U.S. patent application number 11/222108 was filed with the patent office on 2006-01-05 for high-efficiency aav helper functions.
This patent application is currently assigned to Avigen, Inc.. Invention is credited to Peter Colosi, Gary Kurtzman, Georges Natsoulis.
Application Number | 20060003453 11/222108 |
Document ID | / |
Family ID | 22318036 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060003453 |
Kind Code |
A1 |
Natsoulis; Georges ; et
al. |
January 5, 2006 |
High-efficiency AAV helper functions
Abstract
The present invention provides methods and compositions for
producing high titer preparations of recombinant AAV ("rAAV")
virions. The compositions of the present invention include AAV
helper function systems and host cells. The present invention also
includes methods of using AAV helper function vectors that effect
the production of only small amounts of the long forms of Rep
protein, and rAAV virions produced by such methods.
Inventors: |
Natsoulis; Georges; (San
Francisco, CA) ; Kurtzman; Gary; (Villanova, PA)
; Colosi; Peter; (Alameda, CA) |
Correspondence
Address: |
ROBINS & PASTERNAK LLP
1731 EMBARCADERO ROAD
SUITE 230
PALO ALTO
CA
94303
US
|
Assignee: |
Avigen, Inc.
Alameda
CA
|
Family ID: |
22318036 |
Appl. No.: |
11/222108 |
Filed: |
September 8, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10631403 |
Jul 30, 2003 |
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11222108 |
Sep 8, 2005 |
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10026389 |
Dec 18, 2001 |
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10631403 |
Jul 30, 2003 |
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09449581 |
Nov 29, 1999 |
6365403 |
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10026389 |
Dec 18, 2001 |
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09107708 |
Jun 30, 1998 |
6027931 |
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09449581 |
Nov 29, 1999 |
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08688648 |
Jul 29, 1996 |
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09107708 |
Jun 30, 1998 |
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08510790 |
Aug 3, 1995 |
5622856 |
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08688648 |
Jul 29, 1996 |
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Current U.S.
Class: |
435/456 ;
435/239; 435/325 |
Current CPC
Class: |
C12N 7/00 20130101; C12N
2750/14152 20130101; C12N 2750/14122 20130101; C12N 2750/14143
20130101; C07K 2319/00 20130101; C12N 15/86 20130101; C12N 2840/20
20130101; C12N 2800/30 20130101; C12N 2750/14162 20130101; C07K
14/005 20130101 |
Class at
Publication: |
435/456 ;
435/239; 435/325 |
International
Class: |
C12N 7/02 20060101
C12N007/02; C12N 15/861 20060101 C12N015/861 |
Claims
1. A method of producing recombinant AAV virions, comprising the
steps of (a) introducing an AAV vector into a host cell; (b)
introducing an AAV helper function vector comprising an AAV rep
coding region into the host cell, wherein said rep coding region
comprises a nucleotide sequence coding for long forms of Rep
protein and short forms of Rep protein, such that the host cell
produces greater quantities of the short forms of Rep protein than
of the long forms of Rep protein; (c) expressing accessory
functions in the host cell; and (d) culturing the host cell to
produce recombinant AAV virions.
2. A recombinant AAV virion produced according to the method of
claim 1.
3. The method of claim 1, wherein the AAV helper function vector
further comprises an AAV cap coding region.
4. The method of claim 1, wherein the host cell produces at least
ten-fold greater quantities of the short forms of Rep protein than
of the long forms of Rep protein.
5. A host cell comprising an AAV helper function vector, said AAV
helper function vector comprising an AAV rep coding region, wherein
the rep coding region comprises a nucleotide sequence that codes
for long forms of Rep protein and short forms of Rep protein, such
that the host cell produces greater quantities of the short forms
of Rep protein than of the long forms of Rep protein.
6. The host cell of claim 5, wherein the AAV helper function vector
further comprises an AAV cap coding region.
