U.S. patent application number 10/267117 was filed with the patent office on 2003-05-01 for raav compositions for gene therapy.
This patent application is currently assigned to University of Florida Research Foundation. Invention is credited to Byrne, Barry J., Flotte, Terence R., Morgan, Michael, Song, Sihong.
Application Number | 20030082162 10/267117 |
Document ID | / |
Family ID | 22175690 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030082162 |
Kind Code |
A1 |
Flotte, Terence R. ; et
al. |
May 1, 2003 |
rAAV compositions for gene therapy
Abstract
The subject invention concerns materials and methods for gene
therapy. One aspect of the invention pertains to vectors which can
be used to effect genetic therapy in animals or humans having
genetic disorders where expression of high levels of a protein of
interest are required to treat or correct the disorder. The subject
invention also pertains to methods for treating animals or humans
in need of gene therapy to treat or correct a genetic disorder. The
materials and methods of the invention can be used to provide
therapeutically effective levels of a protein that is
non-functional, or that is absent or deficient in the animal or
human to be treated. In one embodiment, the materials and methods
can be used to treat alpha-1-antitrypsin deficiency.
Inventors: |
Flotte, Terence R.;
(Gainesville, FL) ; Song, Sihong; (Gainesville,
FL) ; Byrne, Barry J.; (Gainesville, FL) ;
Morgan, Michael; (Gainesville, FL) |
Correspondence
Address: |
Mark D. Moore, Ph.D.
WILLIAMS, MORGAN & AMERSON, P.C.
Suite 250
7676 Hillmont
Houston
TX
77040
US
|
Assignee: |
University of Florida Research
Foundation
|
Family ID: |
22175690 |
Appl. No.: |
10/267117 |
Filed: |
October 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10267117 |
Oct 8, 2002 |
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09299141 |
Apr 23, 1999 |
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6461606 |
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60083025 |
Apr 24, 1998 |
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Current U.S.
Class: |
424/93.21 ;
514/44R |
Current CPC
Class: |
C12N 2750/14143
20130101; C12N 15/86 20130101; A61K 38/57 20130101; C07K 14/8125
20130101; A61K 48/00 20130101; A61P 3/10 20180101 |
Class at
Publication: |
424/93.21 ;
514/44 |
International
Class: |
A61K 048/00 |
Goverment Interests
[0002] The subject invention was made with government support under
a research project supported by National Institute of Health NHLBI
Grant No. HL 59412. The government has certain rights in this
invention.
Claims
41. A recombinant adeno-associated viral vector comprising a
polynucleotide that encodes an .alpha.-1-antitrypsin
polypeptide.
42. The vector of claim 41, wherein said vector comprises a
promoter operably linked to said polynucleotide.
43. The vector of claim 42, wherein said promoter is selected from
the group consisting of a CMV promoter, a hybrid CMV
enhancer/.beta.-actin promoter, an EF1 promoter, an U1a promoter
and an U1b promoter.
44. The vector of claim 42, wherein said promoter is an inducible
promoter selected from the group consisting of a Tet-inducible
promoter and a VP16-LexA promoter.
45. The vector of claim 42, wherein said vector further comprises
an enhancer.
46. The vector of claim 45, wherein said vector further comprises a
CMV enhancer.
47. The vector of claim 45, wherein said enhancer comprises a
synthetic enhancer.
48. The vector of claim 47, wherein said synthetic enhancer
comprises a muscle-specific enhancer.
49. The vector of claim 41, wherein said vector comprises at least
a first intron sequence.
50. The vector of claim 49, wherein said intron sequence comprises
intron II from a mammalian gene encoding .alpha.-1-antitrypsin.
51. The vector of claim 41, wherein said polynucleotide encodes a
human .alpha.-1-antitrypsin polypeptide.
52. The vector of claim 41, wherein said vector is selected from
the group consisting of DE-AT (SEQ ID NO:3)) E-AT (SEQ ID NO:2),
C-AT (SEQ ID NO:1), C-AT2(SEQ ID NO:7), p43C-AT (SEQ ID NO:4),
p43CB-AT (SEQ ID NO:6), p43C-AT-IN (SEQ ID
Description
CROSS-REFERENCE TO A RELATED APPLICATION
[0001] This application claims priority from provisional
application U.S. Serial No. 60/083,025, filed Apr. 24, 1998.
BACKGROUND OF THE INVENTION
[0003] Alpha-1-antitrypsin (AAT) deficiency is the second most
common monogenic lung disease in man, accounting for approximately
3% of all early deaths due to obstructive pulmonary disease. AAT
protein is normally produced in the liver, secreted into the serum
and circulated to the lung where it protects the fine supporting
network of elastin fibers from degradation by neutrophil elastase.
Current therapy for AAT deficiency includes avoidance of cigarette
smoke exposure and weekly intravenous infusions of recombinant
human AAT (hAAT) protein. Attempts to devise gene therapy
strategies to replace AAT either in the lung itself or within any
of a number of other tissues which are capable of AAT secretion
have been limited by the short duration of expression from some
vectors and by the relatively high circulating levels of AAT which
is required for therapeutic effect. Methods of gene therapy have
been described in U.S. Pat. No. 5,399,346.
[0004] It has recently been demonstrated that adeno-associated
virus (AAV) vectors are capable of stable in vivo expression and
may be less immunogenic than other viral vectors (Flotte et al.,
1996; xiao et al., 1996; Kessler et al., 1996; Jooss et al., 1998).
AAV is a nonpathogenic human parvovirus whose life cycle naturally
includes a mechanism for long-term latency. In the case of
wild-type AAV (wtAAV), this persistence is due to site-specific
integration into a site on human chromosome 19 (the AAVSI site) in
the majority of cells (Kotin et al., 1990), whereas with
recombinant AAV (rAAV) vectors, persistence appears to be due to a
combination of episomal persistence and integration into
non-chromosome 19 locations (Afione et al., 1996; Kearns et al.,
1996). Recombinant AAV latency also differs from that of wtAAV in
that wtAAV is rapidly converted to double-stranded DNA in the
absence of helper virus (e.g., adenovirus) infection, while with
rAAV leading strand synthesis is delayed in the absence of helper
virus (Fisher et al., 1996; Ferrari et al., 1996). U.S. Pat. No.
