a second administration of a rAAV.hFIX
vector is given. In one embodiment, the rAAV.hFIX vector of the
second administration has the same AAV capsid as provided with the
first dosage. In one embodiment, the rAAV.hFIX vector of the second
administration has an AAVrh.10 capsid. In another embodiment, the
rAAV.hFIX vector of the second administration has a different AAV
capsid as the vector of the first dose. In one embodiment, the
rAAV.hFIX vector of the second administration has a tropism for
liver. In one embodiment, the rAAV.hFIX vector of the second
administration has an AAV3B capsid.
[0139] In a further aspect, the invention involves targeting
hepatocytes of the patient.
[0140] In one aspect, the delivery of the first rAAV and the second
rAAV are temporally separated by at least about one month, at least
about three months, or about 1 year to about 10 years.
[0141] The viral vectors described herein may be used in preparing
a medicament for delivering hFIX to a subject (e.g., a human
patient) in need thereof, supplying functional hFIX to a subject,
and/or for treating hemophilia B disease.
[0142] In another aspect, an rAAV.hFIX vector as described herein
is provided for use in treating hemophilia B. In another aspect, a
combination product as described herein (e.g., an rAAVrh.10.hFIX
vector and an rAAV3B.hFIX vector) is provided herein for use in
treating hemophilia B. In another aspect, an rAAV.hFIX vector as
described herein is provided for the manufacture of a medicament
for treating hemophilia B. In another aspect, a combination product
as described herein (e.g., an rAAVrh.10.hFIX vector and an
rAAV3B.hFIX vector) is provided herein for the manufacture of a
medicament for treating hemophilia B.
[0143] The following examples are illustrative only and are not
intended to limit the present invention.
EXAMPLES
6. Example 1: Protocol for Treating Human Subjects
[0144] This Example relates to a gene therapy treatment for
patients with genetically confirmed X-linked hemophilia B due to
mutations in the clotting factor 9 (FIX) gene. In this example, the
gene therapy vector, AAVrh.10.LSP.hFIX, a replication deficient
adeno-associated viral vector rh. 10 (AAVrh.10) expressing hFIX is
administered to patients with hemophilia B. Efficacy of treatment
can be assessed using FIX levels as a surrogate for transgene
expression. Primary efficacy assessments include FIX levels during
the first 12 weeks post treatment, with persistence of effect
followed thereafter for at least 1 year. Long term safety and
persistence of transgene expression may be measured post-treatment
in plasma samples.
[0145] 6.1. Gene Therapy Vector
[0146] The AAVrh.10.LSP.hFIXco vector consists of the AAV vector
active ingredient and a formulation buffer. The external AAV vector
component is a serotype rh.10, T=1 icosahedral capsid consisting of
60 copies of three AAV viral proteins, VP1, VP2, and VP3, at a
predicted ratio of 1:1:10. The capsid contains a single-stranded
DNA recombinant AAV (rAAV) vector genome (FIG. 1).
[0147] The genome contains a human factor IX (FIX) transgene
flanked by the two AAV inverted terminal repeats (ITRs). The
transgene expression cassette comprises an enhancer, promoter,
intron, the codon optimized human factor IX (hFIX) coding sequence
(SEQ ID NO: 2), and polyadenylation (polyA) signal. The ITRs are
the genetic elements responsible for the replication and packaging
of the genome during vector production and are the only viral cis
elements required to generate rAAV. Expression of the human FIX
coding sequence is driven from the hepatocyte-specific
thyroxine-binding globulin (TBG) promoter. Two copies of the alpha
1 microglobulin/bikunin (APB) enhancer element precede the TBG
promoter to stimulate promoter activity. Together these elements
(two copies of the APB enhancer and TBG promoter) are termed "LSP"
as shown in FIG. 1. See, Wang et al, Sustained correction of
bleeding disorder in hemophilia B mice by gene therapy, PNAS,
96:3906-10 (March 1999), which is incorporated herein by reference.
A human beta globin IVS2 intron and Woodchuck hepatitis virus
posttranscriptional regulatory element (WPRE) are present to
further enhance expression and a bovine growth hormone polyA signal
is included to mediate termination of human FIX mRNA transcripts.
The vector is supplied as a suspension of AAVrh.10.TBG.hFIX vector
in formulation buffer. The formulation buffer is 0.001% Pluronic
F-68 in TMN200 (200 mM sodium chloride, 1 mM magnesium chloride, 20
mM Tris, pH 8.0).
[0148] Details of the vector manufacturing and characterization of
the vectors, are described in the sections below.
[0149] 6.2. Patient Population
[0150] Severe hemophilia B patients are the chosen study population
for several reasons. Severe hemophilia B patients are defined as
having less than 1% of normal Factor IX (FIX) activity thus
requiring frequent infusions of FIX to control their bleeding
diathesis. This represents a significant burden with respect to
carrying on a normal life and in addition, the blood levels of FIX
go through the well-known peaks and troughs pattern, which is not
optimal. The fact that FIX blood levels in severe patients is less
than 1% makes it possible to reliably measure even low to moderate
increases in FIX blood levels after AAVrh.10/hFIX has been
administered. Recent clinical trials have borne out the validity of
this approach.
[0151] In one embodiment, the patient has one of the mutations
identified in the hFIX sequence shown in FIG. 30A. In another
embodiment, the patient has two of the mutations identified in the
hFIX sequence shown in FIG. 30A. In another embodiment, the patient
has three or more of the mutations identified in the hFIX sequence
shown in FIG. 30A.
[0152] Patients who are candidates for treatment include adult
males .gtoreq.18 years of age, diagnosed with moderate/severe or
severe hemophilia B. In one embodiment, the patient has a baseline
FIX activity .ltoreq.2% of normal or documented history of FIX
activity .ltoreq.2%. In some embodiments, a patient <18 years of
age can be treated. Candidates for treatment include subjects who
have had at least 3 bleeding episodes per year that require
on-demand treatment with FIX. Other candidates for treatment
include subjects who are treated with a prophylactic regimen of
FIX. Other criteria demonstrating that the subject is appropriate
for treatment includes At least 100 days exposure history to FIX;
no documented history of inhibitors (neutralizing antibodies) to
exogenous FIX; no known allergic reaction to exogenous FIX or any
component of rAAVrh.10.LSP.hFIXco.
[0153] Patients that are treated can have a baseline serum AAVrh.10
neutralizing antibody (NAb) titer .ltoreq.1:5. Subjects may be
permitted to continue their standard of care treatment(s) (e.g.,
recombinant FIX) prior to and concurrently with the gene therapy
treatment at the discretion of their caring physician. In the
alternative, the physician may prefer to stop standard of care
therapies prior to administering the gene therapy treatment and,
optionally, resume standard of care treatments as a co-therapy
after administration of the gene therapy. Desirable endpoints of
the gene therapy regimen are sustained FIX activity levels >5%
of normal after administration of the gene therapy treatment.
[0154] 6.3. Dosing & Route of Administration
[0155] Patients receive a single dose of AAVrh.10.LSP.hFIX
administered via a peripheral vein by infusion. Injection may be a
bolus, or infusion over about 10 or about 60 minutes. The dose of
AAVrh. 10.LSP.hFIX administered to a patient is about
5.times.10.sup.11 GC/kg or 1.6.times.10.sup.12 GC/kg or
5.times.10.sup.12 GC/kg. In order to ensure that empty capsids are
removed from the dose of AAVrh. 10.LSP.hFIX that is administered to
patients, empty capsids are separated from vector particles by
cesium chloride gradient ultracentrifugation or by ion exchange
chromatography during the vector purification process, as discussed
above.
[0156] 6.4. Measuring Clinical Objectives
[0157] Primary assessments are for safety of the administered
product. The following assessments are conducted twice a week for 4
weeks after the administration of the product, weekly from week 6
to week 12, monthly throughout the remainder of the first year and
at 6 month intervals for a total period of 5 years. [0158] a.
Physical examination [0159] b. ECG [0160] c. Biochemical
assessment: Serum electrolytes, BUN, creatinine, calcium,
phosphate, total protein, albumin, LDH, CPK, AST, ALT, alkaline
phosphatase, bilirubin [0161] d. Hematological assessment: CBC and
differential, coagulation profile [0162] e. Urinalysis [0163] f.
Immunological assessment: [0164] g. Serological response to rh 10
capsid and to Factor IX [0165] h. T cell response to rh 10 capsid
and Factor IX antigens [0166] i. Assessment of vector DNA; qPCR
measurements in plasma, urine and saliva
[0167] Secondary assessments are based on measurements of transgene
expression and activity as determined by [0168] a. Plasma Factor IX
levels and Factor IX activity [0169] b. Clinical assessment of
replacement Factor IX requirements and frequency of spontaneous
bleeding episodes
7. Example 2: Manufacture of AAVrh.10.LSP.hFIX
[0170] 7.1. Plasmids used to Produce AAVrh.10.LSP.hFIX
[0171] AAVrh.10.LSP.hFIX is produced by 3 plasmid DNA transfection
of human HEK 293 MCB cells with: [0172] (i) the pDTX.hFIX.101
vector plasmid described in Section 7.2.1 [0173] (ii) an AAV helper
plasmid termed pAAV2.rh10.KanR containing the AAV rep2 and cap rh10
wild-type genes described in Section 7.2.2 and [0174] (iii) a
helper adenovirus plasmid termed pAdDeltaF6(Kan) described in
Section 7.2.3.
[0175] 7.2.1 Cis Plasmid (Vector Genome Expression Construct):
[0176] pENN.AAV.LSP.hFIXco3T.WPRE.bGH.KanR contained the human FIX
expression cassette (FIG. 2; SEQ ID NO: 11. A plasmid including an
alternate FIX sequence of SEQ ID NO: 13 is shown in SEQ ID NO: 12).
A codon optimized sequence of the hFIX-Padua amino acid sequence is
shown in SEQ ID NO: 17. This plasmid encoded the rAAV vector
genome. The polyA signal for the expression cassette was from the
bovine growth hormone gene. The plasmid contained a liver-specific
promoter which consists of two repeats of
alpha-1microglobulin/bikunin enhancer followed by thyroid hormone
binding globulin promoter (TBG). In addition, the plasmid contained
the WPRE, described above.
[0177] To generate the cis plasmid used for production of
AAVrh.10.SLP.hFIX, the human FIX codon optimized cDNA was cloned
into an AAV2 ITR-flanked construct. Expression of the human FIXco
cDNA was driven from the TBG promoter with a human beta globin IVS2
intron. The polyA signal for the expression cassette was from the
bovine growth hormone. Two copies of the alpha 1
microglobulin/bikunin enhancer element preceded the TBG
promoter.
[0178] Description of the Sequence Elements
[0179] 1. Inverted terminal repeats (ITR): AAV ITRs are sequences
that are identical on both ends, but found in opposite orientation.
The AAV2 (GenBank #NC001401) ITR sequences function as both the
origin of vector DNA replication and the packaging signal for the
vector genome, when AAV and adenovirus (ad) helper functions are
provided in trans. As such, the ITR sequences represent the only
cis acting sequences required for vector genome replication and
packaging. The 5' ITR sequence used in the exemplified vector is
shown in SEQ ID NO: 3. The 3' ITR sequence used in the exemplified
vector is shown in SEQ ID NO: 9.
[0180] 2. ABP-TBG liver specific hybrid promoter: The promoter
sequences consist of two copies of the alpha 1
microglobulin/bikunin precursor enhancer element (ABP; Genbank
#X67082.1; SEQ ID NO: 4; FIG. 4) which precedes the 495 bp
hepatocyte-specific thyroxine-binding globulin (TBG; Genbank
#L13470.1 SEQ ID NO: 5; FIG. 5) promoter and is used to drive
high-level, liver specific hFIX gene expression.
[0181] 3. Human beta globin intervening sequence (IVS) 2 intron
(0.57 Kb; SEQ ID NO: 6). The 571 bp intron from the from the human
beta globin intervening sequence 2 (IVS2; Genbank #NC 000011.9) is
present in the vector expression cassette. The intron is
transcribed, but removed from the mature mRNA by splicing, bringing
together the sequences on either side of it. The presence of an
intron in an expression cassette has been shown to facilitate the
transport of mRNA from the nucleus to the cytoplasm, thus enhancing
the accumulation of the steady level of mRNA for translation. This
is a common feature in gene vectors intended for increased level of
gene expression. See, Antoniou et al, Nucleic Acids Research,
26(3):721-9 (1998), which is incorporated by reference.
[0182] 4. Human coagulation factor IX (FIX) cDNA (1.38 Kb; Genbank
#NM000133, complete CDS; SEQ ID NO: 1 shows native sequence; SEQ ID
NO: 2 shows codon optimized sequence; SEQ ID NO: 13 shows alternate
codon optimized sequence). The human coagulation factor 9 (FIX)
cDNA encodes a coagulation factor essential for the formation of
blood clots of 461 amino acids with a predicted molecular weight of
51.7 kD and an apparent molecular weight of 55 kD by SDS-PAGE.
Codon optimized human FIX cDNA sequences were synthesized by
Genart.
[0183] 5. Woodchuck hepatitis virus posttranscriptional regulatory
element: Between the coding sequence and the polyA is the woodchuck
hepatitis virus posttranscriptional regulatory element (Genbank
#J04514; SEQ ID NO: 7) with a single nucleotide mutation in the
woodchuck hepatitis virus X (WHX) protein translation start.
[0184] 6. Bovine growth hormone polyadenylation signal: (0.25 Kb;
SEQ ID NO. 8) The 215 bp bovine growth hormone polyadenylation
signal provides cis sequences for efficient polyadenylation of the
hFIX mRNA. This element functions as a signal for transcriptional
termination, a specific cleavage event at the 3' end of the nascent
transcript followed by addition of a long polyadenyl tail.
[0185] Subsequently the ampicillin resistance gene in
pENN.AAV.LSP.hFIXco3T.WPRE.bGH was excised and replaced with the
kanamycin resistance gene to give
pENN.AAV.LSP.hFIXco3T.WPRE.bGH.KanR (SEQ ID NO: 11).