7. A method of producing recombinant AAV virions, comprising the
steps of: (a) introducing an AAV vector into a host cell; (b)
introducing an AAV helper function vector comprising an AAV rep
coding region into the host cell, wherein said rep coding region
comprises a nucleotide sequence coding for long forms of Rep
protein and short forms of Rep protein, and said AAV helper
function vector causes the host cell to produce an amount of the
long forms of Rep protein that is substantially less than an amount
produced from an AAV helper function vector expressing the long
forms of Rep protein under control of an AAV p5 promoter; (c)
expressing accessory functions in the host cell; and (d) culturing
the host cell to produce recombinant AAV virions.
8. A recombinant AAV virion produced according to the method of
claim 7.
9. The method of claim 7, wherein the AAV helper function vector
further comprises an AAV cap coding region.
10. A host cell comprising an AAV helper function vector, said AAV
helper function vector comprising an AAV rep coding region, wherein
the rep coding region comprises a nucleotide sequence that codes
for long forms of Rep protein and short forms of Rep protein, and
said AAV helper function vector causes the host cell to produce an
amount of the long forms of Rep protein that is substantially less
than an amount produced from an AAV helper function vector
expressing the long forms of Rep protein under control of an AAV p5
promoter.
11. The host cell of claim 10, wherein the AAV helper function
vector further comprises an AAV cap coding region.
12. A method of producing recombinant AAV virions, comprising the
steps of: (a) introducing an AAV vector into a host cell; (b)
introducing an AAV helper function vector comprising AAV rep and
cap coding regions into the host cell to express Rep and Cap gene
products, wherein the rep coding region is under the control of an
inducible promoter that expresses an amount of rep RNA that is
substantially less than an amount expressed from an AAV p5
promoter; (c) expressing accessory functions in the host cell; and
(d) culturing the host cell to produce recombinant AAV virions.
13. A recombinant AAV virion produced according to the method of
claim 12.
14. A method of producing recombinant AAV virions, comprising the
steps of: (a) introducing an AAV vector into a host cell; (b)
introducing an AAV helper construct comprising AAV rep and cap
coding regions into the host cell to express Rep and Cap gene
products, said rep coding region comprising a nucleotide sequence
coding for long forms of Rep protein and short forms of Rep
protein, wherein the rep coding region is regulated by an inducible
promoter such that the host cell produces greater quantities of the
short forms of Rep protein than of the long forms of Rep protein;
(c) expressing accessory functions in the host cell; and (d)
culturing the host cell to produce recombinant AAV virions.
15. A recombinant AAV virion produced according to the method of
claim 14.
16. The method of claim 14, wherein the host cell produces at least
ten-fold greater quantities of the short forms of Rep protein than
of the long forms of Rep protein.
Description
1. CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/631,403, filed Jul. 30, 2003; which is a
continuation of U.S. patent application Ser. No. 10/026,389, filed
Dec. 18, 2001; which is a continuation of U.S. patent application
Ser. No. 09/449,581, filed Nov. 29, 199, now U.S. Pat. No.
6,365,403; which is a continuation of U.S. patent application Ser.
No. 09/107,708, filed Jun. 30, 1998, now U.S. Pat. No. 6,027,931;
which is a continuation-in-part of U.S. patent application Ser. No.
08/688,648, filed Jul. 29, 1996; which is a continuation-in-part of
U.S. patent application Ser. No.08/510,790, filed Aug. 3, 1995, now
U.S. Pat. No. 5,622,856; from which applications priority is
claimed pursuant to 35 U.S.C. .sctn. 120 and all of which
applications are incorporated herein by reference in their
entirety.
2. FIELD OF THE INVENTION
[0002] The present invention relates to adeno-associated virus
(AAV) helper function systems for use in recombinant AAV (rAAV)
virion production and methods of using such systems. More
specifically, the present invention relates to AAV helper functions
that product low amounts of the long form of AAV Rep proteins,
resulting in high-efficiency production of rAAV.
3. TECHNICAL BACKGROUND
Gene Therapy
[0003] Scientists are continually discovering genes that are
associated with human diseases such as diabetes, hemophilia and
cancer. Research efforts have also uncovered genes, such as
erythropoietin (which increases red blood cell production), that
are not associated with genetic disorders but code for proteins
that can be used to treat numerous diseases. However, despite
significant progress in the effort to identify and isolate genes, a
major obstacle facing the biopharmaceutical industry is how to
safely and persistently deliver effective quantities of these
genes' products to patients.
