U.S. patent application number 09/746246 was filed with the patent office on 2001-08-23 for aav capsid vehicles for molecular transfer.
This patent application is currently assigned to University of Pittsburgh. Invention is credited to Ferrari, Forrest K., Samulski, Richard Jude.
Application Number | 20010016355 09/746246 |
Document ID | / |
Family ID | 23022972 |
Filed Date | 2001-08-23 |
United States Patent
Application |
20010016355 |
Kind Code |
A1 |
Samulski, Richard Jude ; et
al. |
August 23, 2001 |
AAV capsid vehicles for molecular transfer
Abstract
The invention relates to the production of AAV capsids which may
be used to transfer native or heterologous molecules into
appropriate host cells. The capsid proteins can be expressed from a
recombinant virus, expression vector, or from a cell line that has
stably integrated the AAV capsid genes or coding sequences. The
invention further provides for the production of AAV capsids in
vitro from the AAV capsid proteins and the construction of packaged
capsids in vitro. The invention further provides for the production
of AAV capsids that have been genetically engineered to express
heterologous epitopes of clinically important antigens to elicit an
immune response.
Inventors: |
Samulski, Richard Jude;
(Chapel Hill, NC) ; Ferrari, Forrest K.;
(Carrboro, NC) |
Correspondence
Address: |
PENNIE AND EDMONDS
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
100362711
|
Assignee: |
University of Pittsburgh
|
Family ID: |
23022972 |
Appl. No.: |
09/746246 |
Filed: |
December 22, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09746246 |
Dec 22, 2000 |
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08268430 |
Jun 30, 1994 |
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6204059 |
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Current U.S.
Class: |
435/456 ;
435/235.1 |
Current CPC
Class: |
A61P 37/00 20180101;
C12N 2750/14122 20130101; A61P 17/00 20180101; Y10S 977/84
20130101; C12N 2750/14123 20130101; A61K 48/00 20130101; C12N 7/00
20130101; A61K 39/00 20130101; C12N 2750/14143 20130101; Y10S
977/918 20130101; A61P 3/08 20180101; C12N 2710/10344 20130101;
C07K 14/005 20130101; Y10S 977/804 20130101; A61P 37/04 20180101;
C12N 15/86 20130101; Y10S 977/92 20130101 |
Class at
Publication: |
435/456 ;
435/235.1 |
International
Class: |
C12N 007/01; C12N
015/861 |
Claims
What is claimed is:
1. An adeno-associated virus capsid vehicle, comprising
adeno-associated virus capsid proteins associated with a molecule
heterologous to adeno-associated virus.
2. The adeno-associate virus capsid vehicle of claim 1 in which the
heterologous molecule is encapsidated in the AAV capsid
proteins.
3. The adeno-associated virus capsid vehicle of claim 1 or 2 in
which the heterologous molecule is a DNA molecule.
4. The adeno-associated virus capsid vehicle of claim 1 or 2 in
which the heterologous molecule is an RNA molecule.
5. The adeno-associated virus capsid vehicle of claim 1 or 2 in
which the heterologous molecule is a protein.
6. The adeno-associated virus capsid vehicle of claim 1 or 2 in
which the heterologous molecule is a peptide.
7. The adeno-associated virus capsid vehicle of claim 1 or 2 in
which the heterologous molecule is a small organic molecule.
8. The adeno-associated virus capsid vehicle of claim 2 in which
the encapsidated molecule further includes adeno-associated virus
genomic DNA.
9. The adeno-associated virus capsid vehicle of claim 2 in which
the encapsidated molecule further includes the adeno-associated
virus packaging signal.
10. The adeno-associated virus capsid vehicle of claim 2 in which
the encapsidated molecule includes the adeno-associated virus ITR
sequence.
11. A method for transferring a molecule into a cell, comprising
contacting the cell with an adeno-associated virus capsid vehicle
in which the molecule, which is heterologous to adeno-associated
virus, is associated with adeno-associated virus capsid
proteins.
12. The method of claim 11 in which the encapsidated molecule is a
DNA molecule.
13. The method of claim 11 in which the encapsidated molecule is an
RNA molecule.
14. The method of claim 11 in which the encapsidated molecule is a
protein.
15. The method of claim 11 in which the encapsidated molecule is a
peptide.
16. The method of claim 11 in which the encapsidated molecule is a
small organic molecule.
17. The method of claim 11 in which the encapsidated molecule
further includes adeno-associated virus genomic DNA.
18. The method of claim 11 in which the encapsidated molecule
further includes the adeno-associated virus packaging signal.
19. The method of claim 11 in which the encapsidated molecule
includes the adeno-associated virus ITR sequence.
20. The method of claim 11 in which the encapsidated
adeno-associated virus capsid vehicle is administered to an
animal.
21. The method of claim 20 in which the animal is a human.
Description
1. INTRODUCTION
[0001] The present invention relates to the production of
adeno-associated virus (AAV) capsids in vivo or in vitro which may
be used to transfer native or heterologous molecules into
appropriate host cells. The invention further relates to the
production of recombinant AAV capsids engineered to carry
heterologous antigens for the stimulation of an immune
response.
2. BACKGROUND OF THE INVENTION
[0002] The current interest in molecular replacement therapy as a
modality for clinical treatment has necessitated the development of
methods to safely and efficiently deliver genetic material or other
molecules to cells. This has been attempted using physical means of
cell permeation or by employing biological agents that naturally
infect a host cell.
2.1. PHYSICAL TRANSFER METHODS
[0003] Methods to transfer DNA into a recipient cell include
standard transfection techniques, mediated by calcium phosphate or
DEAE-dextran, electroporation of accessible cells, and
liposome-mediated transfer. These techniques are utilized in the
research setting but require in vitro handling of the recipient
cells and thus have limited clinical potential.
2.2. VIRAL VECTOR SYSTEMS
[0004] Viral vector systems exploit the efficiency of natural
infection and use of DNA technology to engineer recombinant viruses
that carry heterologous genes into the cell. Most clinical trials
to date have taken this approach (Morgan, R. A. and W. F. Anderson,
Annu. Rev. Biochem. 62:191-217, 1993).