[0186] 7.2.2 AAVrh10 Helper Plasmid pAAV2.Rh10.KanR
[0187] This AAVrh10 helper plasmid (8,036 bp) encodes the 4
wild-type AAV2 rep proteins and the 3 wild-type AAV VP capsid
proteins from serotype rh10. A schematic of the pAAV2.rh10.KanR
plasmid is shown in FIG. 15. A novel AAV sequence was obtained from
the liver tissue DNA of a rhesus monkey and designated AAV serotype
rh10. To create the chimeric packaging construct, the AAV2 cap gene
was removed from plasmid p5E18 and replaced with a PCR fragment of
the AAVrh10 cap gene amplified from a primate liver DNA to give
plasmid p5E18VD2/rh10. Note that the AAV p5 promoter which normally
drives rep expression is moved in this construct from the 5' end of
rep to the 3' end of the rh10 cap gene. This arrangement serves to
introduce a spacer between the promoter and the rep gene (i.e., the
plasmid backbone) to down-regulate expression of rep and increase
the ability to support high titer vector production. The plasmid
backbone in p5E18 is from pBluescript KS. All component parts of
the plasmid have been verified by direct sequencing. Finally the
ampicillin resistance gene was replaced by the kanamycin resistance
gene to give pAAV2/rh10 (Kan).
[0188] 7.2.3 pAdDeltaF6(Kan) Adenovirus Helper Plasmid
[0189] Plasmid pAdDeltaF6(Kan) is 15,774 bp in size. The plasmid
contains the regions of adenovirus genome that are important for
AAV replication, namely E2A, E4, and VA RNA (the adenovirus E1
functions are provided by the 293 cells), but does not contain
other adenovirus replication or structural genes. The plasmid does
not contain the cis elements critical for replication such as the
adenoviral inverted terminal repeats and therefore, no infectious
adenovirus is expected to be generated. It was derived from an E1,
E3 deleted molecular clone of Ad5 (pBHG10, a pBR322 based plasmid).
Deletions were introduced in the Ad5 DNA to remove expression of
unnecessary adenovirus genes and reduce the amount of adenovirus
DNA from 32 kb to .about.12 kb. Finally the ampicillin resistance
gene was replaced by the kanamycin resistance gene to give
pAd.DELTA.F6(kan). The identity of these 3 adenovirus genes were
confirmed by DNA plasmid sequencing performed by Qiagen Genomic
Services on the plasmid source stock that was sent to Aldevron Inc
for plasmid DNA manufacturing. DNA Analysis revealed 100% homology
with the 3 Adenovirus type 5 gene regions (GenBank Accession number
AF369965).
[0190] 7.2.4 Bacterial Master Cell Banks (MCB)
[0191] Bacterial MCBs for the three DNA production plasmids that is
used to support the manufacture of rAAVrh.10.LSP.hFIXco were
produced by Aldevron Inc. Cell banks were made from the expansion
of selected cultures and extensive testing was performed for
qualification of each bacterial MCB following Aldevron SOPs and in
accordance with CBER recommendations.
[0192] 7.2.5 Plasmid DNA Manufacturing
[0193] All plasmids used in the production process were produced by
Aldevron Inc. under its GMP-S.TM. quality system and infrastructure
utilizing the most salient features of cGMP manufacturing;
traceability, document control, and materials segregation.
[0194] 7.2.6 Human Embryonic Kidney (HEK) 293 Master Cell Bank
(MCB)
[0195] HEK 293 cells were originally generated by transforming HEK
cells with sheared adenovirus type 5 DNA by Frank Graham and
colleagues. The cells express the Ela and E1b gene products
required for high-titer rAAV production. HEK293 cells are adherent
and highly transfectable yielding high-titers of rAAV upon DNA
plasmid transfection. The original source of the HEK293 cell seed
was a vial of frozen cells from a research HEK293 cell bank (RCB)
prepared in the GTP Wilson Vector Core HEK293 Cell Bank in June of
2012 (James Wilson laboratory at University of Pennsylvania).
Subsequently, a vial was used to generate a second research bank
One vial from the second research bank was then used to generate
the MCB
[0196] 7.3 Recombinant AAV Vector Manufacturing
[0197] 7.3.1 Overview of the Manufacturing Process
[0198] The rAAVrh.10.LSP.hFIXco DP is produced in a controlled
environment consistent with FDA regulations ("Guidance for
Industry--cGMP for Phase 1 Investigational Drugs", July 2008),
which ensures the safety, identity, quality, purity and strength of
the manufactured biologic. A manufacturing process flow diagram is
shown in FIG. 12 and represents the rAAVrh.10.LSP.hFIXco vector
production process. The major reagents entering into the
preparation of the product have been indicated on the left side of
the diagram. A description of each production and purification step
is also provided. Product manufacturing follows a linear flow of
unit operations and utilizes disposable, closed bioprocessing
circuits unless otherwise specified. rAAVrh.10.LSP.hFIXco is the
sole product manufactured within a specified production suite, with
multiple BDS lots being anticipated that is tested separately prior
to pooling to generate the final DP(s). All solutions are sterile
filtered into sterile containers or are purchased sterile. Filters
used in sterile filtration are filter integrity tested post use.
All steps of the production process involving cell culture, from
cell seeding to supernatant collection are performed aseptically
using sterile, single-use disposable tubing and bag assemblies.
Cells are cultivated in Corning Cell Stacks or Hyperstacks and all
open manipulations re performed in class II biosafety cabinets in
an ISO Class 5 environment. The purification process is performed
in a closed system where possible however, column chromatography
manipulations are not viewed as a completely closed system. To
minimize this risk, single-use disposable flow paths re utilized as
part of the GE ReadyMate column chromatography production skid
platform. After column chromatography purification, the product is
diafiltered with final formulation buffer and sterile filtered to
yield the BDS and frozen at .ltoreq.-60.degree. C. After BDS
testing, the BDS is thawed, pooled, and diluted with sterile
formulation buffer (20 mM Tris pH 8.0, 1 mM MgCl2, 200 mM NaCl,
0.001% Pluronic F68) and Filled at SAFC in their Fill Suite.
Following Fill, the DP undergoes release testing and Quality
Assurance review. The entire production process from cell expansion
to fill is documented in executed Batch Record Documents (BRDs)
that undergoes staff and QA technical review.
[0199] 7.3.2 Description of the Manufacturing Process
[0200] 1. Cell Seeding: A qualified human embryonic kidney 293 cell
line is used for the production process. Cells are cultivated in
medium composed of Dulbecco's Modified Eagle Medium (DMEM),
supplemented with 10% gamma irradiated Fetal Bovine Serum (FBS).
The cells are anchorage dependent and cell disassociation is
accomplished using TrypLE Select, a non-animal cell dissociation
reagent. The cells are maintained at 37.degree. C. (+/-1.degree.
C.), in 5% (+/-0.5%) CO.sub.2 atmosphere.
[0201] 2. Transient Transfection: Following 3 days of growth (DMEM
media+10% FBS), Hyperstack cell culture media is replaced with
fresh, serum free DMEM media and transfected with the 3 production
plasmids using an optimized PEI precipitation method. All plasmids
used in the production process are produced by Aldevron Inc. under
its GMP-S.TM. quality system and infrastructure utilizing the most
salient features of cGMP manufacturing; traceability, document
control, and materials segregation.
[0202] Sufficient DNA plasmid transfection complex is prepared in
the BSC to transfect twenty Corning 36-layer HyperStacks (per BDS
lot). Initially a DNA/PEI mixture is prepared containing 3.0 mg of
pDTX.hFIX.101 vector plasmid, 60 mg of pAdDeltaF6(Kan), 30 mg of
pAAV2.rh10.KanR AAV helper plasmid and GMP grade PEI (PEIPro,
PolyPlus Transfection SA). After mixing well, the solution is
allowed to sit at room temperature for 25 min. and then added to
serum-free media to quench the reaction and then added to the
Corning 36-layer Hyperstacks. The transfection mixture is equalized
between all 36 layers of the Hyperstack and the cells are incubated
at 37.degree. C. (+/-2.degree. C.) in a 5% (+/-0.5%) CO.sub.2
atmosphere for 5 days.
[0203] 3. Cell Media Harvesting: Transfected cells and media are
harvested from each Hypertack using disposable bioprocess bags by
aseptically draining the medium out of the units. Following the
harvest of media, the .about.80 liter volume is supplemented with
MgCl.sub.2 to a final concentration of 2 mM (co-factor for
Benzonase) and Benzonase nuclease (Cat #: 1.016797.0001, Merck
Group) added to a final concentration of 25 units/ml. The product
(in a disposable bioprocess bag) is incubated at 37.degree. C. for
2-3 hr in an incubator to provide sufficient time for enzymatic
digestion of residual cellular and plasmid DNA present in the
harvest as a result of the transfection procedure. This step is
performed to minimize the amount of residual DNA in the final
vector DP. After the incubation period, NaCl is added to a final
concentration of 500 mM to aid in the recovery of the product
during filtration and downstream tangential flow filtration.
[0204] 4. Clarification: Cells and cellular debris are removed from
the product using a depth filter capsule (1.2 .mu.m/0.22 .mu.m)
connected in series as a sterile, closed tubing and bag set that is
driven by a peristaltic pump. The media is passed through a
Sartorius Sartoguard PES capsule filter (1.2 .mu.m/0.22 .mu.m,
Sartorius Stedim Biotech Inc.).
[0205] 5. Large-scale Tangential Flow Filtration: Volume reduction
(10-20 fold) of the clarified product is achieved using Tangential
Flow Filtration (TFF) using a custom sterile, closed bioprocessing
tubing, bag and membrane set produced by Spectrum Labs.
[0206] 6. Final Formulation and Sterile Filtration to yield the
BDS: TFF is used to achieve final formulation on the pooled AEX
fractions with a 100 kDa membrane (Spectrum Labs Inc.). The
filtered Purified Bulk is stored in sterile polypropylene tubes and
frozen at .ltoreq.-60.degree. C. in a quarantine location until
release for Final Fill.
[0207] 7. Final Fill: The frozen BDS is thawed (and pooled if
required) and filled into West Pharmaceutical's "Ready-to-Use"
(pre-sterilized) 2 mL glass vials and 13 mm stoppers and seals at a
fill volume >0.6 mL to <2.0 mL per vial. Individually labeled
vials is labeled to include protocol number, product name, lot
number, allocation number and stored in labeled boxes. Box labels
contain protocol number, product name, lot number, fill volume,
storage temperature, expiration date, route of administration,
client name and warning information. Labeled vials are transferred
to quarantine .ltoreq.-60.degree. C. until release.
[0208] 7.4.1 Proposed In-Process Testing
[0209] Tests are performed on In-Process samples during the
manufacturing and purification processes according to a detailed in
process sampling plan. The test name, description of the test, and
the laboratory in which they are performed are listed in Table 1
below.
TABLE-US-00001 TABLE 1 In-Process Methods Method Method Description
BIOBURDEN Based on the filtration of Sample onto 2 separate
membranes, incubation of the membranes on 2 media types, and
quantification of resulting colonies qPCR GC Titer GC titer
determination based on degradation of non-encapsidated DNA followed
by digestion of viral capsids. Released encapsidated DNA is
quantified by qPCR targeting the the BGH polyA DNA sequence
ENDOTOXIN Kinetic Chromogenic LAL Assay utilizing the cartridge
based system from Charles River Laboratories. Cartridges include
two sample wells to average duplicate results as well as two spike
recovery wells to verify lack of inhibition/enhancement. PURITY
Qualitative analysis of Purity based on SDS-PAGE of samples
MYCOPLASMA qPCR Cellular and Mycoplasma DNA is extracted and
quantified by A260 spectrophotometry. DNA is tested at a
concentration of 120 .mu.g/mL using a qPCR kit capable of
identifying the most common species of mycoplasma. HCP ELISA
Quantification of HEK293 Host Cell Protein by a Cygnus HEK293
ELISA. HC-DNA qPCR Quantification of host cell DNA using qPCR
targeting the 18S gene and HEK293 gDNA BENZONASE ELISA
Quantification of Benzonase using a commercial Benzonase ELISA
LEACHED AVB LIGAND Quantification of leached camilid antibody ELISA
fragment using a commercial AVB ELISA BSA ELISA Quantification of
BSA using a commercial BSA ELISA
[0210] 7.4.2 Proposed Bulk Drug Substance Testing
[0211] Table 2 below provides details of the proposed BDS release
testing performed.
TABLE-US-00002 TABLE 2 BPS Release Test Methods Test Method and
Description Acceptance Criteria Bioburden USP <61> <1
CFU/10 ml Endotoxin USP <85> <5 EU/mL In-Vitro Assay for
Viral Contaminants - Not Detected Test Article is applied to Vero,
MRC-5 & A549 cells and are monitored for viral contaminants
.sup.1 Mycoplasma USP <63>.sup.1 Negative Osmolality USP
<785> Osm 350-450 pH USP <791> 7.0-8.5 Appearance -
Visual inspection for Clear to Slightly Color, Appearance &
Clarity Opaque, Colorless to Faint White Solution GC Titer by qPCR
targeting the .gtoreq.1.0 .times. 1013 GC/mL BGH poly A vector DNA
sequence AAV Purity Determination by Purity >90% with no
SDS-PAGE single impurity >4% AAV Identity - SDS-PAGE Western
Blot Conforms to Reference analysis with anti-AAV antibodies Sample
Potency - FIX expression by human FIX Report Result ELISA following
infection of Huh7 cells in vitro FIX Identity - FIX expression by
human FIX ELISA following infection of Huh7 cells in vitro .sup.1
Test Material for this assay is sampled at the time of Media &
Cell harvest
[0212] Table 3 below provides details of the proposed BDS
characterization testing performed.
TABLE-US-00003 TABLE 3 BDS Characterization Test Methods Test
Method and Description Acceptance Criteria AAV Capsid Protein
ratio: CE-SDS Report Result Empty:Full particle ratio by AUC Report
Result Empty:Full particle ratio by OD 260/280 Report Result GC
titer by OD 260/280 Report Result Infectious Unit Titer: RC-32
cells w/ Report Result qPCR Detection (Bovine GH polyA target)
Replication competent AAV (RCAAV) Report Result detection by triple
passage on HEK293 cells + Ad5 Plasmid DNA (free and packaged) by
Report Result qPCR to Kan gene target HEK293 E1a DNA by qPCR Report
Result HEK 293 HCP by ELISA Report Result AAV Vector Genome
Sequencing 100% Match to expected sequence HC-DNA by qPCR Report
Result Residual Benzonase ELISA Report Result AVB Leached Ligand
ELISA Report Result BSA ELISA Report Result
8. Example 3: AAV.hFIXco in Animal Models
[0213] 8.1 Preliminary Animal Studies for Investigation of
Hemophilia B Gene Therapy
[0214] Several preliminary studies were conducted in animals to
prepare for the formal IND-enabling studies that are described
below. These studies employed either a precursor to our proposed
hFIX expression cassette or the actual proposed hFIX expression
cassette. The AAVrh10 capsid was constant throughout these studies
although in certain studies, comparisons were made to an AAV8
capsid bearing the same hFIX expression cassette. These preliminary
studies included assessments of safety and of the MED. These
studies included the AAV8 vs AAVrh10 expression comparison (data
not shown).