5,658,785 describes adeno-associated virus vectors and methods for
gene transfer to cells.
[0005] Kessler et al. (1996) demonstrated that murine skeletal
myofibers transduced by an rAAV vector were capable of sustained
secretion of biologically active human erythropoietin (hEpo),
apparently without eliciting a significant immune response against
the secreted hepo. See also U.S. Pat. No. 5,858,351 issued to
Podsakoffet al. Likewise, Murphy et al. (1997) have observed the
expression and secretion of sustained levels of leptin in ob/ob
mice after AAV muscle transduction. Brantly et al. (U.S. Pat. No.
5,439,824) disclose methods for increasing expression of AAT using
vectors comprising intron II of the human AAT gene. However, the
level of leptin expression observed was only in the range of 2 to 5
ng/ml. Therapy for AAT deficiency requires serum levels of at least
about 800 .mu.g/ml. Thus, there remains a need in the art for a
means of providing therapeutically beneficial levels of a protein
to a person in need of such treatment.
BRIEF SUMMARY OF THE INVENTION
[0006] The subject invention concerns materials and methods for
gene therapy. One aspect of the invention pertains to vectors which
can be used to provide genetic therapy in animals or humans having
a genetic disorder where relatively high levels of expression of a
protein is required to treat the disorder. The vectors of the
invention are based on adeno-associated virus (AAV). The vectors
are designed to provide high levels of expression of heterologous
DNA contained in the vector. In one embodiment, the vectors
comprise AAV inverted terminal repeat sequences and constitutive or
regulatable promoters for driving high levels of gene expression.
The subject invention also pertains to methods for treating animals
or humans in need of gene therapy, e.g., to correct a genetic
deficiency disorder.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 shows rAAV-AAT vector cassettes used according to the
subject invention. The A-AT and B-AT constructs contain the
promoters from the small nuclear RNA genes, U1a and U1b,
respectively. The C-AT construct contains the CMV promoter, whereas
the E-AT vector uses the human elongation factor 1-.alpha. (ELF in
the figure) promoter. ITR refers to AAV inverted terminal repeat;
An refers to polyA signal; Tk refers to the HSV thymidine kinase
promoter; neo refers to the Tn5 neomycin phosphotransferase
gene.
[0008] FIG. 2 shows hAAT secretion rates in vitro from transiently
transfected murine C2C12 myoblast cell line using expression
vectors according to the subject invention. C-AT does not differ
significantly from E-AT, but both differ from A-AT and B-AT
(p<0.05) AAT expression was detected using an ELISA assay
specific for human AAT.
[0009] FIG. 3 shows hAAT secretion rates in vitro from stably
transduced murine C2C12 myoblast cell line using viral particles
comprising expression vectors according to the subject invention.
The mean rates of secretion from G418-resistant cultures 1 mo after
transduction with either packaged E-AT vector or packaged C-AT
vector are shown. In each instance, a "low" multiplicity
transduction (4.times.10.sup.5 particles/cell) and a high
multiplicity transduction (4.times.10.sup.6 particles/cell) were
performed. E-AT "low" and "high" are greater than "high"
multiplicity C-AT (P=0.02) but are not significantly different from
each other (n=3). AAT expression was detected using an ELISA assay
specific for human AAT.
[0010] FIG. 4 shows additional constructs tested for hAAT
expression. The murine myoblast C2C12 cells were grown in 35-mm
wells with approximately 4.times.10.sup.5 cell per well and were
transfectd with 5 .mu.g of the appropriate plasmid DNA using
SUPERFECT transfection (Qiagen Inc., CA). Secretion of hAAT into
the medium was assessed at 2 days after transfection using an
antigen-capture ELISA. Each bar represents the mean of results from
three experiments (triplicate in each experiment).
[0011] Data from transfection experiments indicate that the
expression from p43CB-AT was at least three times higher than that
from C-AT in vitro.
[0012] FIGS. 5A and 5B show sustained secretion of therapeutic
levels of HAAT using either the C-AT vector or the E-AT vector in
either SCID or C57BL mice. FIG. 5A shows the mean total serum
levels of hAAT observed in groups of either SCID (squares) or C57BL
(circles) mice receiving either low dose (5.times.10.sup.11
particles) (open symbols) or high dose (1.4.times.10.sup.13
particles) (filled symbols) single injections into muscle of the
C-AT vector measured at time points ranging from 1 to 16 wk after
injection. For each strain, the high-dose curve is significantly
different from the low-dose curve (P=1.009 for SCID, P=0.02 for
C57BL), but the strains do not differ from each other. FIG. 5B
shows analogous data with the E-AT vector. None of these
differences were significant.
[0013] FIG. 5C shows long term secretion of HAAT from murine muscle
transduced with C-AT. C57B1/6 or C57B1/6-SCID mice received
3.5.times.10.sup.10 IU, 1.4.times.10.sup.13 particles/mouse. One
year after injection, serum hAAT levels were still 400 .mu.g/ml in
C57B1/6-SCID and 200 .mu.g/ml in C57B1/6. This level are comparable
with the peak levels observed (800 or 400 .mu.g/ml,
respectively).
[0014] FIG. 6 shows an immunoblot of sera taken from several of the
C-AT vector-treated mice at 11 weeks after vector administration.