[0005] Retroviral vectors have been most commonly used, chiefly
because they can facilitate the integration of the carried DNA into
the host cell genome, establishing stable integrants which are
amplified during cellular DNA replication. However, this
capability, while circumventing the limitations of transient gene
expression, can result in the inadvertent activation of host genes
or the interruption of cellular coding sequences due to random
integration.
[0006] Adenoviral vectors can introduce DNA into a cell, but do not
support the integration of the genetic material, which remains in
episomal form in the nucleus and does not co-replicate with the
cellular DNA. While adenoviruses are minor pathogens, their
optimization as clinically relevant transfer vehicles may be
limited to those tissues that are natural hosts for these viruses,
i.e., the lungs (Berkner, K., Curr. Topics. Micro. Immunol.
158:39-66, 1992).
[0007] There is a clear need for safer delivery systems that
combine the efficiency of viral infection with the potential to
deliver genetic material to a targeted integration site in the host
cell genome. There is also the need to be able to construct
molecular delivery vehicles in vitro which can then be packaged in
a cell-free system and which are capable of encapsidating a wide
range of molecular constituents.
2.3. ADENO-ASSOCIATED VIRUS
[0008] AAV is a parvovirus that can assume two pathways upon
infection of a host cell. In the presence of helper virus, AAV will
enter the lytic pathway where the viral genome is transcribed,
replicated, and encapsidated into newly formed viral particles. In
the absence of helper virus function, the AAV genome becomes
integrated as a provirus into a specific region of the host cell
genome, through recombination between the AAV termini and host cell
sequences. Characterization of the proviral integration site and
analysis of flanking cellular sequences indicates specific
targeting of AAV viral DNA into the long arm of human chromosome 19
(Kotin, R. M., et al., Proc. Natl. Acad. Sci. USA 87:2211-2215,
1990; Samulski, R. J., et al., EMBO J. 10:3941-3950, 1991). This
particular feature of AAV reduces the likelihood of insertional
mutagenesis resulting from random integration of viral vector DNA
into the coding region of a host gene. Furthermore, in contrast to
the retroviral LTR sequences, the AAV ITR sequences appear to be
devoid of transcriptional regulatory elements, reducing the risk of
insertional activation of protooncogenes.
[0009] The AAV genome is composed of a linear single stranded DNA
molecule of 4680 nucleotides which contains major open reading
frames coding for the Rep (replication) and Cap (capsid) proteins.
Flanking the AAV coding regions are two 145 nucleotide inverted
termini (ITR) repeat sequences that contain palindromic sequences
that can fold over to form hairpin structures that function as
primers during initiation of DNA replication. In addition to their
role in DNA replication, the ITR sequences have been demonstrated
to be necessary for viral integration, rescue from the host genome
and encapsidation of viral nucleic acid into mature virions
(Muzyczka, N., Curr. Top. Micro. Immunol. 158:97-129, 1992).
[0010] The capsids have icosahedral symmetry and are about 20-24 nm
in diameter. They are composed of three proteins (VP1, VP2, and
VP3, which are approximately 87, 73 and 61 Kd, respectively)
(Muzyczka, N., Curr. Top. Micro. Immunol. 158:97-129, 1992). VP3
represents 90% of the total virion protein; VP2 and VP1 account for
approximately 5% each. All capsid proteins are N-acetylated.
2.4. RECOMBINANT PRODUCTION OF VIRAL CAPSIDS
[0011] Recombinant DNA technology has been used to isolate the
genes for structural proteins of many viruses. For example,
vaccinia virus has been used to carry the structural genes for
human papilloma virus 1 (Hagensee, M. E., et al., J. Virol.
67:315-322, 1993); simian immunodeficiency virus (Gonzalez, S.A.,
et al., Virology 194:548-556, 1993); and Aleutian mink disease
parvovirus (Clemens, D. L., et al., J. Virol. 66:3077-3085, 1992);
capsid formation has been detected in all systems. Baculovirus
vectors have been used for the expression of the structural
proteins of human papilloma virus (Kirnbauer, R., et al., J. Virol.
67:6929-6936, 1993) and B19 parvovirus (Kajigaya, S., et al., Proc.
Natl. Acad. Sci. 88:4646-4650, 1991); these proteins have assembled
into capsids within the infected cells. Baculovirus-mediated
expression of the capsid proteins of adeno-associated virus-2
(Ruffing, M., et al., J. Virol. 66:6922-6930, 1992) resulted in the
formation of capsids with an altered stoichiometry from wild-type
capsids and which failed to localize into the nuclear clusters
observed in a wild-type infection.
[0012] These efforts have been expended to study virus life-cycles
and do not use of any of these systems to encapsidate foreign
genomes or other materials for delivery in vivo.
3. SUMMARY OF THE INVENTION
[0013] The invention relates to the production of AAV capsids which
may be used to transfer native or heterologous molecules into
appropriate host cells. The capsid proteins can be expressed from a
recombinant virus, expression vector, or from a cell line that has
stably integrated the AAV capsid genes or coding sequences. The
invention further provides for the production of AAV capsids in
vitro from the AAV capsid proteins and the construction of packaged
capsids in vitro. The invention further provides for the production
of AAV capsids that have been genetically engineered to express
heterologous epitopes of clinically important antigens to elicit an
immune response.
[0014] Molecules which may be associated with or encapsidated into
capsids include DNA, RNA, proteins, peptides, small organic
molecules, or combinations of the same. The AAV capsids can
accommodate nucleic acids which are quite large e.g., 5000 bp, and
therefore, may be advantageously used for the transfer and delivery
of large genes and genomic sequences. Because the AAV inverted
terminal repeats (ITRs) are responsible for the ability of the AAV
genome to integrate into the host cell genome (Samulski, R. J., et
al., EMBO 10:3941-3950, 1991), these sequences may be used with the
heterologous DNA in order to provide for integration of the
heterologous DNA into the host cell genome and may further
facilitate packaging into an AAV capsid.
[0015] The invention is demonstrated by way of examples in which
the AAV capsid is produced from a recombinant adenovirus engineered
to carry the capsid genes. This system may be particularly
advantageous in AAV gene delivery systems because adenovirus serves
as a natural helper for AAV infection. Upon infection of the host
cell, expression of the capsid proteins is followed by assembly of
the AAV capsid.
4. BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1. Representation of d1324/CMV/Cap construction.
[0017] FIG. 2. Immunoblot of products of d1324/CMV/Cap expression
in 293 cells. Lanes 1-5 were probed with anti-AAV2, lanes 6-10 were
probed with anti-Rep. Lanes 1 and 6: pCAD/Cap; lanes 2 and 7:
d1324/CMV/Cap; lanes 7 and 8: Ad+AAV2; lanes 4 and 9; Ad; lanes 5
and 10: Mock.
[0018] FIG. 3. Results of a one-step growth curve of d1324/CMV/Cap
and Ad-.beta.gal. At the times indicated on the X-axis, virus was
collect from infected 293 cells and a plaque assay was performed to
determine the titer (pfu/ml) shown on the Y-axis. This is the
average result of two experiments.
[0019] FIG. 4. d1324/CMV/Cap Capsid expression time course. 293
cells were infected with equal amounts of d1324/CMV/Cap and
collected at various time points post infection. Capsid expression
was detected by immunoblot. Lanes 1 and 7: Oh post infection; lanes
2 and 8: 6h post infection; lanes 3 and 9: 12h post infection;
lanes 4 and 10: 24h post infection; lanes 6 and 11: 36h post
infection; lanes 7 and 12: 48h post infection. The right-hand side
of the blot shows the results from a parallel Ad+AAVp2
infection.
[0020] FIG. 5. Splicing of AAV2 capsid messages expressed from
d1324/CMV/Cap. mRNA from infected 293 cells was isolated and
synthesized into CDNA. This cDNA was then amplified, in the
presence of .alpha.-.sup.32P-dCTP using the primers CAPL and CAPR1.
The products were separated on 10% polyacrylamide gels and
visualized by autoradiography. Lane 1: Ad+AAV2; lane 2:
d1324/CMV/Cap.
[0021] FIG. 6. Immunofluorescence of AAV2 capsid and Rep proteins.
HeLa cells infected with d1324/CMV/Cap in the absence (left photo)
or presence (right photo) of Rep protein expression.
[0022] FIG. 7. Capsid immunoblot of CsCl density gradient
fractions.
[0023] FIG. 8. Electron micrograph of empty capsids expressed from
d1324/CMV/Cap. Mag. 250,000.
[0024] FIG. 9. 293 cells were transfected/infected (panel 1A, panel
1B) with 10 ug/per 10 cm dish, pAB11 plasmid DNA and 5 pfu wild
type adenovirus. 293 cells were infected with 200 ul viral lysate
from ad/CMV/CAP complementation system (Middle panel A and B) or
from pAd/AAV helper system (Third panel A and B). LacZ
histochemical analysis of infected cells were assayed at 24 hours
(EMBO 5:3133, 1986). Photographs were taken at a magnification of
10x for panel 1A or 20x for the remainder of the panels. Each panel
represents an individual field from an infected dish of cells.
5. DETAILED DESCRIPTION OF THE INVENTION
[0025] The invention relates to methods for producing AAV capsids
which may be used to transfer molecules for molecular replacement
therapy. Methods for the intracellular production of AAV capsids
provided include vector-mediated expression systems and cell-line
expression systems for the generation of capsids. Methods for the
in vitro construction of AAV capsids and for the in vitro packaging
of these capsids are also provided. The invention is also directed
to the production of AAV capsids which are engineered to carry
heterologous epitopes that can elicit an immune response in
vivo.
5.1. AAV CAPSID PROTEINS
[0026] The AAV capsids of the present invention are produced by the
expression of the three capsid genes, VP1, VP2, and VP3, and the
subsequent assembly of these proteins into the AAV capsid
particle.
[0027] The AAV capsid genes are found in the right-hand end of the
AAV genome, and are encoded by overlapping sequences of the same
open reading frame through the use of alternative initiation
codons. A 2.6 kb precursor mRNA is alternatively spliced into two
2.3 kb transcripts. Both VP2 and VP3 can be produced from either
transcript with the use of different translation initiation
signals, while VP1 can only be translated from one of the
transcripts. The fact that overlapping reading frames code for the
three AAV capsid proteins results in the obligatory expression of
all capsid proteins in a wild-type infection.
[0028] In accordance with the invention the open reading frame
which encodes the entire AAV VP1, VP2 and VP3 capsid proteins may
be engineered into expression vectors. The use of a gene sequence
that encodes the three overlapping reading frames may result in a
level and pattern of expression of the capsid proteins that mimics
the wild-type infection and generates wild-type AAV capsids. The
disadvantage of this approach is that the capsid composition cannot
be regulated or altered.
[0029] Alternatively, multiple vectors may be used to separately
introduce each of the capsid genes into expression host cell. The
use of AAV capsid CDNA gene sequences allows for construction of
separate expression vectors which may be introduced into the cell
alone or together, and may also be quantitatively controlled. Such
control may be achieved by the amount of a vector introduced into a
cell, or, alternatively, individual vectors may employ specific
promoters that are chosen for strength of expression of a linked
capsid gene. Thus, the stoichiometry and composition of the AAV
capsids may be regulated. A disadvantage of this approach is that
the natural stoichiometry of the capsid proteins in a wild-type
infection may not be achieved for optimal assembly into
capsids.
[0030] The sequences of the capsid genes are reported in
Srivastava, A., et al., 1983, J. Virol. 45:555-564; Muzyczka, N.,
1992, Curr. Top. Micro Immunol. 158:97129, and Ruffing, M., et al.,
1992, J. Virol. 66:6922-6930, 1992. Sources for the AAV capsid
genes may include the mammalian virus serotypes AAV-1, AAV-2,
AAV-3, AAV-4, and AAV-5, as well as bovine AAV and avian AAV. The
invention contemplates, in addition to the capsid DNA sequences
disclosed therein, (1) any DNA sequence that encodes the same amino
acid sequence for capsid VP1, VP2 and VP3 shown in Srivastava, A.,
et al., supra; Muzyczka, N., supra and Ruffing, M., et al. supra;
(2) any DNA sequence that hybridizes to the complement of the
coding sequences disclosed therein under highly stringent
conditions, e.g., washing in 0.1xSSC/0.1% SDS at 68.degree. C.