[0004] Currently, the protein products of these genes are
synthesized in cultured bacterial, yeast, insect, mammalian, or
other cells and delivered to patients by intravenous injection.
Intravenous injection of recombinant proteins has been successful
but suffers from several drawbacks. First, patients frequently
require multiple injections in a single day in order to maintain
the necessary levels of the protein in the blood stream. Even then,
the concentration of protein is not maintained at physiological
levels--the level of the protein is usually abnormally high
immediately following injection and far below optimal levels prior
to injection. Second, intravenous delivery often cannot deliver the
protein to the target cells, tissues or organs in the body. And, if
the protein reaches its target, it is often diluted to
non-therapeutic levels. Third, the method is inconvenient and
severely restricts the patient's lifestyle. The adverse impact on
lifestyle is especially significant when the patient is a
child.
[0005] These shortcomings have led to the development of gene
therapy methods for delivering sustained levels of specific
proteins into patients. These methods allow clinicians to introduce
DNA coding for a gene of interest directly into a patient (in vivo
gene therapy) or into cells isolated from a patient or a donor (ex
vivo gene therapy). The introduced DNA then directs the patient's
own cells or grafted cells to produce the desired protein product.
Gene delivery, therefore, obviates the need for daily injections.
Gene therapy may also allow clinicians to select specific organs or
cellular targets (e.g., muscle, blood cells, brain cells, etc.) for
therapy.
[0006] DNA may be introduced into a patient's cells in several
ways. There are transfection methods, including chemical methods
such as calcium phosphate precipitation and liposome-mediated
transfection, and physical methods such as electroporation. In
general, transfection methods are not suitable for in vivo gene
delivery. There are also methods that use recombinant viruses.
Current viral-mediated gene delivery methods include retrovirus,
adenovirus, herpes virus, pox virus, and adeno-associated virus
(AAV) vectors. Of the more than 100 gene therapy trials conducted,
more than 95% used viral-mediated gene delivery. C. P. Hodgson,
Bio/Technology 13, 222-225 (1995).
Adeno-Associated Virus-Mediated Gene Therapy
[0007] One viral system that has been used for gene delivery is
adeno-associated virus (AAV). AAV is a parvovirus which belongs to
the genus Dependovirus. AAV has several attractive features not
found in other viruses. First, AAV can infect a wide range of host
cells, including non-dividing cells. Second, AAV can infect cells
from different species. Third, AAV has not been associated with any
human or animal disease and does not appear to alter the biological
properties of the host cell upon integration. Indeed, it is
estimated that 80-85% of the human population has been exposed to
the virus. Finally, AAV is stable at a wide range of physical and
chemical conditions which lends itself to production, storage and
transportation requirements.
[0008] The AAV genome is a linear, single-stranded DNA molecule
containing 4681 nucleotides. The AAV genome generally comprises an
internal non-repeating genome flanked on each end by inverted
terminal repeats (ITRs). The ITRs are approximately 145 base pairs
(bp) in length. The ITRs have multiple functions, including as
origins of DNA replication and as packaging signals for the viral
genome.
[0009] The internal non-repeated portion of the genome includes two
large open reading frames, known as the AAV replication (rep) and
capsid (cap) genes. The rep and cap genes code for viral proteins
that allow the virus to replicate and package the viral genome into
a virion. In particular, a family of at least four viral proteins
are expressed from the AAV rep region, Rep 78, Rep 68, Rep 52, and
Rep 40, named according to their apparent molecular weight. The AAV
cap region encodes at least three proteins, VP1, VP2, and VP3.
[0010] AAV is a helper-dependent virus; that is, it requires
co-infection with a helper virus (e.g., adenovirus, herpesvirus or
vaccinia) in order to form AAV virions. In the absence of
co-infection with a helper virus, AAV establishes a latent state in
which the viral genome inserts into a host cell chromosome, but
infectious virions are not produced. Subsequent infection by a
helper virus "rescues" the integrated genome, allowing it to
replicate and package its genome into infectious AAV virions. While
AAV can infect cells from different species, the helper virus must
be of the same species as the host cell. Thus, for example, human
AAV will replicate in canine cells co-infected with a canine
adenovirus.