Ten microliters of a 1:100 dilution of serum was electrophoresed by
10% SDS/PAGE, blotted, and incubated with 1:1,500 dilution of goat
anti-hAAT-horseradish peroxidase conjugate (Cappel/ICN). Samples
from three high-dose SCID (h1-h3), one high-dose C57B1 (h3), and
three low-dose C57B1 (101-103) were included, along with one
negative control (saline-injected=sal) serum to indicate the level
of reactivity with endogenous mAAT. As a standard, hAAT was added
either to negative-control C57B1 serum (first hAAT lane) or to PBS
(second hAAT) lane to final equivalent serum concentration of 100
.mu.g/ml.
[0015] FIGS. 7A and 7B show that some BALB/c mice mount humoral
immune responses to hAAT, which correlate with lower serum levels
but no observable toxicity. FIG. 7A shows serum hAAT levels and
FIG. 7B shows serum anti-hAAT antibody levels as determined by
ELISA performed on serum taken from mice injected with
1.times.10.sup.11 particles of the C-AT vector. Each set of symbols
represents an individual animal (.quadrature., no. 1; .DELTA., no.
2; .smallcircle., no. 3). Note the inverse correlation between the
presence of antibody and the presence of circulating hAAT.
[0016] FIG. 8 shows the persistence of rAAV-AAT vector DNA in high
molecular weight form. PCR products were amplified from DNA
prepared by Hirt extraction from three SCID mice injected 16 wk
earlier with 5.times.10.sup.11 resistant-particles of C-AT and
analyzed by Southern blot. The high molecular weight Hirt pellet
(genomic DNA lanes) and the low molecular weight supernatant
(episomal DNA lanes) were analyzed separately. Control lanes
include a sample in which an hAAT cDNA plasmid was the template DNA
(+) and a control in which water was the template (-). In this
internal PCR reaction, a 500-bp product is expected regardless of
whether or not the vector genome is integrated.
[0017] FIG. 9 shows serum hAAT in C57B1/6 mice transduced with C-AT
and p43CB-AT. C57B1/6 mice were injected in muscle with C-AT
(3.5.times.10.sup.10 IU/mouse, 1.times.10.sup.12 particles/mouse)
or p43CB-AT (6.times.10.sup.9 IU, 1.times.10.sup.12
particles/mouse). The level of hAAT from p43CB-AT were projected
based on an estimation of the equivalent dosage (infectious unit)
of C-AT.
[0018] FIG. 10 shows enhancement of CMV promoter activity by a
synthetic enhancer in C2C12 cells. The murine myoblast C2C12 cells
were grown in 35-mm wells with approximately 4.times.10.sup.5 cell
per well and were transfected with 5 .mu.g of p43msENC-AT vector
DNA using SUPERFECT transfection (Qiagen Inc, CA). Secretion of
hAAT into the medium was assessed at 2 days after transfection
using an antigen-capture ELISA. Each bar represents the mean of
results from one experiment (triplicate).
[0019] FIG. 11 shows secretion of hAAT from mouse liver cells
(HO15) transfected with different constructs. The murine liver
cells (HO15) were grown in 35-mm wells with approximately
4.times.10.sup.5 cell per well and were transfected with 5 .mu.g of
the plasmid DNA using LIPOFECTAMINE reagents (Life Technologies
Inc, MD). Secretion of hAAT into the medium was assessed at 2 days
after transfection using an antigen-capture ELISA. Each bar
represents the mean of results from two experiments
(triplicate).
[0020] FIG. 12 shows secretion of HAAT from mouse liver cells
(HO15) transfected using different methods. The murine liver cells
(HO15) were grown in 35-mm wells with approximately
4.times.10.sup.5 cell per well and were transfected with 5 .mu.g of
the p43CB-AT vector using SUPERFECT (Qiagen Inc., CA), FuGENE
(Boehringer Mannhem Co, IN), Lipofectin, LIPOFECTAMNE (ife
Technologies Inc, MD) reagents and Calcium phosphate (CA-PO4)
transfection. Secretion of hAAT into the medium was assessed at 2
days after transfection using an antigen-capture ELISA. Each bar
represents the mean of results from one experiment
(triplicate).
[0021] FIG. 13 shows hAAT secretion from mouse liver transduced
with rAAV. C57B1/6 mice were injected with either p43CB-AT, C-AT or
E-AT vector either by portal vein or tail vein injection. PV=portal
vein injection. TV=tail vein injection.
[0022] FIG. 14 shows serum HAAT levels in C57B1/6 mice after
intratracheal (IT) injection of C-AT orp43CB-AT vector. Mice
received either 10.sup.9 IU of C-AT (open circles), 10.sup.9 IU of
p43CB-AT (open triangles) or 10.sup.10 IU of p43CB-AT (open
squares).
[0023] FIG. 15 shows a map and nucleotide sequence for the vector
of the present invention designated as C-AT.
[0024] FIG. 16 shows a map and nucleotide sequence for the vector
of the present invention designated as E-AT.
[0025] FIG. 17 shows a map and nucleotide sequence for the vector
of the present invention designated as DE-AT.
[0026] FIG. 18 shows a map and nucleotide sequence for the vector
of the present invention designated as p43C-AT.
[0027] FIG. 19 shows a map and nucleotide sequence for the vector
of the present invention designated as p43C-AT-IN. This vector
includes intron II from human AAT gene to enhance
transcription.
[0028] FIG. 20 shows a map and nucleotide sequence for the vector
of the present invention designated as p43CB-AT.
[0029] FIG. 21 shows a map and nucleotide sequence for the vector
of the present invention designated as C-AT2.
[0030] FIG. 22 shows a map and nucleotide sequence for the vector
of the present invention designated as p43 msENC-AT. This vector is
similar to p43C-AT but also comprises an enhancer sequence upstream
of the CMV promoter.
[0031] FIG. 23 shows a map and nucleotide sequence for the vector
of the present invention designated as p43rmsENC-AT. This vector is
the same as the p43msENC-AT vector except that the enhancer
sequence is in an opposite orientation.
[0032] FIG. 24 shows a map and nucleotide sequence for the vector
of the present invention designated as p43 msENCB-AT. This vector
is similar to p43CB-AT but also comprises an enhancer sequence
upstream of the CMV promoter.