(Ausubel F. M. et al., eds., 1989, Current Protocols in Molecular
Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley
& sons, Inc., New York, at p. 2.10.3) and encodes a
functionally equivalent gene product; and/or 3) any DNA sequence
that hybridizes to the complement of the coding sequences disclosed
therein under less stringent conditions, such as moderately
stringent conditions, e.g., washing in 0.2xSSC/0.1% SDS at
42.degree. C. (Ausubel et al., 1989, supra) , yet which still
encodes a functionally equivalent gene product.
[0031] The invention also encompasses 1) DNA vectors that contain
any of the coding sequences disclosed herein and/or their
complements (i.e., antisense); 2) DNA expression vectors that
contain any of the coding sequences disclosed herein and/or their
complements (i.e., antisense), operatively associated with a
regulatory element that directs the expression of the coding and/or
antisense sequences; and (3) genetically engineered host cells that
contain any of the coding sequences disclosed herein and/or their
complements (i.e., antisense), operatively-associated with a
regulatory element that directs the expression of the coding and/or
antisense sequences in the host cell. Regulatory elements include
but are not limited to inducible and non-inducible promoters,
enhancers, operators and other elements known to those skilled in
the art that drive and regulate expression. The invention includes
fragments of any of the DNA sequences disclosed herein.
[0032] Alternatives to isolating a capsid gene sequence include,
but are not limited to, chemically synthesizing the gene sequence
from a known sequence or making cDNA to the RNA which encodes the
capsid proteins. Other methods are possible and within the scope of
the invention.
[0033] Nucleic acids which encode derivatives (including fragments)
and analogs of native capsid proteins can also be used in the
present invention, as long as such derivatives and analogs retain
the ability to assemble into an AAV capsid. In particular, capsid
derivatives can be made by altering capsid sequences by
substitutions, additions, or deletions that provide for
functionally active molecules. Furthermore, due to the degeneracy
of nucleotide coding sequences, other DNA sequences which encode
substantially the same or a functionally equivalent AAV capsid
amino acid sequence may be used in the practice of the methods of
the invention. The gene product may contain deletions, additions or
substitutions of amino acid residues within the sequence which
result in silent changes thus producing a bioactive product. Such
amino acid substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity and/or
the ampipathic nature of the residues involved. For example,
negatively charged amino acids include aspartic acid and glutamic
acid; positively charged amino acids include lysine and arginine;
amino acids with uncharged polar head groups or nonpolar head
groups having similar hydrophilicity values include the following:
leucine, isoleucine, valine, glycine, alanine, asparagine,
glutamine, serine, threonine, phenylalanine, tyrosine.
5.2. IN VIVO PACKAGING SYSTEMS
[0034] AAV capsid genes may be expressed from the recombinant
expression vectors or from an engineered cell line so that the
proteins are able to assemble into the capsid within the cell.
5.2.1. RECOMBINANT EXPRESSION OF AAV CAPSID PROTEINS AND CAPSID
FORMATION
[0035] The invention can be facilitated with the use of a number of
viruses or vectors which can be engineered to carry the genes for
the AAV capsid proteins. Recombinant viruses or vectors will be
used to infect or transfect appropriate host cells, and the
expression of the AAV proteins will commence, resulting in the
production of adequate levels of the capsid proteins to facilitate
capsid formation.
[0036] In a preferred embodiment, the virus for the construction of
a recombinant virus is a virus which is a natural helper for
wild-type AAV infection. Such viruses could include herpesviruses
or adenoviruses. Since these viruses are required for gene
expression by a wild-type AAV, their use as the recombinant carrier
for the AAV capsid proteins may be optimal for the production of
appropriate levels and ratios of the three capsid proteins since
they may be facilitating these processes in the wild-type
infection.
[0037] In a specific embodiment, adenovirus is used as the
recombinant virus. Deletion strains of adenovirus can accommodate
the insertion of the heterologous material, i.e., the AAV capsid
coding region, into non-essential regions of the adenovirus such as
E1 or E3. Infection of adenovirus into a complementing host cell
line, such as the 293 line, will allow the expression of the AAV
capsid proteins and the subsequent assembly of these into the
capsid vehicle. Heterologous promoters for the capsid genes may be
used, including but not limited to CMV, pGK, beta actin, RSV, SV40,
and transthyretin liver specific promoter. Host cells may include
AS49, HeLa, Cos-1, KB and Vero.
[0038] Recombinant vaccinia virus can be produced by homologous
recombination between a plasmid carrying the capsid genes and
wild-type vaccinia virus within a host cell. Expression of these
genes by the recombinant virus results in the assembly of the
proteins into the capsids. Host cells may include CV-1, HeLa,
BSC-40, BSC-1 and TK.sub.143B.
[0039] In another embodiment of the invention, baculovirus vectors
may be constructed to carry the AAV capsid coding region by
engineering these genes into the polyhedrin coding region of a
baculovirus vector and producing viral recombinants by transfection
into a baculovirus-infected cell. These viruses can express the AAV
capsid proteins and facilitate the production of the capsids
subsequently. Host cells may include Sf9 and Sf24.
[0040] In another embodiment of the invention, recombinant
expression vectors may be used which are engineered to carry one or
more of the AAV capsid genes into a host cell to provide for
expression of the AAV capsid proteins.
[0041] Such vectors may be introduced into a host cell by
transfection with calcium-phosphate or DEAE-dextran, or by
electroporation or liposome-mediated transfer.
[0042] Recombinant expression vectors include, but are not limited
to, COS cell-based expression vectors such as CDM8 or pDC201, or
CHO cell-based expression vectors such as pED vectors.
[0043] The capsid coding region may be linked to any number of
promoters in an expression vector that can be activated in the
chosen cell line. Additionally, this cassette (capsid genes and
promoter) is carried by a vector that contains a selectable marker
so that cells receiving the vector may be identified.
[0044] Promoters to express the capsid proteins within a cell line
may be drawn from those that are functionally active within the
host cell. They may include, but are not limited to, the CMV
promoter, the SV40 early promoter, the herpes TK promoter, and
others well known in recombinant DNA technology. Inducible
promoters may be used, including but not limited to, the
metallothionine promoter (MT), the mouse mammary tumor virus
promoter (MMTV), and others known to those skilled in the art.