[0215] 8.2 AAVrh10 Gene Therapy in the Mouse Models of Hemophilia
B
[0216] 8.2.1 Evaluation of rAAVrh.10.LSP.hFIXco in a Factor IX
Knockout Mouse Model
[0217] A Factor IX knockout mouse model was developed as an
appropriate animal model for studying the efficacy of delivery of
Factor IX by way of AAV gene therapy vectors and was used
previously by numerous investigators for research studies and for
IND-enabling studies. See, Wang, Lili, et al. "A factor
IX-deficient mouse model for hemophilia B gene therapy."
Proceedings of the National Academy of Sciences 94.21 (1997):
11563-11566. This model is a reasonable approximation of a severe
hemophilia B patient because there is no Factor IX protein produced
and the animals have a severe clotting dysfunction. The last 164
amino acids at the C terminus of the Factor IX protein and the 3'
untranslated region are deleted. There is no evidence of truncated
Factor IX mRNA or protein. The Factor IX knockout mouse was
backcrossed to C57Bl/6 strain background and was maintained by
homozygous female mating with hemizygous male.
[0218] rAAVrh.10.LSP.hFIXco (whose vector genome is nt 1 to nt 3951
of SEQ ID NO: 11) was evaluated in the Factor IX knockout mouse
model described above to verify Factor IX activity of the vector
and to provide a preliminary assessment of the minimal effective
dose (MED). The MED in this study was based on achieving
therapeutic levels of hFIX (5% of normal). The vector was assessed
at six doses after intravenous administration: 8.times.10.sup.7,
2.7.times.10.sup.8, 2.7.times.10.sup.9, 2.7.times.10.sup.10,
2.7.times.10.sup.11 and 8.times.10.sup.11 GC/mouse.
[0219] Mice were bled at 2 and 4 weeks following vector
administration and Factor IX antigen and activity levels were
determined by hFIX ELISA and aPTT, respectively. The following
observations were noted:
[0220] 1. Factor IX knockout mice receiving rAAVrh.10.LSP.hFIXco at
the dose of 2.7.times.10.sup.9 GC/mouse achieved normal levels of
hFIX activity.
[0221] 2. MED for rAAVrh. 10.LSP.hFIXco lied between
2.7.times.10.sup.8 and 2.7.times.10.sup.9 GC/mouse
(1.35.times.10.sup.10 GC/kg-1.35.times.10.sup.11 GC/kg).
[0222] 3. hFIX activity reached a plateau of close to 1000% of
normal level at the dose of 2.7.times.10.sup.10 GC/mouse, while
hFIX antigen reached a plateau of 1000% of normal at the dose of
2.7.times.10.sup.11 GC/mouse.
[0223] 4. Mice with super physiological levels of hFIX appeared
normal, and no animal death was observed at any of the six dose
groups (see FIG. 11).
[0224] 8.2.2 Evaluation of rAAVrh.10.LSP.hFIXco in C57Bl/6 Mice
[0225] A dose-response study was conducted in C57Bl/6 male mice.
rAAVrh. I0.LSP.hFIXco was assessed at four doses after intravenous
administration: 7.times.10.sup.8, 2.3.times.10.sup.9,
7.times.10.sup.9, and 2.3.times.10.sup.10 GC/mouse. Factor IX
levels were observed above therapeutic levels (5% of normal; 100%=5
ug/ml) at a dose of 2.3.times.10.sup.9 GC/mouse
(1.1.times.10.sup.11 GC/kg), and above normal levels at
2.3.times.10.sup.10 GC/mouse (1.1.times.10.sup.12 GC/kg). See FIG.
14. The hFIX antigen levels in C57Bl/6 mice were about 3-fold lower
than those in Factor IX knockout mice.
[0226] 8.3 Observed Differences in Gene Expression Between Mice and
Non-Human Primate (NHP)
[0227] 8.3.1 Introduction
[0228] An important factor in comparing relative expression of AAV
vectors in different species is the rationale for scaling the dose.
The method for dosing in AAV gene therapy studies directed to liver
is based on total mass of the organism. The only complete data set
of mouse, non-human primate (NHP) and human that is available for
liver directed gene therapy is with AAV8 in patients with
hemophilia B. Review of these data is nonetheless complicated due
to changes in the quantitative assessment of vector titer reported
by the inventors during the development of the product. A review of
the published data suggested a 10-20-fold reduction in expression
when comparing mice to monkeys and expression that is similar to,
or slightly reduced, when comparing monkeys to humans.
[0229] Studies comparing transgene expression in mice and nonhuman
primates with vectors based on AAV8 and AAVrh10 are summarized
below. The same method of assessing vector titer was identical
throughout except as otherwise noted. Both methods were based on
TaqMan qPCR although the standard method yielded results that were
2-3-fold lower than the optimized method.
[0230] 8.3.2 Relative Expression of EGFP in Mice and Macaques for
AAV8 and AAVrh10
[0231] Following systemic injection of 3.times.10.sup.12 GC/kg AAV8
or AAVrh10.TBG.EGFP in two rhesus macaques each, GFP expression was
lower than that observed in mice receiving a similar dose (data not
shown). Transduction efficiency was quantified by percentage of
GFP-positive area in the liver and GFP intensity (data not shown).
The reduction was 4 and 14-fold lower with AAV8 based on percentage
transduction and intensity, respectively. The reduction was
5.5-fold with AAVrh10 based on percentage transduction.
[0232] 8.4 Animal Studies
[0233] Three animal models are used to further evaluate the safety,
biodistribution and efficacy of rAAVrh.10. LSP.hFIXco: C57Bl/6
wild-type mouse, a Factor IX knockout mouse model, and a non-human
primate model.
[0234] 8.4.1 Hemophilia B Mouse Model
[0235] 1. Animal Model
[0236] The Factor IX knockout mouse model is an appropriate animal
model for studying the efficacy of delivery of Factor IX by way of
AAV gene therapy vectors. This model has been used previously by
numerous investigators for research studies and for IND-enabling
studies. This model is a reasonable approximation of a severe
hemophilia B patient because there is no Factor IX protein produced
and the animals have a severe clotting dysfunction.
[0237] Two cohorts of male FIX-KO mice were included in the study.
The initial cohort (Subset A) was evaluated for 90 days following
dose administration on Day 0 and was terminated on Day 90. A second
cohort (Subset B) was evaluated for 28 days following dose
administration on Day 0 and was terminated on Day 28.
[0238] 2. Administration
[0239] For both Subset A and Subset B, male FIX-KO mice (7/group)
were administered vector doses once on Day 0 by intravenous
injection into the tail vein. The vector was formulated in Vehicle
Buffer composed of 0.001% Pluronic F-68 in TMN200 (200 mM sodium
chloride, 1 mM magnesium chloride, 20 mM Tris, pH 8.0). The vector
dose levels tested were 1.6.times.10.sup.10 GC/kg,
5.0.times.10.sup.10 GC/kg, 1.6.times.10.sup.11 GC/kg,
5.0.times.10.sup.11 GC/kg, 5.0.times.10.sup.12 GC/kg, and
5.0.times.10.sup.13 GC/kg. Each mouse received the test article
formulations at a dose volume of 0.150 mL/mouse. Dose
concentrations were calculated based on the average Day 0 mouse
weight for each dose group.
[0240] For each subset, concurrent control groups were administered
the vehicle once on Day 0, also by intravenous injection into the
tail vein at a dose volume of 0.150 mL/mouse.
[0241] 3. Justification of Gender of the Animal
[0242] Only male FIX-KO mice were used for this study, as
hemophilia B is an X-linked genetic disorder that affects only
males. Proposed clinical trials are carried out in male hemophilia
B patients.
[0243] 4. Coagulation
[0244] Measurements of PT (prothrombin time) in FIX-KO male mouse
plasma were determined using a Stago ST Art Start Hemostasis
Coagulation Analyzer set to PT mode and Dade Innovin Reagent.
Normal PT values for male FIX-KO mice range from 6.8-8.2 seconds
with mean and standard deviation of 7.3 t 0.3 seconds, respectively
(FIG. 7).
[0245] Measurements of hFIX activity in FIX-KO male mouse plasma
were determined by a one-step aPTT-based Factor IX assay using a
Stago ST Art Start Hemostasis Coagulation Analyzer set to aPTT
mode. A standard curve was generated using known concentrations of
human FIX in FIX deficient plasma. Samples were compared to the
standard curve to obtain the relative activity of hFIX within each
plasma sample. Samples at higher concentrations required dilution
in FIX deficient plasma to obtain levels within the interpretable
range. Results were presented as percent of normal human plasma
activity.
[0246] 4. hFIX Protein Levels
[0247] The circulating levels of hFIX in mouse plasma were
determined using an hFIX ELISA using a coating antibody
(Haematologic Technologies) and an HRP-conjugated detecting
antibody (Cedarlane) specific to human factor IX (FIG. 6).
[0248] 5. Anti-hFIX Antibodies
[0249] The presence of murine anti-hFIX antibodies in mouse serum
collected on Days 28 and 90 was determined using an anti-hFIX IgG
ELISA assay.
[0250] 6. Quantification of Vector Genomes and hFIXco mRNA in
Liver
[0251] Sections of the liver were removed and placed into sterile
tubes, snap-frozen on dry ice, and stored at <-65.degree. C. for
QPCR and RT-QPCR studies. DNA and RNA were extracted. QPCR and
RT-QPCR assays were performed on the extracted liver DNA/RNA to
measure vector DNA copies and hFIXco transcript levels in the liver
by real-time PCR (TaqMan Universal Master Mix, Applied
Biosystems).
[0252] 7. Statistical Analysis
[0253] For FIX expression data, cohort average and standard
deviation were calculated and reported. Analysis of variance was
performed using GraphPad Prism 6 to determine any vector-related
effects.
[0254] 8.4.2 Non-Clinical Study of rAAVrh.10.LSP.hFIXco in Rhesus
Macaque
[0255] The primary objective of this non-GLP study is to evaluate
the potential vector related toxicity and biodistribution in rhesus
macaques to support the safety of rAAVrh.10.LSP.hFIXco for the
clinical trial. rAAVrh.10.LSP.hFIXco is examined at
1.0.times.10.sup.13 GC/kg which is 2 fold higher than the proposed
clinical high dose.
[0256] Male rhesus macaques aged 2 to 3 years are used for this
study. Only male animals are used in the study since hemophilia B
is an X-linked genetic disorder. A minimum of three animals per
time point are enrolled into the study. Animals are screened for
pre-existing neutralizing antibodies (NAbs) to AAVrh.10 before
study starts, only animals with neutralizing antibodies
(NAbs)<1:10 are used in this study.
[0257] Animals receive vector via the saphenous vein in a total
volume of 10 ml. After vector administration, the animals are
monitored daily for general observations. At time of necropsy, on
day 90 and 360, the organs (such as brain, lung, muscle, kidney,
heart, spleen, liver, stomach, small intestine, large intestine,
pancreas, lymph node, testis, haired skin, gross lesions if any)
are harvested for a complete gross pathology and histopathology
examination. Additionally, blood is collected for a complete serum
chemistry panel, hematology, and gene expression at selected time
points. hFIX protein levels in the plasma are analyzed by a hFIX
ELISA assay. For immunology, antigen specific T-cell responses are
examined on days 14, 28, and every 28 days using an interferon
gamma ELISPOT assay which allows an examination of antigen specific
T-cells directed against either the capsid or transgene. Humoral
immune responses to AAV capsid at selected time points are examined
using a neutralizing or binding antibody assay. Antibody responses
(inhibitors) to human Factor IX are examined using an ELISA.
[0258] C57Bl/6 and the existence of a large body of data from
multiple sponsors using different AAV vectors makes the safety data
developed in this model relevant. The non-human primate is an
appropriate model in particular for studying the potential immune
responses to AAV vectors and the tolerability of high doses of AAV
vectors in an animal that is closely related to humans.
[0259] 8.5. Testing of Vector
[0260] Characterization assays including serotype identity, empty
particle content and transgene product identity are performed.
Descriptions of all the assays appear below.
[0261] 8.5.1 Genomic Copy (GC) Titer
[0262] An optimized quantitative PCR (oqPCR) assay is used to
determine genomic copy titer by comparison with a cognate plasmid
standard. The oqPCR assay utilizes sequential digestion with DNase
I and Proteinase K, followed by qPCR analysis to measure
encapsidated vector genomic copies. DNA detection is accomplished
using sequence specific primers targeting the hBG polyA region in
combination with a fluorescently tagged probe hybridizing to this
same region. Comparison to the plasmid DNA standard curve allows
titer determination without the need of any post-PCR sample
manipulation. A number of standards, validation samples and
controls (for background and DNA contamination) have been
introduced into the assay. This assay has been qualified by
establishing and defining assay parameters including sensitivity,
limit of detection, range of qualification and intra and inter
assay precision. An internal AAVrh.10 reference lot was established
and used to perform the qualification studies.
[0263] 8.5.2 Vector Capsid Identity: AAV Capsid Mass Spectrometry
of VP3
[0264] Confirmation of the AAV2/rh.10 serotype of the vector is
achieved by an assay based upon analysis of peptides of the VP3
capsid protein by mass spectrometry (MS). The method involves
multi-enzyme digestion (trypsin, chymotrypsin and endoproteinase
Glu-C) of the VP3 protein band excised from SDS-PAGE gels followed
by characterization on a UPLC-MS/MS on a Q-Exactive Orbitrap mass
spectrometer to sequence the capsid protein. A tandem mass
spectrometry (MS) method was developed that allows for
identification of certain contaminant proteins and deriving peptide
sequence from mass spectra.
[0265] 8.5.3 Empty to Full Particle Ratio
[0266] Vector particle profiles are using analytical
ultracentrifugation (AUC) Sedimentation velocity as measured in an
analytical ultracentrifuge is an excellent method for obtaining
information about macromolecular structure heterogeneity,
difference in confirmation and the state of association or
aggregation. Sample was loaded into cells and sedimented at 12000
RPM in a Beckman Coulter Proteomelab XL-I analytical
ultracentrifuge. Refractive index scans were recorded every two
minutes for 3.3 hours. Data are analyzed by a c(s) model (Sedfit
program) and calculated sedimentation coefficients plotted versus
normalized c(s) values. A major peak representing the monomeric
vector should be observed. The appearance of peaks migrating slower
than the major monomeric peak indicates empty/misassembled
particles. The sedimentation coefficient of the empty particle peak
is established using empty AAV8 particle preparations. Direct
quantitation of the major monomeric peak and preceding peaks allow
for the determination of the empty to full particle ratio.