[0033] FIG. 25 shows a map and nucleotide sequence for the vector
of the present invention designated as p43rmsENCB-AT. This vector
is the same as p43msENCB-AT except that the enhancer sequence is in
an opposite orientation.
DETAILED DISCLOSURE OF THE INVENTION
[0034] The subject invention pertains to novel materials and
methods for providing gene therapy to a mammal or human having a
condition or disorder, such as genetic deficiency disorders, where
high levels of expression of a protein are required to treat the
disorder or condition. In one method of the subject invention, a
viral vector is introduced into cells of an animal wherein a
therapeutic protein is produced, thereby providing genetic therapy
for the animal. In one embodiment, a method of the invention
comprises introducing into an animal cell or tissue an effective
amount of viral particles or vector comprising a recombinant genome
which includes heterologous polynucleotide encoding a protein
useful in genetic therapy and that can be expressed by the cell or
tissue. Expression of the heterologous polynucleotide results in
production of the protein. Preferably, the therapeutic protein
encoded by the heterologous polynucleotide is a serum protein. In a
preferred embodiment, vector material comprising the heterologous
polynucleotide is integrated into a chromosome of the cell of the
host animal.
[0035] In one embodiment, a recombinant polynucleotide vector of
the present invention is derived from adeno-associated virus (AAV)
and comprises a constitutive or regulatable promoter capable of
driving sufficient levels of expression of the heterologous DNA in
the viral vector. Preferably, a recombinant vector of the invention
comprises inverted terminal repeat sequences of AAV, such as those
described in WO 93/24641. In a preferred embodiment, a vector of
the present invention comprises polynucleotide sequences of the
pTR-UF5 plasmid. The pTR-UF5 plasmid is a modified version of the
pTRBSUF/UF1/UF2/UFB series of plasmids (Zolotukhin et al., 1996;
Klein et al., 1998). The pTR-UF5 plasmid contains modifications to
the sequence encoding the green fluorescent protein (GFP).
[0036] Promoters useful with the subject invention include, for
example, the cytomegalovirus immediate early promoter (CMV), the
human elongation factor 1-alpha promoter (EF1), the small nuclear
RNA promoters (U1a and U1b), .alpha.-myosin heavy chain promoter,
Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV),
adenovirus major late promoter, .beta.-actin promoter and hybrid
regulatory element comprising a CMV enhancer/.beta.-actin promoter.
These promoters have been shown to be active in a wide range of
mammalian cells.
[0037] The promoters are operably linked with heterologous DNA
encoding the protein of interest. By "operably linked," it is
intended that the promoter element is positioned relative to the
coding sequence to be capable of effecting expression of the coding
sequence.
[0038] Promoters particularly useful for expression of a protein in
muscle cells include, for example, hybrid CMV enhancer/.beta.-actin
promoters, CMV promoters, synthetic promoters and EF1 promoter.
Promoters particularly useful for expression of a protein in liver
cells include, for example, hybrid CMV enhancer/.beta.-actin
promoters and EF1 promoters.
[0039] Also contemplated for use with the vectors of the present
invention are inducible and cell type specific promoters. For
example, Tet-inducible promoters (Clontech, Palo Alto, Calif.) and
VP16-LexA promoters (Nettelbeck et al., 1998) can be used in the
present invention.
[0040] The vectors can also include introns inserted into the
polynucleotide sequence of the vector as a means for increasing
expression of heterologous DNA encoding a protein of interest. For
example, an intron can be inserted between a promoter sequence and
the region coding for the protein of interest on the vector.
Introns can also be inserted in the coding regions. Exemplified in
the present invention is the use of intron H from the hAAT gene in
a subject vector. Transcriptional enhancer elements which can
function to increase levels of transcription from a given promoter
can also be included in the vectors of the invention. Enhancers can
generally be placed in either orientation, 3' or 5', with respect
to promoter sequences. In addition to the natural enhancers,
synthetic enhancers can be used in the present invention. For
example, a synthetic enhancer randomly assembled from
Spc5-12-derived elements including muscle-specific elements, serum
response factor binding element (SRE), myocyte-specific enhancer
factor-1 (NEF-1), myocyte-specific enhancer factor-2 (MEF-2),
transcription enhancer factor-1 (TEF-1) and SP-1 (Li et al., 1999;
Deshpande et al., 1997; Stewart et al., 1996; Mitchell et al.,
1989; Briggs et al., 1986; Pitluk et al., 1991) can be used in
vectors of the invention.
[0041] Heterologous polynucleotide in the recombinant vector can
include, for example, polynucleotides encoding normal, functional
proteins which provide therapeutic replacement for normal
biological function in animals afflicted with genetic disorders
which cause the animal to produce a defective protein, or abnormal
or deficient levels of that protein. Proteins, and the
polynucleotide sequences that encode them, which can be provided by
gene therapy using the subject invention include, but are not
limited to, anti-proteases, enzymes, structural proteins, coagulase
factors, interleulcins, cytokines, growth factors, interferons, and
lymphokines. In an exemplified embodiment, heterologous DNA in a
recombinant AAV vector encodes human alpha-1-antitrypsin
protein.
[0042] As those of ordinary skill in the art will appreciate, any
of a number of different nucleotide sequences can be used, based on
the degeneracy of the genetic code, to produce a protein of
interest for use in the present invention. Accordingly, any
nucleotide sequence which encodes a protein of interest comes
within the scope of this invention. Biologically active fragments
and variants of a protein of interest can easily and routinely be
produced by techniques well known in the art. For example,
time-controlled Bal31 exonuclease digestion of the full-length DNA
followed by expression of the resulting fragments and routine
screening can be used to readily identify expression products
having the desired activity (Wei et al., 1993).