[0045] Selectable markers and their attendant selection agents can
be drawn from the group including but not limited to aminoglycoside
phosphotransferase/G418, hygromycin-B
phosphotransferase/hygromycin-B, and amplifiable selection markers
such as dihydrofolate reductase/methotrexate and others known to
skilled practitioners.
[0046] Other embodiments of the present invention include the use
of procaryotic, insect, plant, and yeast expression systems to
express the AAV capsid proteins. In order to express capsid
proteins the nucleotide sequence coding for the capsid proteins, or
a functional equivalent as described in Section 5.1, supra, are
inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted coding sequences. Methods which are
well known to those skilled in the art can be used to construct
expression vectors containing the capsid protein coding sequences
operatively associated with appropriate transcriptional/transla-
tional control signals. These methods include in vitro recombinant
DNA techniques, synthetic techniques, and in vivo
recombination/genetic recombination. See, for example, the
techniques and vectors described in Maniatis, et al., 1989,
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory, New York and Ausubel et al., 1989, Current Protocolsin
Molecular Biology, Greene Publishing Associates & Wiley
Interscience, New York.
[0047] A variety of prokaryotic, insect, plant and yeast expression
vector systems (i.e.-vectors which contain the necessary elements
for directing the replication, transcription, and translation of
capsid coding sequences) may be utilized equally well by those
skilled in the art, to express capsid coding sequences. These
include but are not limited to microorganisms such as bacteria
transformed with recombinant bacteriophage DNA, plasmid DNA or
cosmid DNA expression vectors containing the capsid coding
sequences; yeast transformed with recombinant yeast expression
vectors containing the capsid coding sequences; insect cell systems
infected with recombinant virus expression vectors (e.g.,
baculovirus) containing the capsid coding sequences; plant cell
systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing the capsid coding sequences.
[0048] The expression elements of these vectors may vary in their
strength and specificities. Depending on the host/vector system
utilized, any one of a number of suitable transcription and
translation elements may be used.
[0049] Specific initiation signals are also required for sufficient
translation of inserted protein coding sequences. These signals
include the ATG initiation codon and adjacent sequences. These
exogenous translational control signals and initiation sequences
can be of a variety of origins, both natural and synthetic. For
example, E. coli expression vectors will contain translational
control sequences, such as an appropriately positioned ribosome
binding site and initiation ATG. The efficiency of expression may
be enhanced by the inclusion of transcription attenuation
sequences, enhancer elements, etc.
[0050] An alternative expression system which could be used to
express AAV capsid proteins is an insect system. In one such
system, Autographa californica nuclear polyhidrosis virus (AcNPV)
is used as a vector to express foreign genes. The virus grows in
Spodoptera frugiperda cells. The AAV capsid coding sequences may be
cloned into non-essential regions (for example the polyhedrin gene)
of the virus and placed under control of an AcNPV promoter (for
example the polyhedrin promoter). Successful insertion of the AAV
capsid coding sequences will result in inactivation of the
polyhedrin gene and production of non-occluded recombinant virus
(i.e., virus lacking the proteinaceous coat coded for by the
polyhedrin gene). These recombinant viruses are then used to infect
Spodoptera frugiperda cells in which the inserted gene is
expressed.
[0051] In any of these embodiments, the capsid proteins may
assemble into an AAV capsid intracellularly, or, alternatively, the
proteins may be isolated from the expression system for
construction of the AAV capsids in vitro.
[0052] Detection of the AAV capsid proteins produced in the above
embodiments of the invention can be performed by standard
techniques including Northern analysis to detect expression of
mRNA, and protein-based detection techniques such as immunoblotting
or immunoprecipitation. Detection of the AAV capsids can be
accomplished by subjecting a lysate from infected cells to
isopycnic centrifugation to concentrate the viral particles at the
proper gradient density. Further confirmation of the presence of
the viral capsids can be ascertained by transmission electron
microscopy to visualize and measure the particles.
5.2.2. CELL LINES ENGINEERED TO PRODUCE AAV CAPSIDS
[0053] A cell line may be engineered that will natively express the
three AAV capsid proteins, which will then assemble into an AAV
capsid.
[0054] To engineer an AAV-capsid producing cell line, cells are
tranfected with a vector into which the AAV capsid open reading
frame has been inserted. Alternatively, each capsid protein coding
region may be engineered into separate vectors and used to
transfect host cells. Transfection may be accomplished with any of
the standard techniques in the art. Alternatively, a cell line can
be established with the use of viral vectors that are capable of
integrating DNA into the host cell genome. Examples of these
vectors include those derived from retroviruses or AAV.
[0055] Cell lines which may be chosen for integration include but
are not limited to HeLa, COS, NIH 3T3, and others well known to
those skilled in the art. The capsid coding region may be linked to
any number of heterologous promoters that can be activated in the
chosen cell line. Additionally, this insertion cassette (capsid
genes and promoter) may be linked to a gene coding for a selectable
marker, in which case the integration of the capsid coding region
with the linked marker will confer the particular phenotype
afforded by the marker to a stably transfected cell. Thus, the
cells that have successfully integrated the capsid genes will be
selectable. Alternatively, the selectable marker may be transfected
on a separate plasmid.
[0056] Promoters to express the capsid proteins within a cell line
may be drawn from those that are functionally active within the
host cell. They may include, but are not limited to, the CMV
promoter, the SV40 early promoter, the herpes TK promoter, and
others well known in recombinant DNA technology. Inducible
promoters may be used, including but not limited to, the
metallothionine promoter (MT), the mouse mammary tumor virus
promoter (MMTV), and others known to those skilled in the art.
[0057] Selectable markers and their attendant selection agents can
be drawn from the group including but not limited to aminoglycoside
phosphotransferase/G418, hygromycin-B
phosphotransferase/hygromycin-B, and amplifiable selection markers
such as dihydrofolate reductase/methotrexate and others known to
skilled practitioners.
[0058] Stable expressing cell lines may also be constructed by
linking the AAV ITR sequence to an expression cassette containing
the capsid coding region with the appropriate transcriptional
signals to allow for integration into the host cell genome.
[0059] Standard recombinant DNA techniques may be used to construct
the recombinant viruses and vectors (Ausubel, F. et al., eds.,
Current Protocols in Molecular Biology, Wiley & Sons, New York,
1994).