[0267] 8.5.4 Infectious Titer
[0268] The infectious unit (IU) assay is used to determine the
productive uptake and replication of vector in RC32 cells (rep2
expressing HeLa cells). Briefly, RC32 cell in 96 well plates are
co-infected by serial dilutions of vector and a uniform dilution of
Ad5 with 12 replicates at each dilution of rAAV. Seventy-two hours
after infection the cells are lysed, qPCR is performed to detect
rAAV vector amplification over input. An end-point dilution TCID50
calculation (Spearman-Karber) is performed to determine a
replicative titer expressed as IU/ml. Since "infectivity" values
are dependent on particles coming into contact with cells, receptor
binding, internalization, transport to the nucleus and genome
replication, they are influenced by assay geometry and the presence
of appropriate receptors and post-binding pathways in the cell line
used. Receptors and post-binding pathways critical for AAV vector
import are usually maintained in immortalized cell lines and thus
infectivity assay titers are not an absolute measure of the number
of "infectious" particles present. However, the ratio of
encapsidated GC to "infectious units" (described as GC/IU ratio)
can be used as a measure of product consistency from lot to
lot.
[0269] 8.6 Readministration with Second Vector
[0270] 8.6.1 Readministration of AAV3B or AAV5
[0271] The efficiency of vector readministration using AAV3B or
AAV5 in rhesus macaques previously treated with AAVrh10 or AAV8
vectors was evaluated. Vectors as shown in Table 4 were produced as
previously described in which the vector was recovered from the
supernatant following triple transfection in HEK293 cells and
purified on an iodixanol gradient. Vector titer was determined by a
digital PCR method.
[0272] Twenty four male rhesus macaques (3-5 years old) were
enrolled into study in 8 groups (n=3/group; Table 1) based on the
status of pre-existing NAb. Macaques were injected on day zero with
1.0.times.10.sup.13 GC/kg of the AAV vector as shown in Table 4. At
week 12, macaques received a second injection with
1.0.times.10.sup.13 GC/kg of the AAV vector as shown in Table 4.
Liver biopsies were performed at week 2 and week 14, and a necropsy
was performed at week 26.
TABLE-US-00004 TABLE 4 Cohort and Vector Summary Animal Cohort ID
1st Injection 2nd injection G1A RA0931 PBS AAV3B.T8G.rhAFP RA1388
RQ9745 G1B RA0923 PBS AAV5.TBG.rhAFP RA1275 RQ9383 G2A RA0985
AAVrh10.TBG.rhCG.WPRE AAV3B.TBG.rhAFP RQ9638 RQ9746 G2B RA0992
AAVrh10.TBG.rhCG.WPRE AAV5.TBG.rhAFP RA1322 RA1417 G3A RA1234
AAV8.TBG.rhCG.WPRE AAV3B.TBG.rhAFP RQ9737 RQ9751 G3B RA1339
AAV8.TBG.rhCG.WPRE AAV5.TBG.rhAFP RA1390 RQ9805 G4 RA0548
AAV3B.TBG.rhCG.WPRE N/A RA0658 RQ9840 G5 RA0968 AAV5.TBG.rhCG.WPRE
N/A RA1208 RA1239
[0273] Expression levels of transgenes (rhCG--rhesus chorionic
gonadotropin b subunit; rhAFP--rhesus alpha fetoprotein) in the
serum were measured by enzyme-linked immunosorbent assay (ELISA).
To measure vector DNA copies in liver, QPCR assays were performed
on total cellular DNA extracted from liver samples collected during
liver biopsy and necropsy. AAV NAb assay was performed as
previously described. Liver sections were stained with an anti-CG
antibody for imaging.
[0274] FIG. 16 shows a comparison of rhCG expression levels by
AAVrh10, AAV8, AAV3B and AAV5 vectors (first vector injection).
FIG. 17 shows expression of rhCG in the liver at different time
points. FIG. 18A-18D shows rhCG vector DNA copies in liver at
different time points by AAVrh10, AAV8, AAV3B and AAV5 vectors.
FIGS. 19A and 19B show rhAFP levels after readministration (second
vector injection) with AAV3B or AAV5 vectors expressing rhAFP. FIG.
20A and FIG. 20B show rhAFP vector genome copies in liver for the
listed vectors. FIG. 21 shows differential AAV Nab response in
macaques.
[0275] In naive animals, clade E vectors (AAVrh10 & AAV8)
demonstrated the highest levels of periportal gene transfer with
AAV5 vectors having the lowest. The periportal zone is nearest to
the entering vascular supply, receives the most oxygenated blood,
and is an important region of the liver for metabolic processes.
AAVrh10 and AAV5 elicited higher levels of neutralizing antibodies
(NAb) than AAV8 and AAV3B. Significant animal-to-animal variation
in transgene expression was noted with AAV3B in seronegative
animals.
[0276] Within the short time frame tested, NAb elicited from
AAVrh10 inhibited subsequent in vivo transduction with the
serologically distinct AAV3B serotype. Prior exposure to AAV8 did
not interfere with AAV3B transduction.
[0277] 8.7 Further Animal Studies
[0278] 8.7.1 Comparison of AAVrh10.hFIXco3T and
AAVrh10.hFIXco3T-Padua (SEQ ID NO: 16.
[0279] FIX knock out mice were treated as follows:
TABLE-US-00005 TABLE 5 Group (n = 6-7 Dose (QPCR) mice/group)
Vector GC/mouse G1 DTX101 3x10e7 G2 (AAVrh10.hFIXco3T) 1x10e8 G3
1x10e9 G4 1x10e10 G5 1x10e11 G6 3x10e11 G7 AAVrh10.hFIXco3T- 3x10e7
G8 Padua 1x10e8 G9 1x10e9 G10 1x10e10 G11 1x10e11 G12 3x10e11
[0280] Mice were bled at weeks 2, 4 and 6 and tested for hFIX
antigen by ELISA and activity (APTT), as discussed in Example 8.4.
At week 6, mice were euthanized by terminal bleeds (superchem and
CBC performed). Tissues were harvested for histology & liver
genome copies.
[0281] FIX antigen and activity levels at 2, 4, and 6 weeks are
shown in FIGS. 22-24 respectively. Vector genome copies in liver at
6 weeks are shown in FIG. 25. FIX-KO mice treated with
AAVrh0.hFIXco3T-Padua achieved similar hFIX antigen levels but 7-8
fold higher hFIX activity. FIX-KO mice treated with
1.times.10.sup.8 GC of AAVrh10.hFIXco3T-Padua achieved above
therapeutic levels (5% of normal) of hFIX activity. hFIX antigen
levels plateaued at the dose of 1.times.10.sup.11 GC/mouse, while
hFIX activity levels plateaued at 1.times.1010 GC/mouse, likely due
to the limitation of other factors or cofactor.
[0282] A time course of prothrombin time (PT) was performed at 2,
4, and 6 weeks (FIG. 26). Elevation of PT was observed in animals
treated with high doses of vector (0.times.1011 or 3.times.1011
GC/mouse), likely due to the over production of hFIX and exhaustion
of post translational modification pathways which are shared by
other factors in the coagulation pathway.
[0283] In summary, DTX101 and hFIXco3T-Padua treated mice expressed
similar levels of hFIX antigen. hFIXco3T-Padua treated mice had 7-8
fold higher FIX activity than DTX101-treated mice. Based on
activity levels, MED for DTX101 is between 1E8-1E9 GC/mouse in
hemophilia B mouse, MED for hFIX-Padua is <1E8 GC/mouse.
Abnormal PT was observed in mice treated with either vector at
doses .gtoreq.1E11 GC/mouse. DTX101 and hFIX-Padua treated mice
showed similar serum chemistry and hematologic parameters at w6.
Histology analysis on H&E stained tissue sections from the 3E11
GC dose groups showed similar findings in liver (minimal-mild
hepatitis) and heart (mild myocardial degeneration). In lung, 2 out
of 6 Padua (3E11 GC)-treated mice showed focal alveolar edema.
[0284] 8.8 Human Clinical Studies
[0285] Six patients were administered AAVrh10.hFIXco3T vector gene
therapy intravenously (i.v.) and composed the low-dose
(1.6.times.10.sup.12 GC/kg) and mid-dose cohort
(5.0.times.10.sup.12 GC/kg) of the hemophilia B clinical trial.
Table 7 below provides Enzyme-Linked ImmunoSpot; ELISPOT results
representing SFUs (spot forming units) per million lymphocytes at
various time-points throughout the study. AAV vector injections
were performed on a rolling basis as subjects were enrolled in the
trial. The ELISPOT results represent T-cell responses against
specific peptide pools from the AAV capsid of interest (AAVrh.10)
and transgene (FIX). All lymphocytes used in the ELISPOT assay were
isolated from peripheral blood and positive ELISPOT (T-cell)
responses are noted in bold font with an asterisk.
[0286] Intracellular cytokine staining (ICS) of CD4+ and CD8+
peripheral blood mononuclear cells (PBMCs) was performed at various
time-points from six human patients making up the low- and mid-dose
cohort of the trial discussed in the paragraph above. The graphs
depict the percentage of CD4+(FIG. 27A) and CD8+(FIG. 27B)
lymphocytes expressing lysosomal-associated membrane protein 1
(LAMP-1; CD107a), interferon-gamma (IFN.gamma.), tumor necrosis
factor alpha (TNF.alpha.), interleukin-2 (IL-2), or a combination
as noted (IFN.gamma.+ TNF.alpha.). No substantial percentages of T
cells were detected as expressing the cytokines described above in
a PBMC culture stimulated with AAV (top panels of FIG. 27A and FIG.
27B) or Factor IX (bottom panels of FIG. 27A and FIG. 27B) except
that Factor IX challenged PBMC of one patient collected on week 6
post treatment with medium dose of the AAV.hFIXco3T vector showed
about 1% IL-2 positive CD4+ T cells of the total memory T cells.
These results indicate that memory T cells recognizing AAV or
Factor IX were not induced and generated after administering
AAV.hFlXco3T at both dosages thus no obvious immunogenicity was
observed.
[0287] Patients from the low- and mid-dose cohort were screened
prior to and after AAV vector administration for neutralizing
antibodies (NAbs) and Immunoglobulin-G (IgG) responses to the AAV
capsid of interest (AAVrh.10) from isolated serum. All subjects
except for one (Subject #3) showed NAbs below the limit of
detection on the day of AAV vector administration (Day 0). All
results are reported as the reciprocal of serum dilution. Serum
from the same patients were analyzed using a Luminex multiplex
system that allowed simultaneous testing against 41 different
analytes linked with inflammation. The resulting data was plotted
as a heatmap. Any analyte showing an increase in activity was coded
in red while decreases were coded in blue in FIG. 29. The NAb titer
of the six patients is shown below in Table 6.
TABLE-US-00006 TABLE 6 Dose Low High Subject 1 2 3 4 5 6 AAVrh10
<5 <5 10 <5 <5 <5 Nab
[0288] Each subject's mutation and MHC Class I binding prediction
was examined for any unique indicators. Each subject's mutation is
denoted by a bolded letter in the FIX amino acid sequence. Only 5
mutations are noted because Subject #1007001's mutation is a
non-coding mutation (FIG. 30A). Using prediction software, the MHC
Class I binding affinity to various alleles was predicted (FIG.
30B).
TABLE-US-00007 TABLE 7 AAVrh.10 AAVrh.10 AAVrh.10 FIX FIX Subject
Medium Pool A Pool B Pool C Pool A Pool B Number Study Visit
(SFU/1E+6 (SFU/1E+6 (SFU/1E+6 (SFU/1E+6 (SFU/1E+6 (SFU/1E+6
(XXXXXXX) (Day or Week) PBMCs) PBMCs) PBMCs) PBMCs) PBMCs) PBMCs)
1001001 Day 0 Predose 10 18 13 23 13 10 1001001 Week 16 0 5 5 3 8 3
1001001 Week 6 5 0 3 5 0 8 1001001 Week 8 0 5 0 0 3 8 1001001 Week
32 3 0 8 3 23 3 1001001 Week 40 25 43 33 33 60 50 1001001 Week 48
10 5 13 20 10 10 1001002 Day 0 Predose 5 8 5 0 5 10 1001002 Week 6
13 10 18 8 3 3 1001002 Week 8 3 3 10 8 0 3 1001002 Week 16 0 0 5 0
3 3 1001002 Week 32 73 80 108 120 33 23 1001002 Week 40 8 23 13 20
13 15 1002002 Day 0 Predose 125 170 168 153 170 145 1002002 Week 6
25 38 53 20 20 33 1002002 Week 8 23 33 83* 73* 18 45 1007001 Dry 0
Predose 35 38 43 45 50 55 1007001 Week 6 8 38 50* 63* 28 18 1007001
Week 8 15 15 25 38 20 28 1007001 Week 16 13 15 15 25 25 10 4401002
Day 0 Predose 23 18 55 15 23 50 4401002 Week 6 15 40 133* 148* 13
25 4401002 Week 8 8 35 60* 85* 28 20 4401002 Week 12 0 3 13 10 8 3
4402003 Day 0 Predose PBMC Isolation Unsuccessful 4402003 Week 6 3
43 25 28 0 3 Bold* = Positive Response to Specific Peptide Pool
Positive Results must meet 2 criteria - 1) >40 SFU/1E+6 PBMCs
& 2) At least 3 times the Negative Control (Medium; Column
C).
Sequence Listing Free Text
[0289] The following information is provided for sequences
containing free text under numeric identifier <223>.
TABLE-US-00008 SEQ ID NO: (containing free text) Free text under
<223> 2 <223> constructed sequence 3 <223>
constructed sequence 4 <223> constructed sequence 5
<223> constructed sequence 6 <223> constructed sequence
7 <223> constructed sequence 8 <223> constructed
sequence 9 <223> constructed sequence 11 <223>
constructed sequence 12 <223> constructed sequence 13
<223> constructed sequence 14 <223> AAVrh.10 capsid 15
<223> constructed sequence 16 <223> constructed
sequence 17 <223> constructed sequence
[0290] All publications cited in this specification are
incorporated herein by reference. Similarly, the SEQ ID NOs which
are referenced herein and which appear in the appended Sequence
Listing are incorporated by reference. Also incorporated by
reference are U.S. Provisional Patent Application No. 62/323,375,
filed Apr. 15, 2016, US Provisional Patent Application No.