[0043] As used herein, the terms "polynucleotide" and
"polynucleotide sequence" refer to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form,
and unless otherwise limited, would encompass known analogs of
natural nucleotides that can function in a similar manner as
naturally-occurring nucleotides. Polynucleotide sequences can
include both DNA strand sequences, such as that which is
transcribed into RNA, and RNA sequences. The polynucleotide
sequences include both full-length sequences as well as shorter
sequences derived from the full-length sequences. It is understood
that a particular polynucleotide sequence includes sequences, such
as degenerate codons of the native sequence or sequences, which may
be introduced to provide codon preference in a specific host cell.
Polynucleotides of the invention encompass both the sense and
antisense strands as either individual strands or in the
duplex.
[0044] The polynucleotides of the subject invention also encompass
equivalent and variant sequences containing mutations in the
exemplified sequences. These mutations can include, for example,
nucleotide substitutions, insertions, and deletions as long as the
variant sequence functions in a manner similar to the exemplified
sequences.
[0045] The gene therapy methods of the invention can be performed
by ex vivo or in vivo treatment of the patient's cells or tissues.
Cells and tissues contemplated within the scope of the invention
include, for example, muscle, liver, lung, skin and other cells and
tissues that are capable of producing and secreting serum proteins.
The vectors of the invention can be introduced into suitable cells,
cell lines or tissue using methods known in the art. The viral
particles and vectors can be introduced into cells or tissue in
vitro or in vivo. Methods contemplated include transfection,
transduction, injection and inhalation. For example, vectors can be
introduced into cells using liposomes containing the subject
vectors, by direct transfection with vectors alone, electroporation
or by particle bombardment. In an exemplified embodiment, muscle
cells are infected in vivo by injection of viral particles
comprising recombinant vector into muscle tissue of an animal. In
another embodiment, liver cells are infected in vivo by injection
of recombinant virus into either the portal vein or peripheral
veins.
[0046] The methods and materials of the subject invention can be
used to provide genetic therapy for any conditions or diseases
treatable by protein or cytokine infusion such as, for example,
alpha-1-antitrypsin deficiency, hemophilia, adenosine deaminase
deficiency, and diabetes. The methods and materials of the subject
invention can also be used to provide genetic therapy for treating
conditions such as, for example, cancer, autoimmune diseases,
neurological disorders, immunodeficiency diseases, and bacterial
and viral infections. For example, the present invention can be
used to provide genetic therapy to a patient wherein cells from the
patient are transformed to express and produce interleukins such as
interleukin-2.
[0047] Using the materials and methods of the subject invention,
the skilled artisan can for the first time provide therapeutically
effective levels of a serum protein through genetic therapy. In a
preferred embodiment, the therapeutically effective level of serum
protein that can be obtained using the subject materials and
methods is at least about 1 .mu.g/ml of protein in serum.
Preferably, the level of serum protein that can be obtained using
the present invention is at least about 100 .mu.g/ml of protein in
the serum. Most preferably, the level of serum protein that can be
obtained by the present invention is at least about 500 .mu.g/ml of
protein in the serum.
[0048] Animals that can be treated with the materials and methods
of the invention include mammals such as bovine, porcine, equine,
ovine, feline and canine mammals. Preferably, the mammals are
primates such as chimpanzees and humans.
[0049] The subject invention also concerns cells containing
recombinant vectors of the present invention. The cells can be, for
example, animal cells such as mammalian cells. Preferably, the
cells are human cells. More preferably, the cells are human
myofibers or myoblasts, hepatocytes or lung cells. In a preferred
embodiment, a recombinant vector of the present invention is stably
integrated into the host cell genome. Cell lines containing the
recombinant vectors are also within the scope of the invention.
[0050] In an exemplified embodiment, recombinant AAV vectors
comprising the human AAT gene (hAAT) using either the CMV promoter
(AAV-C-AT) or the human elongation factor 1-alpha (EF1) promoter
(AAV-E-AT) to drive expression were constructed and packaged using
standard techniques. A murine myoblast cell line, C2C12, was
transduced with each vector and expression of HAAT into the medium
was measured by ELISA. In vitro, the EF1 promoter construct
resulted in 10-fold higher hAAT expression than the CMV promoter
construct. In vivo transduction was performed by injecting doses of
up to 1.4.times.10.sup.13 Dnase-resistant particles of each vector
into skeletal muscles of a number of different strains of mice
(including C57B1/6, Balb/c, and SCID). In vivo, the CMV promoter
construct resulted in higher levels of expression, with sustained
serum levels up to 800 .mu.g/ml in SCID mice, approximately
10,000-fold higher than those previously observed with proteins
secreted from AAV vectors in muscle. At lower doses in both C57B1/6
and SCID mice, expression was delayed for several weeks, but was
sustained for over 10 weeks without declining. Thus, increasing
dosage AAV vector via transduction of skeletal muscle provides a
means for replacing AAT or other serum proteins.
[0051] Transduction of muscle using the vectors of the subject
invention presents several advantages in that it is stable,
non-toxic, and relatively nonimmunogenic. Furthermore, certain
transcription promoters, such as the CMV promoter, which appear to
be markedly down-regulated in other contexts have been found to
remain active over time as used in the subject invention. Using the
materials and methods of the subject invention, microgram/ml serum
levels of a therapeutic protein can be achieved. In an exemplified
embodiment, the levels of in vivo protein expression achieved
represent a 10,000-fold or more increase over previously published
results. In addition, a dose-effect relationship was demonstrable
within the range of doses used, providing for further increases in
expression levels as vector dose is increased.
[0052] In another embodiment of the invention, recombinant AAV
vectors i.e., C-AT, p43C-AT, P43CB-AT, E-AT and DE-AT comprising
the human AAT gene (hAAT) using were constructed and packaged using
standard techniques. A murine liver cell line, HO15, was
transfected with each vector and expression of hAAT into the medium
was measured by ELISA. In vitro, transduction with the p43CB-AT
vector exhibited the highest level of hAAT expression. In vivo, the
p43CB-AT vector also gave higher levels of expression. Portal vein
administration appeared to be the more efficient route of
administration as mice injected in this manner exhibited higher
levels of expression than those receiving peripheral vein
injections. Transduction of liver offers the same advantages as for
muscle, but hepatocytes may be more efficient at secretion of
protein.