[0060] Detection of the expression of the capsid genes can be
performed by standard techniques including Northern analysis,
immunoblotting, and immunoprecipitation. Detection of the
production of the viral capsids can be accomplished by subjecting a
cell lysate to isopycnic centrifugation wherein the viral particles
will band according to their density. Further confirmation of the
presence of the viral capsids can be ascertained by transmission
electron microscopy to measure and visualize the particles.
[0061] Sources for the AAV capsid genes may include the mammalian
virus serotypes AAV-1, AAV-2, AAV-3, AAV-4, and AAV-5, as well as
bovine AAV and avian AAV.
5.3. MANIPULATIONS OF AAV CAPSID VEHICLES
[0062] AAV capsids are constructed in vitro from the AAV capsid
proteins and these vehicles can be used to deliver molecular
constituents into a target cell. In another embodiment, the AAV
capsids are recovered from a cell and packaged in vitro with the
desired constituents. In a yet another embodiment, AAV capsid
produced intracellularly can be packaged in vivo with DNA
introduced into the same cell or other intracellular molecular
constituents.
5.3.1. INTRACELLULAR FORMATION OF PACKAGED CAPSIDS
[0063] In this embodiment of the present invention, the AAV capsids
produced intracellularly by any of the methods described in Section
5.2., supra, may be packaged within the cell with any number of
molecular constituents. DNA introduced into the cell which
expresses the capsid proteins may be packaged into an AAV capsid,
and this DNA may be advantageously linked to the AAV ITR to
increase the efficiency of packaging. Other molecular constituents,
such as RNA, proteins, peptides, or small organic molecules that
are either introduced into the cell or are naturally found
intracellularly may also be packaged into the AAV capsid within the
cell.
5.3.2. IN VITRO PACKAGING OF AAV CAPSIDS
[0064] In this embodiment, AAV capsids are isolated by standard
methods for the recovery of AAV virions. Cells engineered to
express the AAV capsid proteins as described in Sections 5.1 and
5.2 supra, or cells infected with recombinant viruses carrying the
AAV capsid genes or cells infected with wild-type AAV in a
helper-virus background are collected by centrifugation and
subjected to freeze-thaw cycles that separate viral material from
the cells. The lysate is subjected to isopycnic centrifugation
(CsCl) and the particles are recovered from the appropriate band on
the gradient. This sample is subjected to a second round of CsC1
centrifugation, and fractions from the gradient are recovered and
analyzed for the presence of the AAV capsid proteins by Western
Blot analysis. The gradient density of these enriched fractions
will determine the nature of the viral particles that are banded.
Fractions which show the presence of the three capsid proteins are
those that are enriched with the viral capsids. Further
confirmation of capsid production can be obtained by subjecting an
aliquot of the enriched fractions to transmission electron
microscopy to visualize the AAV capsids.
[0065] The capsids may be dissembled by known techniques for the
dissociation of macromolecular protein structures, including
denaturation with urea or guanidium hydrochloride, heat, or pH
manipulation. Where structural integrity is dependent on disulfide
bond formation, mercaptoethanol or any thiol reducing agent will
cause covalent disulfide bond rupture.
[0066] Reassembly of the AAV capsid with the desired constituents
is accomplished by co-incubation of the capsid proteins with the
materials to be packaged. Favorable conditions for reassembly
include manipulations of pH, temperature, and buffer conditions
that are well known to one skilled in the art.
[0067] In the specific embodiment in which DNA is to be packaged,
the molecule may be linked to the AAV ITR signal for optimal
encapsidation and for integration of the DNA into the host cell
genome.
5.3.3. IN VITRO ASSEMBLY OF AAV CAPSIDS
[0068] In this embodiment of the invention, the three AAV capsid
proteins may be isolated in vitro and combined to form the AAV
capsid. The proteins may be recovered from lysates of
virus-infected cells or from pure preparations of AAV virus.
Alternatively, the proteins may be recovered from cells infected
with recombinant virus carrying the AAV capsid genes, or from cells
engineered to stably express the capsid proteins.
[0069] Recovery of the capsid proteins from any of the above
sources may be accomplished by the use of known techniques for
protein isolation, i.e., affinity chromatography, ion-exchange
chromatography, gel-filtration chromatography, or HPLC (Creighton,
T. E., Proteins, W. H. Freeman and Company, New York, 1984).
[0070] With the AAV capsid proteins so isolated, they may be
combined in vitro so as to mimic the levels of the proteins found
in the AAV virion and therefore facilitate the reconstitution of
the capsid. In this embodiment of the invention, VP3 is the major
constituent of the in vitro reaction since it accounts for about
90% of the virion protein, and VP2 and VP1 are each present is
lesser amounts, about 5% each, corresponding to their quantitative
presence as a component of the virion. Alternatively, the proteins
may be combined in an equimolar ratio for the formation of the
capsid. Optimization for the formation of the capsids may rely on
the parameters of pH, temperature, or buffer conditions, and are
known to those skilled in the art.
[0071] The capsid proteins may be also combined with the
constituents to be packaged into the viral particle to allow for
assembly and packaging simultaneously. In this embodiment, the
constituent may be native or heterologous DNA to which the AAV
packaging signal is attached. Alternatively, the constituents may
include DNA, RNA, proteins, or peptides which can be associated
with, or encapsidated into the assembling capsid. Capsid proteins
and the constituents may be combined simultaneously in vitro for
the formation of packaged AAV capsids ready for transfer.
5.4. ENCAPSIDATED COMPONENTS
[0072] Molecules which may be packaged by the AAV capsids and
subsequently transferred into celIs include recombinant AAV
genomes, which advantageously may then integrate into the target
cell genome, and other heterologous DNA molecules. RNA, proteins
and peptides, or small organic molecules, or combinations of the
same, may also be encapsidated and transferred. Native molecular
constituents are defined as those found in a wild-type AAV
infection such as the AAV DNA genome, AAV RNA or AAV viral
proteins. Heterologous molecules are defined as those that are not
naturally found in an AAV infection; i.e., those not encoded by the
AAV genome.