62/331,064, filed May 3, 2016, and U.S. Provisional Patent
Application No. 62/428,804, filed Dec. 1, 2016. While the invention
has been described with reference to particular embodiments, it
will be appreciated that modifications can be made without
departing from the spirit of the invention. Such modifications are
intended to fall within the scope of the appended claims.
Sequence CWU 1
1
1912802DNAHomo sapiens 1accactttca caatctgcta gcaaaggtta tgcagcgcgt
gaacatgatc atggcagaat 60caccaggcct catcaccatc tgccttttag gatatctact
cagtgctgaa tgtacagttt 120ttcttgatca tgaaaacgcc aacaaaattc
tgaatcggcc aaagaggtat aattcaggta 180aattggaaga gtttgttcaa
gggaaccttg agagagaatg tatggaagaa aagtgtagtt 240ttgaagaagc
acgagaagtt tttgaaaaca ctgaaagaac aactgaattt tggaagcagt
300atgttgatgg agatcagtgt gagtccaatc catgtttaaa tggcggcagt
tgcaaggatg 360acattaattc ctatgaatgt tggtgtccct ttggatttga
aggaaagaac tgtgaattag 420atgtaacatg taacattaag aatggcagat
gcgagcagtt ttgtaaaaat agtgctgata 480acaaggtggt ttgctcctgt
actgagggat atcgacttgc agaaaaccag aagtcctgtg 540aaccagcagt
gccatttcca tgtggaagag tttctgtttc acaaacttct aagctcaccc
600gtgctgagac tgtttttcct gatgtggact atgtaaattc tactgaagct
gaaaccattt 660tggataacat cactcaaagc acccaatcat ttaatgactt
cactcgggtt gttggtggag 720aagatgccaa accaggtcaa ttcccttggc
aggttgtttt gaatggtaaa gttgatgcat 780tctgtggagg ctctatcgtt
aatgaaaaat ggattgtaac tgctgcccac tgtgttgaaa 840ctggtgttaa
aattacagtt gtcgcaggtg aacataatat tgaggagaca gaacatacag
900agcaaaagcg aaatgtgatt cgaattattc ctcaccacaa ctacaatgca
gctattaata 960agtacaacca tgacattgcc cttctggaac tggacgaacc
cttagtgcta aacagctacg 1020ttacacctat ttgcattgct gacaaggaat
acacgaacat cttcctcaaa tttggatctg 1080gctatgtaag tggctgggga
agagtcttcc acaaagggag atcagcttta gttcttcagt 1140accttagagt
tccacttgtt gaccgagcca catgtcttcg atctacaaag ttcaccatct
1200ataacaacat gttctgtgct ggcttccatg aaggaggtag agattcatgt
caaggagata 1260gtgggggacc ccatgttact gaagtggaag ggaccagttt
cttaactgga attattagct 1320ggggtgaaga gtgtgcaatg aaaggcaaat
atggaatata taccaaggta tcccggtatg 1380tcaactggat taaggaaaaa
acaaagctca cttaatgaaa gatggatttc caaggttaat 1440tcattggaat
tgaaaattaa cagggcctct cactaactaa tcactttccc atcttttgtt
1500agatttgaat atatacattc tatgatcatt gctttttctc tttacagggg
agaatttcat 1560attttacctg agcaaattga ttagaaaatg gaaccactag
aggaatataa tgtgttagga 1620aattacagtc atttctaagg gcccagccct
tgacaaaatt gtgaagttaa attctccact 1680ctgtccatca gatactatgg
ttctccacta tggcaactaa ctcactcaat tttccctcct 1740tagcagcatt
ccatcttccc gatcttcttt gcttctccaa ccaaaacatc aatgtttatt
1800agttctgtat acagtacagg atctttggtc tactctatca caaggccagt
accacactca 1860tgaagaaaga acacaggagt agctgagagg ctaaaactca
tcaaaaacac tactcctttt 1920cctctaccct attcctcaat cttttacctt
ttccaaatcc caatccccaa atcagttttt 1980ctctttctta ctccctctct
cccttttacc ctccatggtc gttaaaggag agatggggag 2040catcattctg
ttatacttct gtacacagtt atacatgtct atcaaaccca gacttgcttc
2100cgtagtggag acttgctttt cagaacatag ggatgaagta aggtgcctga
aaagtttggg 2160ggaaaagttt ctttcagaga gttaagttat tttatatata
taatatatat ataaaatata 2220taatatacaa tataaatata tagtgtgtgt
gtatgcgtgt gtgtagacac acacgcatac 2280acacatataa tggaagcaat
aagccattct aagagcttgt atggttatgg aggtctgact 2340aggcatgatt
tcacgaaggc aagattggca tatcattgta actaaaaaag ctgacattga
2400cccagacata ttgtactctt tctaaaaata ataataataa tgctaacaga
aagaagagaa 2460ccgttcgttt gcaatctaca gctagtagag actttgagga
agaattcaac agtgtgtctt 2520cagcagtgtt cagagccaag caagaagttg
aagttgccta gaccagagga cataagtatc 2580atgtctcctt taactagcat
accccgaagt ggagaagggt gcagcaggct caaaggcata 2640agtcattcca
atcagccaac taagttgtcc ttttctggtt tcgtgttcac catggaacat
2700tttgattata gttaatcctt ctatcttgaa tcttctagag agttgctgac
caactgacgt 2760atgtttccct ttgtgaatta ataaactggt gttctggttc at
280221386DNAArtificial Sequenceconstructed sequence 2atgcagcgcg
tgaacatgat tatggccgag agccctggcc tgatcaccat ctgcctgctg 60ggctacctgc
tgagcgccga gtgcaccgtg tttctggacc acgagaacgc caacaagatc
120ctgaaccggc ccaagcggta caacagcggc aagctggaag agttcgtgca
gggcaacctg 180gaacgcgagt gcatggaaga gaagtgcagc ttcgaagagg
ccagagaggt gttcgagaac 240accgagcgga ccaccgagtt ctggaagcag
tacgtggacg gcgaccagtg cgagagcaac 300ccctgtctga acggcggcag
ctgcaaggac gacatcaaca gctacgagtg ctggtgcccc 360ttcggcttcg
agggcaagaa ctgcgagctg gacgtgacct gcaacatcaa gaacggcagg
420tgcgagcagt tctgcaagaa cagcgccgac aacaaggtcg tgtgctcctg
caccgagggc 480tacagactgg ccgagaacca gaagtcctgc gagcccgccg
tgcctttccc ttgtggaaga 540gtgtccgtgt cccagaccag caagctgacc
agagccgaga cagtgttccc cgacgtggac 600tacgtgaaca gcaccgaggc
cgagacaatc ctggacaaca tcacccagag cacccagtcc 660ttcaacgact
tcaccagagt cgtgggcggc gaggacgcca agcctggaca gttcccctgg
720caggtggtgc tgaacggaaa ggtggacgcc ttttgcggcg gcagcatcgt
gaacgagaag 780tggatcgtga cagccgccca ctgcgtggaa accggcgtga
agattacagt ggtggccggc 840gagcacaaca tcgaggaaac cgagcacaca
gagcagaaac ggaacgtgat cagaatcatc 900ccccaccaca actacaacgc
cgccatcaac aagtacaacc acgatatcgc cctgctggaa 960ctggacgagc
ccctggtgct gaatagctac gtgaccccca tctgtatcgc cgacaaagag
1020tacaccaaca tctttctgaa gttcggcagc ggctacgtgt ccggctgggg
cagagtgttt 1080cacaagggca gatccgctct ggtgctgcag tacctgagag
tgcctctggt ggaccgggcc 1140acctgtctga gaagcaccaa gttcaccatc
tacaacaaca tgttctgcgc cggctttcac 1200gagggcggca gagatagctg
tcagggcgat tctggcggcc ctcacgtgac agaggtggaa 1260ggcaccagct
ttctgaccgg catcatcagc tggggcgagg agtgcgccat gaaggggaag
1320tacggcatct acaccaaggt gtccagatac gtgaactgga tcaaagaaaa
gaccaagctg 1380acatga 13863168DNAArtificial Sequenceconstructed
sequence 3ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg
ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa
ctccatcact 120aggggttcct tgtagttaat gattaacccg ccatgctact tatctacg
1684106DNAArtificial Sequenceconstructed sequence 4agttaatttt
taaaaagcag tcaaaagtcc aagtgccctt gcgagcattt actctctctg 60tttgctctgg
ttaataatct caggagcaca aacattcctt actagt 1065496DNAArtificial
Sequenceconstructed sequence 5ccagtgtgct ggaattcggc ttttttaggg
ctggaagcta cctttgacat catttcctct 60gcgaatgcat gtataatttc tacagaacct
attagaaagg atcacccagc ctctgctttt 120gtacaacttt cccttaaaaa
actgccaatc ccactgctgt ttggcccaat agtgagaact 180ttttcctgct
gcctcttggt gcttttgcct atggccccta ttctgcctgc tgaagacact
240cttgccagca tggacttaaa cccctccagc tctgacaatc ctctttctct
tttgttttac 300atgaagggtc tggcagccaa agcaatcact caaagttcaa
accttatcat tttttgcttt 360gttcctcttg gccttggttt tgtacatcag
ctttgaaaat accatcccag ggttaatgct 420ggggttaatt tataactgag
agtgctctag ttctgcaata caggacatgc tataaaaatg 480gaaagatgtt gctttc
4966572DNAArtificial Sequenceconstructed sequence 6agcttacttg
tggtaccgag ctcggatcct gagaacttca gggtgagtct atgggaccct 60tgatgttttc
tttccccttc ttttctatgg ttaagttcat gtcataggaa ggggagaagt
120aacagggtac acatattgac caaatcaggg taattttgca tttgtaattt
taaaaaatgc 180tttcttcttt taatatactt ttttgtttat cttatttcta
atactttccc taatctcttt 240ctttcagggc aataatgata caatgtatca
tgcctctttg caccattcta aagaataaca 300gtgataattt ctgggttaag
gcaatagcaa tatttctgca tataaatatt tctgcatata 360aattgtaact
gatgtaagag gtttcatatt gctaatagca gctacaatcc agctaccatt
420ctgcttttat tttatggttg ggataaggct ggattattct gagtccaagc
taggcccttt 480tgctaatcat gttcatacct cttatcttcc tcccacagct
cctgggcaac gtgctggtct 540gtgtgctggc ccatcacttt ggcaaagaat tg
5727542DNAArtificial Sequenceconstructed sequence 7aatcaacctc
tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60ccttttacgc
tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
120atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
tgaggagttg 180tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
ttgctgacgc aacccccact 240ggttggggca ttgccaccac ctgtcagctc
ctttccggga ctttcgcttt ccccctccct 300attgccacgg cggaactcat
cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360ttgggcactg
acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
420gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
ttcggccctc 480aatccagcgg accttccttc ccgcggcctg ctgccggctc
tgcggcctct tccgcgtctt 540cg 5428215DNAArtificial
Sequenceconstructed sequence 8gcctcgactg tgccttctag ttgccagcca
tctgttgttt gcccctcccc cgtgccttcc 60ttgaccctgg aaggtgccac tcccactgtc
ctttcctaat aaaatgagga aattgcatcg 120cattgtctga gtaggtgtca
ttctattctg gggggtgggg tggggcagga cagcaagggg 180gaggattggg
aagacaatag caggcatgct gggga 2159168DNAArtificial
Sequenceconstructed sequence 9cgtagataag tagcatggcg ggttaatcat
taactacaag gaacccctag tgatggagtt 60ggccactccc tctctgcgcg ctcgctcgct
cactgaggcc gggcgaccaa aggtcgcccg 120acgcccgggc tttgcccggg
cggcctcagt gagcgagcga gcgcgcag 16810461PRTHomo sapiens 10Met Gln
Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr1 5 10 15Ile
Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu 20 25
30Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn
35 40 45Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu
Cys 50 55 60Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe
Glu Asn65 70 75 80Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val
Asp Gly Asp Gln 85 90 95Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser
Cys Lys Asp Asp Ile 100 105 110Asn Ser Tyr Glu Cys Trp Cys Pro Phe
Gly Phe Glu Gly Lys Asn Cys 115 120 125Glu Leu Asp Val Thr Cys Asn
Ile Lys Asn Gly Arg Cys Glu Gln Phe 130 135 140Cys Lys Asn Ser Ala
Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly145 150 155 160Tyr Arg
Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe 165 170
175Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala
180 185 190Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu
Ala Glu 195 200 205Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser
Phe Asn Asp Phe 210 215 220Thr Arg Val Val Gly Gly Glu Asp Ala Lys
Pro Gly Gln Phe Pro Trp225 230 235 240Gln Val Val Leu Asn Gly Lys
Val Asp Ala Phe Cys Gly Gly Ser Ile 245 250 255Val Asn Glu Lys Trp
Ile Val Thr Ala Ala His Cys Val Glu Thr Gly 260 265 270Val Lys Ile
Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu 275 280 285His
Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn 290 295
300Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu
Glu305 310 315 320Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr
Pro Ile Cys Ile 325 330 335Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu
Lys Phe Gly Ser Gly Tyr 340 345 350Val Ser Gly Trp Gly Arg Val Phe
His Lys Gly Arg Ser Ala Leu Val 355 360 365Leu Gln Tyr Leu Arg Val
Pro Leu Val Asp Arg Ala Thr Cys Leu Arg 370 375 380Ser Thr Lys Phe
Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His385 390 395 400Glu
Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val 405 410
415Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly
420 425 430Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys
Val Ser 435 440 445Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu
Thr 450 455 460117209DNAArtificial Sequenceconstructed sequence
11ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
120aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta
gccatgctct 180aggaagatcg gaattcgccc ttaagctagg ggggatccac
tagtactcga gacctaggag 240ttaattttta aaaagcagtc aaaagtccaa
gtgcccttgc gagcatttac tctctctgtt 300tgctctggtt aataatctca
ggagcacaaa cattccttac tagttctagg agttaatttt 360taaaaagcag
tcaaaagtcc aagtgccctt gcgagcattt actctctctg tttgctctgg
420ttaataatct caggagcaca aacattcctt actagttcta gagcggccgc
cagtgtgctg 480gaattcggct tttttagggc tggaagctac ctttgacatc
atttcctctg cgaatgcatg 540tataatttct acagaaccta ttagaaagga
tcacccagcc tctgcttttg tacaactttc 600ccttaaaaaa ctgccaatcc
cactgctgtt tggcccaata gtgagaactt tttcctgctg 660cctcttggtg