[0053] The dosage of recombinant vector or the virus to be
administered to an animal in need of such treatment can be
determined by the ordinarily skilled clinician based on various
parameters such as mode of administration, duration of treatment,
the disease state or condition involved, and the like. Typically,
recombinant virus of the invention is administered in doses between
10.sup.5 and 10.sup.14 infectious units. The recombinant vectors
and virus of the present invention can be prepared in formulations
using methods and materials known in the art. Numerous formulations
can be found in Remington's Pharmaceutical Sciences, 15.sup.th
Edition (1975).
[0054] All publications and patents cited herein are expressly
incorporated by reference.
Materials and Methods
[0055] Construction of rAAV plasmids. The rAAV-AAT vector plasmids
used for these experiments are depicted diagrammatically (FIG. 1).
Briefly, the plasrmid pN2FAT (Garver et al., 1987) plasmid was
digested with Xhol to release 1.8-kb fragment containing the human
AAT cDNA along with the SV40 promoter and a polyadenylation signal.
This fragment was subcloned into a plasmid, pBlueScript
(Stratagene) and, after the removal of the SV40 promoter by Hind
III digestion and religation, the hAAT cDNA with its polyA signal
was released by XbaI and XhoI digestion. This 1.4-kb XbaI-XhoI
fragment was then cloned in to the pTR-UF5 (an AAV-inverted
terminal repeat-containing vector) plasmid (Zolotukhin et al.,
1996) between the XbaI site 3' to the CMV promoter and the XhoI
site 5' to the polyoma virus enhancer/HSVthyrnidine kinase promoter
cassette, which drives neo in that construct. This yielded the
pAAV-CMV-AAT construct (C-AT). Analogous constructs using the
promoter from the small nuclear RNA proteins, U1a and U1b, (to give
the A-AT and B-AT constructs, respectively) and human elongation
factor 1-alpha (EF1) promoter (to give the E-AT construct) were
constructed by substituting each of these promoter cassettes in
place of the CMV promoter, between the KpnI and XbaI sites.
[0056] The construct, DE-AT derived from E-AT by deletion of the
silencer (352 bp) by SAC II-cut (Wakabayashi-Ito et al., 1994).
C-AT2 is similar with C-AT except there are SV40 intron and poly
(A) sequences flanking the cDNA of HAAT. The p43C-AT was
constructed by insertion of hAAT cDNA to an AAV-vector plasmid
(p43), which has CMV promoter, intron and poly (A) sequences. The
p43CB-AT is derived by replacement of CMV promoter with CMV
enhancer and chicken P-actin promoter sequences. The p43C-AT-IN is
derived from p43C-AT by insertion of intron 11 sequences of hAAT
gene to HAAT cDNA (Brantly et al., 1995).
[0057] Packaging of rAAV vectors. Vectors were packaged using a
modification of the method described by Ferrari et al. (1997).
Briefly, plasmids containing the AAV rep and cap genes (Li et al.,
1997) and the Ad genes (E2a, E4 and VA-RNA) were co-transfected
along with the appropriate AAV-AAT vector plasmid into 293 cells
grown in Cell Factories (Nunc). Cells were harvested by
trypsinization and disrupted by freeze-thaw lysis to release vector
virions which were then purified by iodixanol gradient
ultracentrifugation followed by heparin sepharose affinity column
purification. Alternatively, recombinant virus can be prepared
according to methods described in Zolotukhin et al. (1999).
[0058] Vector preparations had their physical titer assessed by
quantitative competitive PCR and their biological titer assessed by
infectious center assay. The presence of wild-type AAV was also
assessed using these same assays with appropriate internal AAV
probes. The highdose C-AT stock had a particle-titer of
2.0.times.10.sup.14 particles/ml and an infectious titer of
5.0.times.10.sup.11 infectious units (i.u.)/ml (particle to i.u.
ratio=400:1). The low-dose C-AT measured 8.times.10.sup.12
particles/ml and 1.2.times.10.sup.10 i.u./ml (particle to
i.u.=667:1). For the E-AT experiments, the titers were
1.times.10.sup.13 particles/ml and 2.5.times.10.sup.10 i.u./ml
(particle to i.u.=400:1). The low-dose C-AT stock had a wt-like AAV
particle titer (i.e., positive AAV genome PCR) equal to 0.1 times
the recombinant titer but no detectable infectious wtAAV. The other
two preparations had wt-like AAV particle titers <10.sup.-5
times the recombinant titer and no detectable infectious wtAAV.
[0059] In vitro transfection and transduction experiments. The
C2C12 murine myoblast line was used for in vitro transfection and
transduction experiments. Cells were grown in 35-mm wells with
approximately 4.times.10.sup.5 cells per well and transfected with
5 .mu.g of each plasmid DNA using SUPERFECT (Qiagen Corp.).
Secretion of hAAT into the medium was assessed at 2 days after
transfection using an antigen-capture ELISA assay with standards
(Brantly et al., 1991). An SV40 promoter luciferase-expression
plasmid, pGL2 (Promega), was used as an internal control. For
transduction experiments, cells were grown under similar conditions
and were transduced with vector at multiplicities of infection
ranging from 4.times.10.sup.5 to 4.times.10.sup.6 particles per
cell. Cells were then passaged in the presence of geneticin sulfate
(350 .mu.g/ml) and geneticin-resistant clones were isolated for
hAAT secretion studies.