[0073] In a preferred embodiment of the present invention, the
segment of DNA to be encapsidated may be linked to the AAV ITR
sequences which contain the viral packaging signals and introduced
into a host cell in which the AAV capsids are produced, and this
segment may then be packaged into the AAV capsid. Such segments of
DNA may encode genes or heterologous viral genomes. The inclusion
of the packaging signal increases the efficiencies of
encapsidation.
[0074] In an embodiment that allows for the integration of the
packaged DNA into the host cell genome, the DNA may be linked to
the AAV integration sequences (ITRs) that will target these
sequences for integration into the host cell chromosome 19.
5.5. ASSOCIATION OF HETEROLOGOUS MOLECULES WITH AAV CAPSIDS
[0075] The invention is further directed to the association of
therapeutically useful molecules with the outside of AAV capsids
for efficient transfer of said molecules into host target cells.
Such associated molecules may include DNA, RNA, proteins or
peptides. In an embodiment of the invention the therapeutically
useful molecules can, be covalently linked to the capsid proteins.
Alternatively, AAV capsid proteins may be genetically engineered to
code for fusion capsid proteins to which associating molecules may
bind.
5.6. RECOMBINANT AAV CAPSIDS AS EPITOPE CARRIERS
[0076] The invention is further directed to the production of AAV
capsids by any of the above methods that are engineered to carry a
heterologous epitope within any of the three capsid proteins, VP1,
VP2 or VP3. In this embodiment, DNA encoding a capsid protein is
engineered by standard techniques in molecular biology, including
but not limited to site-directed mutagenesis or polymerase chain
reaction (PCR) mutagenic techniques, to incorporate a heterologous
sequence that encodes an epitope from a clinically relevant
antigen. This will result in the expression of a capsid fusion
protein. The foreign is epitope preferably engineered into a region
of the capsid protein that does not interfere with capsid
formation.
[0077] Examples of antigens which may be the source of these
epitopes include those from bacterial, viral or cellular origin.
Antigens from bacteria include those from Salmonella,
Staphylococcus, Streptococcus, cholera and mycobacterium (TB).
Examples of antigens from viruses include the env protein of HIV,
HA protein of influenza, hepatitis surface antigen, herpes
glycoprotein, and the surface antigen of human papilloma virus.
Examples of antigens from cellular sources include those identified
as tumor-specific antigens in cancer, for example the
carcinoembryonic (CEA) antigen found in colon cancer or the PSA
antigen found in prostate cancer. Additionally, antigens
corresponding to anti-immunoglobulin sequences that could be used
to raise antibodies that would neutralize those in autoimmune
disorders, including but not limited to multiple sclerosis, lupus
erythematosus, diabetes, and scleroderma are within the scope of
the invention.
5.7. USE OF AAV VEHICLES
[0078] The AAV capsid vehicles can be administered to a patient at
therapeutically effective doses. A therapeutically effective dose
refers to that amount of the compound sufficient to result in
amelioration of symptoms of disease.
[0079] Toxicity and therapeutic efficacy of the AAV capsid vehicles
can be determined by standard pharmaceutical procedures in cell
cultures or experimental animals, e.g., for determining the
LDS.sub.50 (the dose lethal to 50% of the population) and the
ED.sub.50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD.sub.50/ED.sub.50. Doses which exhibit large therapeutic indices
are preferred. While doses that exhibit toxic side effects may be
used, care should be taken to design a delivery system that targets
such AAV capsid vehicles to the site of treatment in order to
minimize damage to untreated cells and reduce side effects.
[0080] The data obtained from cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such capsid vehicles lies preferably within a
range of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage. form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound which achieves a half-maximal infection or a half-maximal
inhibition) as determined in cell culture. Such information can be
used to more accurately determine useful doses in humans. Levels in
plasma may be measured, for example, by high performance liquid
chromatography.
[0081] Pharmaceutical compositions comprising the AAV capsid
vehicles, for use in accordance with the present invention, may be
formulated in conventional manner using one or more physiologically
acceptable carriers or excipients. For example, the AAV capsid
vehicles may be suspended in a carrier such as PBS (phosphate
buffered saline).
[0082] The AAV capsid vehicles and their physiologically acceptable
salts and solvates may be formulated for administration by
inhalation or insufflation (either through the mouth or the nose)
or for oral, buccal, parenteral or rectal administration.
[0083] For administration by inhalation, the AAV capsid vehicles
for use according to the present invention are conveniently
delivered in the form of an aerosol spray presentation from
pressurized packs or a nebulizer, with the use of a suitable
propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules.and
cartridges of e.g.gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of a therapeutic compound and
a suitable powder base such as lactose or starch.
[0084] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g. magnesium stearate, talc or silica);
disintegrants (e.g. potato starch or sodium starch glycolate); or
wetting agents (e.g. sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous
vehicles (e.g. almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g. methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0085] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound.
[0086] For buccal administration the compositions may take the form
of tablets or lozenges formulated in conventional manner.
[0087] The AAV capsids may be formulated for parenteral
administration by injection e.g. by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form e.g. in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0088] The AAV capsid vehicles may also be formulated in rectal
compositions such as suppositories or retention enemas, e.g.
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0089] In addition to the formulations described previously, the
AAV capsid vehicles may also be formulated as a depot preparation.
Such long acting formulations may be administered by implantation
(for example, subcutaneously or intramuscularly) or by
intramuscular injection. Thus, for example, the therapeutic
compounds may be formulated with suitable polymeric or hydrophobic
materials (for example as an emulsion in an acceptable oil) or ion
exchange resins, or as sparingly soluble derivatives, for example,
as a sparingly soluble salt.
[0090] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example,
comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0091] 6. EXAMPLE: PRODUCTION OF AAV CAPSIDS
[0092] FROM RECOMBINANT ADENOVIRUS
6.1. CONSTRUCTION OF DL324/CMV CAP
[0093] The AAV2 capsid coding region of psub201 (Samulski, R. J.,
et al., J. Virol. 61:3096-3101, 1987) was cloned into pCMV-Ad
(Dolph, P. J., J.Virol. 62:2059-2066, 1988) to derive a plasmid
(pCAD/Cap) in which the capsid genes were under the control of the
CMV promoter. This plasmid was co-transfected with the linearized
DNA fragment of adenovirus d1324 into 293 cells to create
d1324/CMV/Cap, a recombinant adenovirus containing the AAV2 capsid
genes (FIG. 1). Plaques resulting from the infection of this virus
into 293 cells were picked and screened for the expression of the
AAV2 capsid proteins. One isolate (d1324/CMV/CapFF) was expanded
into a viral stock for further analysis (FIG. 2). Correct splicing
of the capsid messages occurred to give rise to the three capsid
proteins (VP1, VP2, and VP3) in levels that mimicked those seen in
a wild-type infection (5% VP1, 5%VP2, and 90% VP3).