cttttgccta tggcccctat tctgcctgct gaagacactc ttgccagcat
720ggacttaaac ccctccagct ctgacaatcc tctttctctt ttgttttaca
tgaagggtct 780ggcagccaaa gcaatcactc aaagttcaaa ccttatcatt
ttttgctttg ttcctcttgg 840ccttggtttt gtacatcagc tttgaaaata
ccatcccagg gttaatgctg gggttaattt 900ataactgaga gtgctctagt
tctgcaatac aggacatgct ataaaaatgg aaagatgttg 960ctttctgaga
gatcagctta catgtggtac cgagctcgga tcctgagaac ttcagggtga
1020gtctatggga cccttgatgt tttctttccc cttcttttct atggttaagt
tcatgtcata 1080ggaaggggag aagtaacagg gtacacatat tgaccaaatc
agggtaattt tgcatttgta 1140attttaaaaa atgctttctt cttttaatat
acttttttgt ttatcttatt tctaatactt 1200tccctaatct ctttctttca
gggcaataat gatacaatgt atcatgcctc tttgcaccat 1260tctaaagaat
aacagtgata atttctgggt taaggcaata gcaatatttc tgcatataaa
1320tatttctgca tataaattgt aactgatgta agaggtttca tattgctaat
agcagctaca 1380atccagctac cattctgctt ttattttatg gttgggataa
ggctggatta ttctgagtcc 1440aagctaggcc cttttgctaa tcatgttcat
acctcttatc ttcctcccac agctcctggg 1500caacgtgctg gtctgtgtgc
tggcccatca ctttggcaaa gaattgatct cgagtaactg 1560agccgccacc
atgcagcgcg tgaacatgat tatggccgag agccctggcc tgatcaccat
1620ctgcctgctg ggctacctgc tgagcgccga gtgcaccgtg tttctggacc
acgagaacgc 1680caacaagatc ctgaaccggc ccaagcggta caacagcggc
aagctggaag agttcgtgca 1740gggcaacctg gaacgcgagt gcatggaaga
gaagtgcagc ttcgaagagg ccagagaggt 1800gttcgagaac accgagcgga
ccaccgagtt ctggaagcag tacgtggacg gcgaccagtg 1860cgagagcaac
ccctgtctga acggcggcag ctgcaaggac gacatcaaca gctacgagtg
1920ctggtgcccc ttcggcttcg agggcaagaa ctgcgagctg gacgtgacct
gcaacatcaa 1980gaacggcagg tgcgagcagt tctgcaagaa cagcgccgac
aacaaggtcg tgtgctcctg 2040caccgagggc tacagactgg ccgagaacca
gaagtcctgc gagcccgccg tgcctttccc 2100ttgtggaaga gtgtccgtgt
cccagaccag caagctgacc agagccgaga cagtgttccc 2160cgacgtggac
tacgtgaaca gcaccgaggc cgagacaatc ctggacaaca tcacccagag
2220cacccagtcc ttcaacgact tcaccagagt cgtgggcggc gaggacgcca
agcctggaca 2280gttcccctgg caggtggtgc tgaacggaaa ggtggacgcc
ttttgcggcg gcagcatcgt 2340gaacgagaag tggatcgtga cagccgccca
ctgcgtggaa accggcgtga agattacagt 2400ggtggccggc gagcacaaca
tcgaggaaac cgagcacaca gagcagaaac ggaacgtgat 2460cagaatcatc
ccccaccaca actacaacgc cgccatcaac aagtacaacc acgatatcgc
2520cctgctggaa ctggacgagc ccctggtgct gaatagctac gtgaccccca
tctgtatcgc 2580cgacaaagag tacaccaaca tctttctgaa gttcggcagc
ggctacgtgt ccggctgggg 2640cagagtgttt cacaagggca gatccgctct
ggtgctgcag tacctgagag tgcctctggt 2700ggaccgggcc acctgtctga
gaagcaccaa gttcaccatc tacaacaaca tgttctgcgc 2760cggctttcac
gagggcggca gagatagctg tcagggcgat tctggcggcc ctcacgtgac
2820agaggtggaa ggcaccagct ttctgaccgg catcatcagc tggggcgagg
agtgcgccat 2880gaaggggaag tacggcatct acaccaaggt gtccagatac
gtgaactgga tcaaagaaaa 2940gaccaagctg acatgataaa agcttggatc
caatcaacct ctggattaca aaatttgtga 3000aagattgact ggtattctta
actatgttgc tccttttacg ctatgtggat acgctgcttt 3060aatgcctttg
tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
3120atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
gtggcgtggt 3180gtgcactgtg tttgctgacg caacccccac tggttggggc
attgccacca cctgtcagct 3240cctttccggg actttcgctt tccccctccc
tattgccacg gcggaactca tcgccgcctg 3300ccttgcccgc tgctggacag
gggctcggct gttgggcact gacaattccg tggtgttgtc 3360ggggaaatca
tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
3420gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
cccgcggcct 3480gctgccggct ctgcggcctc ttccgcgtct tcgagatctg
cctcgactgt gccttctagt 3540tgccagccat ctgttgtttg cccctccccc
gtgccttcct tgaccctgga aggtgccact 3600cccactgtcc tttcctaata
aaatgaggaa attgcatcgc attgtctgag taggtgtcat 3660tctattctgg
ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
3720aggcatgctg gggactcgag ttaagggcga attcccgata aggatcttcc
tagagcatgg 3780ctacgtagat aagtagcatg gcgggttaat cattaactac
aaggaacccc tagtgatgga 3840gttggccact ccctctctgc gcgctcgctc
gctcactgag gccgggcgac caaaggtcgc 3900ccgacgcccg ggctttgccc
gggcggcctc agtgagcgag cgagcgcgca gccttaatta 3960acctaattca
ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
4020acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
aagaggcccg 4080caccgatcgc ccttcccaac agttgcgcag cctgaatggc
gaatgggacg cgccctgtag 4140cggcgcatta agcgcggcgg gtgtggtggt
tacgcgcagc gtgaccgcta cacttgccag 4200cgccctagcg cccgctcctt
tcgctttctt cccttccttt ctcgccacgt tcgccggctt 4260tccccgtcaa
gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
4320cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
cgccctgata 4380gacggttttt cgccctttga cgttggagtc cacgttcttt
aatagtggac tcttgttcca 4440aactggaaca acactcaacc ctatctcggt
ctattctttt gatttataag ggattttgcc 4500gatttcggcc tattggttaa
aaaatgagct gatttaacaa aaatttaacg cgaattttaa 4560caaaatcatg
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
4620ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
gctcaagtca 4680gaggtggcga aacccgacag gactataaag ataccaggcg
tttccccctg gaagctccct 4740cgtgcgctct cctgttccga ccctgccgct
taccggatac ctgtccgcct ttctcccttc 4800gggaagcgtg gcgctttctc
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 4860tcgctccaag
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
4920cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
tggcagcagc 4980cactggtaac aggattagca gagcgaggta tgtaggcggt
gctacagagt tcttgaagtg 5040gtggcctaac tacggctaca ctagaagaac
agtatttggt atctgcgctc tgctgaagcc 5100agttaccttc
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
5160cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
ctcaagaaga 5220tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
gaaaactcac gttaagggat 5280tttggtcatg agattatcaa aaaggatctt
cacctagatc cttttgatcc tccggcgttc 5340agcctgtgcc acagccgaca
ggatggtgac caccatttgc cccatatcac cgtcggtact 5400gatcccgtcg
tcaataaacc gaaccgctac accctgagca tcaaactctt ttatcagttg
5460gatcatgtcg gcggtgtcgc ggccaagacg gtcgagcttc ttcaccagaa
tgacatcacc 5520ttcctccacc ttcatcctca gcaaatccag cccttcccga
tctgttgaac tgccggatgc 5580cttgtcggta aagatgcggt tagcttttac
ccctgcatct ttgagcgctg aggtctgcct 5640cgtgaagaag gtgttgctga
ctcataccag gcctgaatcg ccccatcatc cagccagaaa 5700gtgagggagc
cacggttgat gagagctttg ttgtaggtgg accagttggt gattttgaac
5760ttttgctttg ccacggaacg gtctgcgttg tcgggaagat gcgtgatctg
atccttcaac 5820tcagcaaaag ttcgatttat tcaacaaagc cgccgtcccg
tcaagtcagc gtaatgctct 5880gccagtgtta caaccaatta accaattctg
attagaaaaa ctcatcgagc atcaaatgaa 5940actgcaattt attcatatca
ggattatcaa taccatattt ttgaaaaagc cgtttctgta 6000atgaaggaga
aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
6060cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
aaaataaggt 6120tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
tgagaatggc aaaagcttat 6180gcatttcttt ccagacttgt tcaacaggcc
agccattacg ctcgtcatca aaatcactcg 6240catcaaccaa accgttattc
attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc 6300tgttaaaagg
acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
6360catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
gctgttttcc 6420cggggatcgc agtggtgagt aaccatgcat catcaggagt
acggataaaa tgcttgatgg 6480tcggaagagg cataaattcc gtcagccagt
ttagtctgac catctcatct gtaacatcat 6540tggcaacgct acctttgcca
tgtttcagaa acaactctgg cgcatcgggc ttcccataca 6600atcgatagat
tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
6660aatcagcatc catgttggaa tttaatcgcg gcctcgagca agacgtttcc
cgttgaatat 6720ggctcataac accccttgta ttactgttta tgtaagcaga
cagttttatt gttcatgatg 6780atatattttt atcttgtgca atgtaacatc
agagattttg agacaccatg ttctttcctg 6840cgttatcccc tgattctgtg
gataaccgta ttaccgcctt tgagtgagct gataccgctc 6900gccgcagccg
aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
6960tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
cacgacaggt 7020ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
tgtgagttag ctcactcatt 7080aggcacccca ggctttacac tttatgcttc
cggctcgtat gttgtgtgga attgtgagcg 7140gataacaatt tcacacagga
aacagctatg accatgatta cgccagattt aattaaggcc 7200ttaattagg
7209127748DNAArtificial Sequenceconstructed sequence 12cagcagctgc
gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg 60acctttggtc
gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
120atcactaggg gttccttgta gttaatgatt aacccgccat gctacttatc
tacgtagcca 180tgctctagct aggcccgggg gatccactag tactcgagac
ctaggagtta atttttaaaa 240agcagtcaaa agtccaagtg cccttgcgag
catttactct ctctgtttgc tctggttaat 300aatctcagga gcacaaacat
tccttactag ttctaggagt taatttttaa aaagcagtca 360aaagtccaag
tgcccttgcg agcatttact ctctctgttt gctctggtta ataatctcag
420gagcacaaac attccttact agttctagag cggccgccag tgtgctggaa
ttcggctttt 480ttagggctgg aagctacctt tgacatcatt tcctctgcga
atgcatgtat aatttctaca 540gaacctatta gaaaggatca cccagcctct
gcttttgtac aactttccct taaaaaactg 600ccaatcccac tgctgtttgg
cccaatagtg agaacttttt cctgctgcct cttggtgctt 660ttgcctatgg
cccctattct gcctgctgaa gacactcttg ccagcatgga cttaaacccc
720tccagctctg acaatcctct ttctcttttg ttttacatga agggtctggc
agccaaagca 780atcactcaaa gttcaaacct tatcattttt tgctttgttc
ctcttggcct tggttttgta 840catcagcttt gaaaatacca tcccagggtt
aatgctgggg ttaatttata actgagagtg 900ctctagttct gcaatacagg
acatgctata aaaatggaaa gatgttgctt tctgagagat 960cagcttacat
gtggtaccga gctcggatcc tgagaacttc agggtgagtc tatgggaccc
1020ttgatgtttt ctttcccctt cttttctatg gttaagttca tgtcatagga
aggggagaag 1080taacagggta cacatattga ccaaatcagg gtaattttgc
atttgtaatt ttaaaaaatg 1140ctttcttctt ttaatatact tttttgttta
tcttatttct aatactttcc ctaatctctt 1200tctttcaggg caataatgat
acaatgtatc atgcctcttt gcaccattct aaagaataac 1260agtgataatt
tctgggttaa ggcaatagca atatttctgc atataaatat ttctgcatat
1320aaattgtaac tgatgtaaga ggtttcatat tgctaatagc agctacaatc
cagctaccat 1380tctgctttta ttttatggtt gggataaggc tggattattc
tgagtccaag ctaggccctt 1440ttgctaatca tgttcatacc tcttatcttc
ctcccacagc tcctgggcaa cgtgctggtc 1500tgtgtgctgg cccatcactt
tggcaaagaa ttgatctcga gaaagctaac aacaaagaac 1560aacaaacaac
aatcaggata acaagaacga aacaataaca gccaccatgc agagggtgaa
1620catgatcatg gctgagagcc ctggcctgat caccatctgc ctgctgggct
acctgctgtc 1680tgctgagtgc actgtgttcc tggaccatga gaatgccaac
aagatcctga acaggcccaa 1740gagatacaac tctggcaagc tggaggagtt
tgtgcagggc aacctggaga gggagtgcat 1800ggaggagaag tgcagctttg
aggaggccag ggaggtgttt gagaacactg agaggaccac 1860tgagttctgg
aagcagtatg tggatgggga ccagtgtgag agcaacccct gcctgaatgg
1920gggcagctgc aaggatgaca tcaacagcta tgagtgctgg tgcccctttg
gctttgaggg 1980caagaactgt gagctggatg tgacctgcaa catcaagaat
ggcagatgtg agcagttctg 2040caagaactct gctgacaaca aggtggtgtg
cagctgcact gagggctaca ggctggctga 2100gaaccagaag agctgtgagc
ctgctgtgcc attcccatgt ggcagagtgt ctgtgagcca 2160gaccagcaag
ctgaccaggg ctgaggctgt gttccctgat gtggactatg tgaacagcac
2220tgaggctgaa accatcctgg acaacatcac ccagagcacc cagagcttca
atgacttcac 2280cagggtggtg gggggggagg atgccaagcc tggccagttc
ccctggcaag tggtgctgaa 2340tggcaaggtg gatgccttct gtgggggcag
cattgtgaat gagaagtgga ttgtgactgc 2400tgcccactgt gtggagactg
gggtgaagat cactgtggtg gctggggagc acaacattga 2460ggagactgag
cacactgagc agaagaggaa tgtgatcagg atcatccccc accacaacta
2520caatgctgcc atcaacaagt acaaccatga cattgccctg ctggagctgg
atgagcccct 2580ggtgctgaac agctatgtga cccccatctg cattgctgac
aaggagtaca ccaacatctt 2640cctgaagttt ggctctggct atgtgtctgg
ctggggcagg gtgttccaca agggcaggtc 2700tgccctggtg ctgcagtacc
tgagggtgcc cctggtggac agggccacct gcctgaggag 2760caccaagttc
accatctaca acaacatgtt ctgtgctggc ttccatgagg ggggcaggga
2820cagctgccag ggggactctg ggggccccca tgtgactgag gtggagggca
ccagcttcct 2880gactggcatc atcagctggg gggaggagtg tgccatgaag
ggcaagtatg gcatctacac 2940caaagtctcc agatatgtga actggatcaa
ggagaagacc aagctgacct gaaataagct 3000tatcgataat caacctctgg