[0060] In vivo injection of AAV-C-AT and AAV-E-AT vectors into
murine muscle. Mice strains (C57B1/6, SCID, and Balb/c) were
obtained from Jackson Laboratories (Bar Harbor, Me.) and were
handled under specific pathogen-free conditions under a protocol
approved by the University of Florida Institutional Animal Care and
Use Committee. Animals were anesthetized by metaphane inhalation
and aliquots of vector were injected percutaneously into the
quadriceps femoris muscles of both hind limbs. The volume of vector
ranged from 50 to 100 .mu.l per injection site and the total amount
of virus injected per animal ranged from 5.times.10.sup.10 to
1.4.times.10.sup.13 Dnase-resistant particles.
[0061] Antigen capture ELISA assay for hAAT expression. Microtiter
plates (Immulon 4, Dynex Technologies, Chantilly, Va.) were coated
with 100 .mu.l of a 1:200 dilution of goat anti-human AAT
(CAPPEL/ICN) in Vollers buffer (Na2CO3=2.76 g, NaHCO3=1.916 g,
NaN3=0.2 g, d.H2O=1 liter, Adjust PH=9.6) overnight at 4.degree. C.
After washing, standards and unknown samples containing HAAT were
incubated in the plates at 37.degree. C. for 1 hour. After blocking
in 3% BSA in PBS-Tween 20 at 37.degree. C. for 1 hour, a second
antibody (1:1000 dilution of rabbit anti-human AAT, Boehringer
Mannheim) was reacted with the captured antigen at 37.degree. C.
for 1 hour. Detection was performed using a third antibody
incubation (1:800 dilution of goat anti-rabbit IgG-peroxidase
conjugate, 37.degree. C.) followed by ophenylenediamine (OPD,
Sigma) detection and measurement of the absorbance at 490 nm.
[0062] ELISA assay for anti-hAAT and anti-AAV VP3 antibodies. Wells
were coated with antigen (1 .mu.g of hAAT or 100 ng of VP3) at
4.degree. C. overnight, blocked with 3% BSA and then reacted with
dilutions of either test serum or with positive control antibodies
at 37.degree. C. for 1 hour. After washing, a goat-anti-mouse
IgG-peroxidase conjugate was used as a secondary antibody (1:1500
dilution) to detect bound anti-AAT antibody, using a standard OPD
reaction, as described above. Antibody levels were quantitated by
comparison with a standard curve generated by reacting dilutions of
known positive monoclonal antibodies against VP3 and hAAT.
[0063] Lymphocvte proliferation assays to detect cell-mediated
immune responses. Lymphocyte proliferation assays were performed in
order to detect T cell responses to the hAAT and VP3 antigens.
Freshly isolated splenocytes were grown in primary culture in 96
well plates coated with 0, 0, 1, 1, and 10 .mu.g of either HAAT or
VP3 in RPMI-C+ medium. On day three, a pulse of .sup.3H-thyrmidine
was added, and the cells were harvested on day 4 for lysis and
scintillation counting. Phytohemagglutinin (PHA) was used as a
mitogen for positive control wells. A stimulation index was
calculated for each antigen dosage level by dividing the counts per
minute (cpm) of .sup.3H-thymidine incorporated in the
antigen-stimulated cells by the cpm in a control (unstimulated)
well.
[0064] Following are examples which illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
EXAMPLE 1
In vitro Studies in Murine C2C12 Myoblasts
[0065] In order to determine the relative strength of a number of
constitutively active promoters in the context of AAV-AAT vectors,
packageable AAV-AAT expression vectors containing one of the CMV,
EF1, U1a or U1b promoters (FIG. 1) were constructed. Each of these
constructs were transfected in to the murine C2C12 myoblast cell
line. Both the EF1 and the CMV promoter were active for AAT
expression, with EF1 construct (AAV-E-AT) expressing 850
ng/10.sup.5 cells/day and the CMV construct (AAV-C-AT) expressing
approximately 670 ng/10.sup.5 cells/day, as measured by a
human-specific ELISA assay for AAT (FIG. 2). This difference was
not statistically significant. The levels of expression from the
U1a and U1b constructs were undetectable.
[0066] In order to better characterize the level and duration of
expression in the setting of vector transduction, cultures of C2C12
cells were transduced with either AAV-E-AT or AAV-C-AT at
multiplicities of infection ranging from 4.times.10.sup.5 to
4.times.10.sup.6 Dnase-resistant particles per cell. Cells were
then selected for expression of the neo gene (present in each of
the AAV constructs) by growth in G418-containing medium. Several
cell clones and pooled cell populations were independently analyzed
for AAT expression at four weeks post-transduction (FIG. 3). There
was a clear trend toward higher levels of expression at higher
multiplicities of infection, and the E-AT construct expressed at
least 10-fold greater quantities under all conditions in these
long-term cultures. The most active E-AT clone expressed hAAT at a
rate of over 1400 ng/10.sup.5 cells/day.
EXAMPLE 2
In vivo Expression of hAAT from Murine Skeletal Muscle
[0067] In order to determine whether the AAV-AAT constructs would
be active in vivo in skeletal muscle, doses of vector were injected
into the quadriceps femoris muscle of mice. Circulating serum
levels of HAAT were then measured for 11 to 15 weeks after the
initial injection. Four saline-injected animals from each mouse
strain served as controls. In the case of the C-AT vector (FIG.
5A), levels of expression were sufficient to achieve serum levels
in excess of 800 .mu.g/ml in SCID mice after a single injection of
1.4.times.10.sup.13 particles. A dose-effect relationship was
observed, with expression levels in SCID being at least 20-fold
lower at the 5.times.10.sup.11 particle dose. The levels of
expression increased over the first several weeks after injection
and were stable thereafter until the time of sacrifice. Since HAAT
has a half-life of less than 1 week, this indicated continuous
expression. Levels from C57B1/6 mice were comparable, and also
achieved values close to the therapeutic range. In similar studies,
two of three Balb/c mice injected with 1.times.10.sup.11 particles
of the C-AT vector did not express hAAT at detectable levels. Both
of these were found to have developed high levels of anti-hAAT
antibodies.