6.2. GROWTH CHARACTERISTICS OF DL324/CMV/CAPFF
[0094] The recombinant adenovirus was tested for its ability to
exhibit wild-type growth in 293 cells. Cells were infected at an
MOI of 200 particles per cell, sufficient to cause one step growth.
The control virus was recombinant adenovirus containing the B-gal
gene, d1324-Bgal. Results over a 48-hr period demonstrated that the
Ad-AAV, recombinant virus had similar one-step growth
characteristics to the control virus (FIG. 3).
6.3. TEMPORAL EXPRESSION OF THE CAPSID PROTEINS FROM AD/AAV
VIRUS
[0095] To assess if the capsid proteins were expressed in a
temporal manner similar to wild-type AAV, 293 cells were infected
with the recombinant virus and samples were collected over a 48-hr
course of infection. Immunoblotting demonstrated that the temporal
expression of the AAV capsid proteins lagged only slightly behind
that of wild-type AAV (FIG. 4).
6.4. mRNA PROCESSING IN AN AD/AAV INFECTION
[0096] To determine if the capsid RNA was correctly spliced to
yield the appropriate mRNAs corresponding to the three capsid
proteins, RT-PCR performed on mRNAs isolated from infected 293
cells revealed the presence of the correctly spliced mRNAs,
identical to those seen in a wild-type infection (FIG. 5).
6.5. SUBCELLULAR LOCALIZATION OF THE AAV CAPSID PROTEINS EXPRESSED
FROM ADIAAV VIRUS
[0097] To determine if the capsid proteins expressed from the
recombinant virus were correctly localized, immunofluorescence of
HeLa cells infected with the virus was performed. Using an anti-AAV
capsid antibody (Hoggan, R., et al., Proc. Natl. Acad. Sci. USA
55:1460-1474, 1966), distinct areas of staining concentration
within the nucleus were seen, similar to that visualized in a
wild-type AAV infection (FIG. 6).
6.6. FORMATION OF EMPTY VIRAL PARTICLES IN THE AD/AAV INFECTION
[0098] Viral lysates from 253 cells infected with d1324/CMV/Cap
were banded by isopycnic centrifugation in a CsCl density gradient.
Fractions from the gradient were immunoblotted with anti-AAV capsid
antibody to localize the presence of the AAV capsid proteins (FIG.
7). Density measurements were calculated for the fractions (14-30)
that exhibited the highest levels of AAV capsid proteins. The
density of these fractions was 1.31 g/ml, which agrees with density
measurements reported for empty viral particles (Myers, M. W. and
Carter, B. J., Virology 102:71-82, 1980).
[0099] Further characterization by transmission electron microscopy
demonstrated that the average diameter of these particles was 20
nm, typical for wild-type AAV2 particles (FIG. 8).
7. EXAMPLE: PACKAGING OF AAV VECTOR DNA MOLECULE USING ADENOVIRUS
HYBRID CARRYING AAV CAPSID GENES
[0100] The following subsection below describes experiments
demonstrating the in vivo encapsidation of AAV vector DNA into
viral capsids in cells infected with the recombinant Ad/CMV/CAP
virus.
[0101] 7.1. MATERIALS AND METHODS
7.1.1. PLASMIDS AND VIRUS
[0102] Plasmid pAB11 is the psub201 plasmid, previously described
in Samulski et al., 1987 J. Virol. 61:3096-3101, carrying an
inserted lacZ reporter gene. Plasmid pAd/AAV codes for both the REP
and CAP proteins (Samulski et al., 1989, J. Virol. 63:3822-3828).
Plasmid pUHD RepA is a psub201 derivative expressing only the AAV
Rep proteins under the control of the tetracycline repressor
promoter. The pUGD construct contains the AAV coding sequences for
REP which includes AAV nucleotides 321-2234. The tetracycline
repressor promoter inducer plasmid, pUHD 15-1 is described in
Gossen and Buyard, 1992, Proc. Natl Acad. Sci. USA
89:5547-5551.
7.1.2. TRANSFECTION AND INFECTION OF CELLS
[0103] Human 293 cells were transfected with 2 ug pAB11 and 12
.mu.g pUHD 15-1. After 12 hours the monolayer cell culture was
infected with Ad/CMV/CAP at 5 plaque forming units (PFU) per cell.
48 hours post-infection viral lysates were made. The control
experiment consisted of transfection of 293 cells with 2 .mu.g
pAB11, 12 .mu.G pAd/AAV followed by infection with 5 pfu per cell
wild type adenovirus (WT300).
[0104] 200.lambda. of the above lysates were used to infect a new
monolayer of 293 cells. 24 hours post-infection the cells were
stained for beta galactosidase activity. In addition, as an
additional control, wild type Adenovirus was mixed with the pAB11
plasmid DNA.
7.2. RESULTS
[0105] After staining the infected cells for beta galactosidase
activity, positive staining was observed with viral lysates
utilizing the Ad/CMV/CAP helper virus as a source of AAV capsid
protein and with the pAd/AAV helper plasmid which express both rep
and cap genes (FIG. 9, middle panel A and middle panel B). There
was no detection of beta galactosidase activity in any cells
infected with wild type Adenovirus and pAB11.
[0106] These results indicate that AAV lacZ DNA may be rescued from
the plasmid, replicated and packaged into AAV particles using
either the traditional packaging system or the Ad/CMV/CAP virus
stock. These results clearly demonstrate the potential for
generating infectious AAV particles using the adenovirus hybrid
virus as a source for producing AAV capsid proteins.
[0107] The present invention is not to be limited in scope by the
specific embodiments described which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and descried herein will become apparent
to those skilled in the art from the foregoing descriptions and
accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
* * * * *