attacaaaat ttgtgaaaga ttgactggta ttcttaacta 3060tgttgctcct
tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
3120ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
ctctttatga 3180ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
actgtgtttg ctgacgcaac 3240ccccactggt tggggcattg ccaccacctg
tcagctcctt tccgggactt tcgctttccc 3300cctccctatt gccacggcgg
aactcatcgc cgcctgcctt gcccgctgct ggacaggggc 3360tcggctgttg
ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
3420gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
acgtcccttc 3480ggccctcaat ccagcggacc ttccttcccg cggcctgctg
ccggctctgc ggcctcttcc 3540gcgtcttcgc cttcgccctc agacgagtcg
gatctccctt tgggccgcct ccccgcatcg 3600ataccgtcga cctcgaatcg
aattcctgca gcccggggga tccactagtt ctagagcggc 3660caaacccgct
gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
3720tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
ctaataaaat 3780gaggaaattg catcgcattg tctgagtagg tgtcattcta
ttctgggggg tggggtgggg 3840caggacagca agggggagga ttgggaagac
aatagcaggc atgctgggga tgcggtgggc 3900tctatggctt ctgaggcgga
aagaaccagg atcctagagc atggctacgt agataagtag 3960catggcgggt
taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct
4020ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
cccgggcttt 4080gcccgggcgg cctcagtgag cgagcgagcg cgcagctggc
gtaatagcga agaggcccgc 4140accgatcgcc cttcccaaca gttgcgcagc
ctgaatggcg aatggaattc cagacgattg 4200agcgtcaaaa tgtaggtatt
tccatgagcg tttttcctgt tgcaatggct ggcggtaata 4260ttgttctgga
tattaccagc aaggccgata gtttgagttc ttctactcag gcaagtgatg
4320ttattactaa tcaaagaagt attgcgacaa cggttaattt gcgtgatgga
cagactcttt 4380tactcggtgg cctcactgat tataaaaaca cttctcagga
ttctggcgta ccgttcctgt 4440ctaaaatccc tttaatcggc ctcctgttta
gctcccgctc tgattctaac gaggaaagca 4500cgttatacgt gctcgtcaaa
gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 4560gcgggtgtgg
tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
4620cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
tcaagctcta 4680aatcgggggc tccctttagg gttccgattt agtgctttac
ggcacctcga ccccaaaaaa 4740cttgattagg gtgatggttc acgtagtggg
ccatcgccct gatagacggt ttttcgccct 4800ttgacgttgg agtccacgtt
ctttaatagt ggactcttgt tccaaactgg aacaacactc 4860aaccctatct
cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
4920ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
attaacgttt 4980acaatttaaa tatttgctta tacaatcttc ctgtttttgg
ggcttttctg attatcaacc 5040ggggtacata tgattgacat gctagtttta
cgattaccgt tcatcgattc tcttgtttgc 5100tccagactct caggcaatga
cctgatagcc tttgtagaga cctctcaaaa atagctaccc 5160tctccggcat
gaatttatca gctagaacgg ttgaatatca tattgatggt gatttgactg
5220tctccggcct ttctcacccg tttgaatctt tacctacaca ttactcaggc
attgcattta 5280aaatatatga gggttctaaa aatttttatc cttgcgttga
aataaaggct tctcccgcaa 5340aagtattaca gggtcataat gtttttggta
caaccgattt agctttatgc tctgaggctt 5400tattgcttaa ttttgctaat
tctttgcctt gcctgtatga tttattggat gttggaattc 5460ctgatgcggt
attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
5520ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
gccaacaccc 5580gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
cttacagaca agctgtgacc 5640gtctccggga gctgcatgtg tcagaggttt
tcaccgtcat caccgaaacg cgcgagacga 5700aagggcctcg tgatacgcct
atttttatag gttaatgtca tgataataat ggtttcttag 5760acgtcaggtg
gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
5820atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
tcaataatat 5880tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
cccttattcc cttttttgcg 5940gcattttgcc ttcctgtttt tgctcaccca
gaaacgctgg tgaaagtaaa agatgctgaa 6000gatcagttgg gtgcacgagt
gggttacatc gaactggatc tcaacagcgg taagatcctt 6060gagagttttc
gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
6120ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
catacactat 6180tctcagaatg acttggttga gtactcacca gtcacagaaa
agcatcttac ggatggcatg 6240acagtaagag aattatgcag tgctgccata
accatgagtg ataacactgc ggccaactta 6300cttctgacaa cgatcggagg
accgaaggag ctaaccgctt ttttgcacaa catgggggat 6360catgtaactc
gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
6420cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
aactggcgaa 6480ctacttactc tagcttcccg gcaacaatta atagactgga
tggaggcgga taaagttgca 6540ggaccacttc tgcgctcggc ccttccggct
ggctggttta ttgctgataa atctggagcc 6600ggtgagcgtg ggtctcgcgg
tatcattgca gcactggggc cagatggtaa gccctcccgt 6660atcgtagtta
tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
6720gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
ttactcatat 6780atactttaga ttgatttaaa acttcatttt taatttaaaa
ggatctaggt gaagatcctt 6840tttgataatc tcatgaccaa aatcccttaa
cgtgagtttt cgttccactg agcgtcagac 6900cccgtagaaa agatcaaagg
atcttcttga gatccttttt ttctgcgcgt aatctgctgc 6960ttgcaaacaa
aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
7020actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
tgtccttcta 7080gtgtagccgt agttaggcca ccacttcaag aactctgtag
caccgcctac atacctcgct 7140ctgctaatcc tgttaccagt ggctgctgcc
agtggcgata agtcgtgtct taccgggttg 7200gactcaagac gatagttacc
ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 7260acacagccca
gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
7320tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
aagcggcagg 7380gtcggaacag gagagcgcac gagggagctt ccagggggaa
acgcctggta tctttatagt 7440cctgtcgggt ttcgccacct ctgacttgag
cgtcgatttt tgtgatgctc gtcagggggg 7500cggagcctat ggaaaaacgc
cagcaacgcg gcctttttac ggttcctggc cttttgctgg 7560ccttttgctc
acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
7620gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
cgagtcagtg 7680agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
tccccgcgcg ttggccgatt 7740cattaatg 7748131386DNAArtificial
Sequenceconstructed sequence 13atgcagaggg tgaacatgat catggctgag
agccctggcc tgatcaccat ctgcctgctg 60ggctacctgc tgtctgctga gtgcactgtg
ttcctggacc atgagaatgc caacaagatc 120ctgaacaggc ccaagagata
caactctggc aagctggagg agtttgtgca gggcaacctg 180gagagggagt
gcatggagga gaagtgcagc tttgaggagg ccagggaggt gtttgagaac
240actgagagga ccactgagtt ctggaagcag tatgtggatg gggaccagtg
tgagagcaac 300ccctgcctga atgggggcag ctgcaaggat gacatcaaca
gctatgagtg ctggtgcccc 360tttggctttg agggcaagaa ctgtgagctg
gatgtgacct gcaacatcaa gaatggcaga 420tgtgagcagt tctgcaagaa
ctctgctgac aacaaggtgg tgtgcagctg cactgagggc 480tacaggctgg
ctgagaacca gaagagctgt gagcctgctg tgccattccc atgtggcaga
540gtgtctgtga gccagaccag caagctgacc agggctgagg ctgtgttccc
tgatgtggac 600tatgtgaaca gcactgaggc tgaaaccatc ctggacaaca
tcacccagag cacccagagc 660ttcaatgact tcaccagggt ggtggggggg
gaggatgcca agcctggcca gttcccctgg 720caagtggtgc tgaatggcaa
ggtggatgcc ttctgtgggg gcagcattgt gaatgagaag 780tggattgtga
ctgctgccca ctgtgtggag actggggtga agatcactgt ggtggctggg
840gagcacaaca ttgaggagac tgagcacact gagcagaaga ggaatgtgat
caggatcatc 900ccccaccaca actacaatgc tgccatcaac aagtacaacc
atgacattgc cctgctggag 960ctggatgagc ccctggtgct gaacagctat
gtgaccccca tctgcattgc tgacaaggag 1020tacaccaaca tcttcctgaa
gtttggctct ggctatgtgt ctggctgggg cagggtgttc 1080cacaagggca
ggtctgccct ggtgctgcag tacctgaggg tgcccctggt ggacagggcc
1140acctgcctga ggagcaccaa gttcaccatc tacaacaaca tgttctgtgc
tggcttccat 1200gaggggggca gggacagctg ccagggggac tctgggggcc
cccatgtgac tgaggtggag 1260ggcaccagct tcctgactgg catcatcagc
tggggggagg agtgtgccat gaagggcaag 1320tatggcatct acaccaaagt
ctccagatat gtgaactgga tcaaggagaa gaccaagctg 1380acctga
138614738PRTUnknownAAVrh.10 capsid 14Met Ala Ala Asp Gly Tyr Leu
Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp
Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln
Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr
Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala
Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln
Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90
95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu
Glu Pro 115 120 125Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro
Gly Lys Lys Arg 130 135 140Pro Val Glu Pro Ser Pro Gln Arg Ser Pro
Asp Ser Ser Thr Gly Ile145 150 155 160Gly Lys Lys Gly Gln Gln Pro
Ala Lys Lys Arg Leu Asn Phe Gly Gln 165 170 175Thr Gly Asp Ser Glu
Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro 180 185 190Pro Ala Gly
Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly 195 200 205Gly
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser 210 215
220Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg
Val225 230 235 240Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr
Tyr Asn Asn His 245 250 255Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser
Gly Gly Ser Thr Asn Asp 260 265 270Asn Thr Tyr Phe Gly Tyr Ser Thr
Pro Trp Gly Tyr Phe Asp Phe Asn 275 280 285Arg Phe His Cys His Phe
Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn 290 295 300Asn Asn Trp Gly
Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn305 310 315 320Ile
Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala 325 330
335Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln
340 345 350Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro
Pro Phe 355 360 365Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr
Leu Thr Leu Asn 370 375 380Asn Gly Ser Gln Ala Val Gly Arg Ser Ser
Phe Tyr Cys Leu Glu Tyr385 390 395 400Phe Pro Ser Gln Met Leu Arg
Thr Gly Asn Asn Phe Glu Phe Ser Tyr 405 410 415Gln Phe Glu Asp Val
Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser 420 425 430Leu Asp Arg
Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu 435 440 445Ser
Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Gln Gln Leu Leu 450 455
460Phe Ser Gln Ala Gly Pro Asn Asn Met Ser Ala Gln Ala Lys Asn
Trp465 470 475 480Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser
Thr Thr Leu Ser 485 490 495Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr
Gly Ala Thr Lys Tyr His 500 505 510Leu Asn Gly Arg Asp Ser Leu Val
Asn Pro Gly Val Ala Met Ala Thr 515 520 525His Lys Asp Asp Glu Glu
Arg Phe Phe Pro Ser Ser Gly Val Leu Met 530 535 540Phe Gly Lys Gln
Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Ser Val545 550 555 560Met
Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr 565 570
575Glu Gln Tyr
Gly Val Val Ala Asp Asn Leu Gln Gln Gln Asn Ala Ala 580 585 590Pro
Ile Val Gly Ala Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val 595 600
605Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile
610 615 620Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly
Gly Phe625 630 635 640Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile
Lys Asn Thr Pro Val 645 650 655Pro Ala Asp Pro Pro Thr Thr Phe Ser
Gln Ala Lys Leu Ala Ser Phe 660 665 670Ile Thr Gln Tyr Ser Thr Gly
Gln Val Ser Val Glu Ile Glu Trp Glu 675 680 685Leu Gln Lys Glu Asn
Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr 690 695 700Ser Asn Tyr
Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Asp705 710 715
720Gly Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg
725 730 735Asn Leu15727DNAArtificial Sequenceconstructed sequence
15agttaatttt taaaaagcag tcaaaagtcc aagtgccctt gcgagcattt actctctctg
60tttgctctgg ttaataatct caggagcaca aacattcctt actagttcta ggagttaatt
120tttaaaaagc agtcaaaagt ccaagtgccc ttgcgagcat ttactctctc
tgtttgctct 180ggttaataat ctcaggagca caaacattcc ttactagttc
tagagcggcc gccagtgtgc 240tggaattcgg cttttttagg gctggaagct
acctttgaca tcatttcctc tgcgaatgca 300tgtataattt ctacagaacc
tattagaaag gatcacccag cctctgcttt tgtacaactt 360tcccttaaaa
aactgccaat cccactgctg tttggcccaa tagtgagaac tttttcctgc