[0068] Surprisingly, expression levels from the AAV-E-AT vector
after in vivo injection were modestly lower than those seen with
the C-AT vector (FIG. 5B), with maximal levels of approximately 250
ng/ml at the 5.times.10.sup.11 dose at and beyond 7 weeks in SCID
mice. When the dose was further increased to 1.times.10.sup.12
particles, levels of approximately 1200 ng/ml were observed. These
levels were stable for one year post-injection (FIG. 5C). Levels
observed in SCID and immune competent C57B1/6 mice were
similar.
EXAMPLE 3
Imunologic Studies
[0069] In studies in Balb/c mice, antibody levels against hAAT were
high in 2 of 3 animals injected. The one which did not have
circulating anti-hAAT was the only animal with levels of hAAT
expression similar to those in the C57B1/6 and SCID groups. The
high-dose C57C-AT injection group had detectable levels of antibody
directed against VP3, but not hAAT.
[0070] In order to determine whether any cell-mediated immune
responses were mounted, lymphocyte proliferation assays were
performed using either hAAT or AAV-VP3 for antigenic stimulation of
primary splenic lymphocytes harvested at the time of animal
sacrifice, 16 weeks post-vector injection. Using this method, no
immune responses were detectable in any of the mice.
EXAMPLE 4
Lack of Toxicity from Direct Vector Injection
[0071] In order to determine whether there was any direct toxicity,
inflammation, or neoplastic change associated with vector
injection, animals underwent complete necropsies. Histopathologic
examination was performed on 5 .mu.m sections taken from the site
of vector injection and from a panel of other organs, including the
brain, heart, lungs, trachea, pancreas, spleen, liver, kidney, and
jejunum. No histologic abnormalities were observed in any of these
sites, even among those mice which developed humanol immune
responses against hAAT.
EXAMPLE 5
Molecular Evidence of AAV-AAT Vector Persistence
[0072] To confirm the presence of vector DNA, a vector-specific PCR
(neo primers 5'-TATGGGATCGGCCATTGAAC-3', and
5'-CCTGATGCTCTTC-GTCCAGA-3', was performed on DNA extracted from 3
SCID mice 16 weeks after injection with the C-AT vector, and PCR
products were analyzed by Southern blot analysis with a
.sup.32P-labeled vectorspecific probe (FIG. 8). The state of vector
DNA was analyzed using the Hirt procedure (Carter et al, 1983) to
separate the low molecular weight episomal DNA from the high
molecular weight fraction, which would contain integrated forms and
large concatemers. In each case, vector DNA was present in the high
molecular weight DNA fraction, whereas in only one of the animals
was there a signal in the episomal fraction. This result indicates
that by 16 weeks most of the vector DNA in our animals was either
integrated or in large concatemers.
EXAMPLE 6
In vivo Expression of HAAT from Murine Liver
[0073] Portal vein or tail vein injections were performed on 18
female C57BL/6 mice 8-10 weeks of age. The injection volume was 100
.mu.l per mouse.
[0074] Each group had the following parameters:
[0075] 1. Group 1: 100 .mu.l of PBS n=4.
[0076] 2. Group 2: 100 .mu.l of p43CB-AT (3.times.10.sup.10
IU/animal) n=3.
[0077] 3. Group 3: 100 .mu.l of p43CB-AT (4.times.10.sup.9
IU/animal) n=4.
[0078] 4. Group 4: 100 .mu.l of C-AT (4.times.10.sup.9 IU/animal)
n--2.
[0079] 5. Group 5: 100 .mu.l of E-AT (4.times.10.sup.9 IU/animal)
n=4.
[0080] 6. Group 6: EATM TV=100 .mu.l by tail vein injection of E-AT
(4.times.10.sup.9 IU/animal) n--3.
[0081] 7. Group 0: 100 .mu.l of PBS by tail vein injection n=2.
[0082] A total of 22 animals were used in this study.
[0083] All animals were anesthetized with 2-2-2 tribromoethanol
(Avertin) using a working solution of 20 mg/ml at a dosage of 0.5
mg/g IP. A 2 cm ventral midline abdominal incision was made from
the pubic symphysis extending cranially to the xyphoid process
through skin and muscle layers. The portal vein was exposed by
retracting the intestines and associated mesentery to the left side
of the aninal. Additionally, the quadrate and right medial lobes of
the liver were retracted cranially. Intestines and peritoneal
cavity were continuously lavaged with 0.9% NaC.l
[0084] Virus or PBS was delivered into the portal vein using a 30 g
needle attached to a 100 ul capillary pipette using mouth delivery
via rubber tubing and a Drummond self-locking double layer 0.8 um
filter. A small piece of Gel-Foam (0.5.times.0.5 cm) was applied to
the injection site before the needle was removed from the portal
vein. The needle was retracted from beneath the Gel-Foam and the
piece was held in place with forceps while the intestines were
replaced into the peritoneal cavity.
[0085] The muscle and skin were closed in one layer using 2 simple
interrupted 3-0 nylon sutures on an FS-1 cutting needle. Surgeries
were performed on a thermoregulated operating board designed to
maintain a temperature of 37 degrees. For recovery from anesthesia,
the animals were placed under a heat lamp adjusted to maintain an
ambient temperature of approximately 37 degrees and given
subcutaneous fluid if there was a significant amount of blood loss
during surgery.
[0086] Serum levels of hAAT in the mice were measured two weeks
after injection. Serum levels of about 200-150 .mu.g/ml HAAT were
detected in mice receiving the p43CB-AT vector (FIG. 13). Studies
using the E-AT vector show that injection of vector by portal vein
led to greater levels of hAAT secretion as compared to E-AT
administered by tail vein injection.
EXAMPLE 7
In vivo Expression of hAAT from Murine Lung
[0087] Mice were injected intratracheally with either C-AT or
p43CB-AT vector. Serum levels of HAAT in the mice were measured at
day 3, 14 and 31 after injection (FIG. 14). The p43CB-AT vector
mediated high levels of expression of hAAT in lung.
[0088] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims.
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