420tgcctcttgg tgcttttgcc tatggcccct attctgcctg ctgaagacac
tcttgccagc 480atggacttaa acccctccag ctctgacaat cctctttctc
ttttgtttta catgaagggt 540ctggcagcca aagcaatcac tcaaagttca
aaccttatca ttttttgctt tgttcctctt 600ggccttggtt ttgtacatca
gctttgaaaa taccatccca gggttaatgc tggggttaat 660ttataactga
gagtgctcta gttctgcaat acaggacatg ctataaaaat ggaaagatgt 720tgctttc
727167198DNAArtificial Sequenceconstructed sequence 16ctgcgcgctc
gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ggtcgcccgg
cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
120aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta
gccatgctct 180aggaagatcg gaattcgccc ttaagctagg ggggatccac
tagtactcga gacctaggag 240ttaattttta aaaagcagtc aaaagtccaa
gtgcccttgc gagcatttac tctctctgtt 300tgctctggtt aataatctca
ggagcacaaa cattccttac tagttctagg agttaatttt 360taaaaagcag
tcaaaagtcc aagtgccctt gcgagcattt actctctctg tttgctctgg
420ttaataatct caggagcaca aacattcctt actagttcta gagcggccgc
cagtgtgctg 480gaattcggct tttttagggc tggaagctac ctttgacatc
atttcctctg cgaatgcatg 540tataatttct acagaaccta ttagaaagga
tcacccagcc tctgcttttg tacaactttc 600ccttaaaaaa ctgccaatcc
cactgctgtt tggcccaata gtgagaactt tttcctgctg 660cctcttggtg
cttttgccta tggcccctat tctgcctgct gaagacactc ttgccagcat
720ggacttaaac ccctccagct ctgacaatcc tctttctctt ttgttttaca
tgaagggtct 780ggcagccaaa gcaatcactc aaagttcaaa ccttatcatt
ttttgctttg ttcctcttgg 840ccttggtttt gtacatcagc tttgaaaata
ccatcccagg gttaatgctg gggttaattt 900ataactgaga gtgctctagt
tctgcaatac aggacatgct ataaaaatgg aaagatgttg 960ctttctgaga
gatcagctta catgtggtac cgagctcgga tcctgagaac ttcagggtga
1020gtctatggga cccttgatgt tttctttccc cttcttttct atggttaagt
tcatgtcata 1080ggaaggggag aagtaacagg gtacacatat tgaccaaatc
agggtaattt tgcatttgta 1140attttaaaaa atgctttctt cttttaatat
acttttttgt ttatcttatt tctaatactt 1200tccctaatct ctttctttca
gggcaataat gatacaatgt atcatgcctc tttgcaccat 1260tctaaagaat
aacagtgata atttctgggt taaggcaata gcaatatttc tgcatataaa
1320tatttctgca tataaattgt aactgatgta agaggtttca tattgctaat
agcagctaca 1380atccagctac cattctgctt ttattttatg gttgggataa
ggctggatta ttctgagtcc 1440aagctaggcc cttttgctaa tcatgttcat
acctcttatc ttcctcccac agctcctggg 1500caacgtgctg gtctgtgtgc
tggcccatca ctttggcaaa gaattgatct cgagtaactg 1560agccgccacc
atgcagcgcg tgaacatgat tatggccgag agccctggcc tgatcaccat
1620ctgcctgctg ggctacctgc tgagcgccga gtgcaccgtg tttctggacc
acgagaacgc 1680caacaagatc ctgaaccggc ccaagcggta caacagcggc
aagctggaag agttcgtgca 1740gggcaacctg gaacgcgagt gcatggaaga
gaagtgcagc ttcgaagagg ccagagaggt 1800gttcgagaac accgagcgga
ccaccgagtt ctggaagcag tacgtggacg gcgaccagtg 1860cgagagcaac
ccctgtctga acggcggcag ctgcaaggac gacatcaaca gctacgagtg
1920ctggtgcccc ttcggcttcg agggcaagaa ctgcgagctg gacgtgacct
gcaacatcaa 1980gaacggcagg tgcgagcagt tctgcaagaa cagcgccgac
aacaaggtcg tgtgctcctg 2040caccgagggc tacagactgg ccgagaacca
gaagtcctgc gagcccgccg tgcctttccc 2100ttgtggaaga gtgtccgtgt
cccagaccag caagctgacc agagccgaga cagtgttccc 2160cgacgtggac
tacgtgaaca gcaccgaggc cgagacaatc ctggacaaca tcacccagag
2220cacccagtcc ttcaacgact tcaccagagt cgtgggcggc gaggacgcca
agcctggaca 2280gttcccctgg caggtggtgc tgaacggaaa ggtggacgcc
ttttgcggcg gcagcatcgt 2340gaacgagaag tggatcgtga cagccgccca
ctgcgtggaa accggcgtga agattacagt 2400ggtggccggc gagcacaaca
tcgaggaaac cgagcacaca gagcagaaac ggaacgtgat 2460cagaatcatc
ccccaccaca actacaacgc cgccatcaac aagtacaacc acgatatcgc
2520cctgctggaa ctggacgagc ccctggtgct gaatagctac gtgaccccca
tctgtatcgc 2580cgacaaagag tacaccaaca tctttctgaa gttcggcagc
ggctacgtgt ccggctgggg 2640cagagtgttt cacaagggca gatccgctct
ggtgctgcag tacctgagag tgcctctggt 2700ggaccgggcc acctgtctgc
tgagcaccaa gttcaccatc tacaacaaca tgttctgcgc 2760cggctttcac
gagggcggca gagatagctg tcagggcgat tctggcggcc ctcacgtgac
2820agaggtggaa ggcaccagct ttctgaccgg catcatcagc tggggcgagg
agtgcgccat 2880gaaggggaag tacggcatct acaccaaggt gtccagatac
gtgaactgga tcaaagaaaa 2940gaccaagctg acatgataaa agcttggatc
caatcaacct ctggattaca aaatttgtga 3000aagattgact ggtattctta
actatgttgc tccttttacg ctatgtggat acgctgcttt 3060aatgcctttg
tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
3120atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
gtggcgtggt 3180gtgcactgtg tttgctgacg caacccccac tggttggggc
attgccacca cctgtcagct 3240cctttccggg actttcgctt tccccctccc
tattgccacg gcggaactca tcgccgcctg 3300ccttgcccgc tgctggacag
gggctcggct gttgggcact gacaattccg tggtgttgtc 3360ggggaaatca
tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
3420gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
cccgcggcct 3480gctgccggct ctgcggcctc ttccgcgtct tcgagatctg
cctcgactgt gccttctagt 3540tgccagccat ctgttgtttg cccctccccc
gtgccttcct tgaccctgga aggtgccact 3600cccactgtcc tttcctaata
aaatgaggaa attgcatcgc attgtctgag taggtgtcat 3660tctattctgg
ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
3720aggcatgctg gggactcgag ttaagggcga attcccgata aggatcttcc
tagagcatgg 3780ctacgtagat aagtagcatg gcgggttaat cattaactac
aaggaacccc tagtgatgga 3840gttggccact ccctctctgc gcgctcgctc
gctcactgag gccgggcgac caaaggtcgc 3900ccgacgcccg ggctttgccc
gggcggcctc agtgagcgag cgagcgcgca gccttaatta 3960acctaattca
ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
4020acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
aagaggcccg 4080caccgatcgc ccttcccaac agttgcgcag cctgaatggc
gaatgggacg cgccctgtag 4140cggcgcatta agcgcggcgg gtgtggtggt
tacgcgcagc gtgaccgcta cacttgccag 4200cgccctagcg cccgctcctt
tcgctttctt cccttccttt ctcgccacgt tcgccggctt 4260tccccgtcaa
gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
4320cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
cgccctgata 4380gacggttttt cgccctttga cgttggagtc cacgttcttt
aatagtggac tcttgttcca 4440aactggaaca acactcaacc ctatctcggt
ctattctttt gatttataag ggattttgcc 4500gatttcggcc tattggttaa
aaaatgagct gatttaacaa aaatttaacg cgaattttaa 4560caaaatcatg
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
4620ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
gctcaagtca 4680gaggtggcga aacccgacag gactataaag ataccaggcg
tttccccctg gaagctccct 4740cgtgcgctct cctgttccga ccctgccgct
taccggatac ctgtccgcct ttctcccttc 4800gggaagcgtg gcgctttctc
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 4860tcgctccaag
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
4920cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
tggcagcagc 4980cactggtaac aggattagca gagcgaggta tgtaggcggt
gctacagagt tcttgaagtg 5040gtggcctaac tacggctaca ctagaagaac
agtatttggt atctgcgctc tgctgaagcc 5100agttaccttc ggaaaaagag
ttggtagctc ttgatccggc aaacaaacca ccgctggtag 5160cggtggtttt
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
5220tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
gttaagggat 5280tttggtcatg agattatcaa aaaggatctt cacctagatc
cttttgatcc tccggcgttc 5340agcctgtgcc acagccgaca ggatggtgac
caccatttgc cccatatcac cgtcggtact 5400gatcccgtcg tcaataaacc
gaaccgctac accctgagca tcaaactctt ttatcagttg 5460gatcatgtcg
gcggtgtcgc ggccaagacg gtcgagcttc ttcaccagaa tgacatcacc
5520ttcctccacc ttcatcctca gcaaatccag cccttcccga tctgttgaac
tgccggatgc 5580cttgtcggta aagatgcggt tagcttttac ccctgcatct
ttgagcgctg aggtctgcct 5640cgtgaagaag gtgttgctga ctcataccag
gcctgaatcg ccccatcatc cagccagaaa 5700gtgagggagc cacggttgat
gagagctttg ttgtaggtgg accagttggt gattttgaac 5760ttttgctttg
ccacggaacg gtctgcgttg tcgggaagat gcgtgatctg atccttcaac
5820tcagcaaaag ttcgatttat tcaacaaagc cgccgtcccg tcaagtcagc
gtaatgctct 5880gccagtgtta caaccaatta accaattctg attagaaaaa
ctcatcgagc atcaaatgaa 5940actgcaattt attcatatca ggattatcaa
taccatattt ttgaaaaagc cgtttctgta 6000atgaaggaga aaactcaccg
aggcagttcc ataggatggc aagatcctgg tatcggtctg 6060cgattccgac
tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
6120tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
aaaagcttat 6180gcatttcttt ccagacttgt tcaacaggcc agccattacg
ctcgtcatca aaatcactcg 6240catcaaccaa accgttattc attcgtgatt
gcgcctgagc gagacgaaat acgcgatcgc 6300tgttaaaagg acaattacaa
acaggaatcg aatgcaaccg gcgcaggaac actgccagcg 6360catcaacaat
attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
6420cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
tgcttgatgg 6480tcggaagagg cataaattcc gtcagccagt ttagtctgac
catctcatct gtaacatcat 6540tggcaacgct acctttgcca tgtttcagaa
acaactctgg cgcatcgggc ttcccataca 6600atcgatagat tgtcgcacct
gattgcccga cattatcgcg agcccattta tacccatata 6660aatcagcatc
catgttggaa tttaatcgcg gcctcgagca agacgtttcc cgttgaatat
6720ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
gttcatgatg 6780atatattttt atcttgtgca atgtaacatc agagattttg
agacaccatg ttctttcctg 6840cgttatcccc tgattctgtg gataaccgta
ttaccgcctt tgagtgagct gataccgctc 6900gccgcagccg aacgaccgag
cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa 6960tacgcaaacc
gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
7020ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
ctcactcatt 7080aggcacccca ggctttacac tttatgcttc cggctcgtat
gttgtgtgga attgtgagcg 7140gataacaatt tcacacagga aacagctatg
accatgatta cgccagattt aattaagg 7198171386DNAArtificial
Sequenceconstructed sequence 17atgcagcgcg tgaacatgat tatggccgag
agccctggcc tgatcaccat ctgcctgctg 60ggctacctgc tgagcgccga gtgcaccgtg
tttctggacc acgagaacgc caacaagatc 120ctgaaccggc ccaagcggta
caacagcggc aagctggaag agttcgtgca gggcaacctg 180gaacgcgagt
gcatggaaga gaagtgcagc ttcgaagagg ccagagaggt gttcgagaac
240accgagcgga ccaccgagtt ctggaagcag tacgtggacg gcgaccagtg
cgagagcaac 300ccctgtctga acggcggcag ctgcaaggac gacatcaaca
gctacgagtg ctggtgcccc 360ttcggcttcg agggcaagaa ctgcgagctg
gacgtgacct gcaacatcaa gaacggcagg 420tgcgagcagt tctgcaagaa
cagcgccgac aacaaggtcg tgtgctcctg caccgagggc 480tacagactgg
ccgagaacca gaagtcctgc gagcccgccg tgcctttccc ttgtggaaga
540gtgtccgtgt cccagaccag caagctgacc agagccgaga cagtgttccc
cgacgtggac 600tacgtgaaca gcaccgaggc cgagacaatc ctggacaaca
tcacccagag cacccagtcc 660ttcaacgact tcaccagagt cgtgggcggc
gaggacgcca agcctggaca gttcccctgg 720caggtggtgc tgaacggaaa
ggtggacgcc ttttgcggcg gcagcatcgt gaacgagaag 780tggatcgtga
cagccgccca ctgcgtggaa accggcgtga agattacagt ggtggccggc
840gagcacaaca tcgaggaaac cgagcacaca gagcagaaac ggaacgtgat
cagaatcatc 900ccccaccaca actacaacgc cgccatcaac aagtacaacc
acgatatcgc cctgctggaa 960ctggacgagc ccctggtgct gaatagctac
gtgaccccca tctgtatcgc cgacaaagag 1020tacaccaaca tctttctgaa
gttcggcagc ggctacgtgt ccggctgggg cagagtgttt 1080cacaagggca
gatccgctct ggtgctgcag tacctgagag tgcctctggt ggaccgggcc
1140acctgtctgc tgagcaccaa gttcaccatc tacaacaaca tgttctgcgc
cggctttcac 1200gagggcggca gagatagctg tcagggcgat tctggcggcc
ctcacgtgac agaggtggaa 1260ggcaccagct ttctgaccgg catcatcagc
tggggcgagg agtgcgccat gaaggggaag 1320tacggcatct acaccaaggt
gtccagatac gtgaactgga tcaaagaaaa gaccaagctg 1380acatga
13861899DNAHomo sapiens 18gttaattttt aaaaagcagt caaaagtcca
agtggccctt ggcagcattt actctctctg 60tttgctctgg ttaataatct caggagcaca
aacattcct 9919471DNAHomo sapiens 19tagggctgga agctaccttt gacatcattt
cctctgcgaa tgcatgtata atttctacag 60aacctattag aaaggatcac ccagcctctg
cttttgtaca actttccctt aaaaaactgc 120caattccact gctgtttggc
ccaatagtga gaactttttc ctgctgcctc ttggtgcttt 180tgcctatggc
ccctattctg cctgctgaag acactcttgc cagcatggac ttaaacccct
240ccagctctga caatcctctt tctcttttgt tttacatgaa gggtctggca
gccaaagcaa 300tcactcaaag ttcaaacctt atcatttttt gctttgttcc
tcttggcctt ggttttgtac 360atcagctttg aaaataccat cccagggtta
atgctggggt taatttataa ctaagagtgc 420tctagttttg caatacagga
catgctataa aaatggaaag atgttgcttt c 471
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References