[0011] AAV has been engineered to deliver genes of interest by
deleting the internal non-repeating portion of the AAV genome and
inserting a heterologous gene between the ITRs. The heterologous
gene is typically functionally linked to a heterologous promoter
(constitutive, cell-specific, or inducible) capable of driving gene
expression in the patient's target cells under appropriate
conditions. Termination signals, such as polyadenylation sites, can
also be included.
[0012] To produce infectious rAAV containing the heterologous gene,
a suitable producer cell line is transfected with an AAV vector
containing a heterologous gene. AAV helper functions and accessory
functions, which are typically derived from a helper virus such as
adenovirus, are then expressed in the producer cell. Once these
factors come together, the heterologous gene is replicated and
packaged as though it were a wild-type AAV genome, forming a
recombinant virion. When a patient's cells are infected with the
resulting rAAV virions, the heterologous gene enters and is
expressed in the patient's cells. Because the patient's cells lack
the rep and cap genes and the adenovirus accessory function genes,
the rAAV are replication defective; that is, they cannot further
replicate and package their genomes. Similarly, without a source of
rep and cap genes, wild-type AAV cannot be formed in the patient's
cells.
Methods of Producing rAAV
[0013] In the earliest attempts to generate rAAV virions,
researchers cotransfected an AAV vector carrying heterologous DNA
with a wild-type AAV genome. Alternatively, cells transfected with
an AAV vector were infected with wild-type AAV particles. In both
types of protocols, accessory functions were provided by infection
with a helper virus such as adenovirus. The wild-type AAV genome
provides the necessary rep and cap functions, but significant
amounts of wild-type AAV are produced by these methods. Moreover,
the rAAV titers produced are usually not sufficient for
therapy.
[0014] In order to increase viral titers, several groups have
focused their attention on Rep protein expression. For example, one
group of researchers constructed a plasmid containing the rep and
cap genes linked to an SV40 origin of replication. See, e.g., U.S.
Pat. No. 5,693,531. The SV40 origin of replication allows high copy
number episomal replication of the plasmid in cells that express
SV40 large T antigen. As a result, large amounts of Rep and Cap
gene products are produced.
[0015] Many other researchers have replaced the AAV p5 promoter
with heterologous promoters and enhancers in an effort to increase
rAAV titers. For example, one group of researchers replaced the p5
promoter with the HIV LTR. U.S. Pat. No. 5,658,776. In 293 cells,
which express the adenoviral E1A gene product, the HIV LTR provides
strong constitutive expression of the Rep gene products. The
authors emphasize that use of the HIV LTR avoids the problem of Rep
autoregulating expression from the p5 promoter, and conlude that in
previously used methods, levels of Rep expression are limiting in
the production of rAAV.
[0016] All of these approaches reflect a widely held notion that
expressing large amounts of the Rep gene products will allow the
production of high titer rAAV. The long forms of Rep, however, have
toxic effects on many cell types.
[0017] From the foregoing, it will be appreciated that it would be
an advancement in the art to provide AAV helper functions that
allow high efficiency production of rAAV. It would be a further
advancement in the art to provide such helper functions that
produce only small quantities of the long forms of the Rep
proteins.
[0018] Such AAV helper functions and methods of their use are
disclosed herein.
BRIEF SUMMARY OF THE INVENTION
[0019] The present invention relates to AAV helper function systems
for rAAV production and methods of using such systems. In certain
embodiments, a method of the present invention includes the steps
of (1) introducing an AAV vector into a suitable host cell; (2)
introducing an AAV helper function vector into the host cell such
that the host cell produces only small amounts of the long forms of
Rep protein; (3) expressing accessory functions in the host cell;
and (4) culturing the host cell to produce rAAV virions. In certain
preferred embodiments, the amount of the long forms of Rep protein
produced are substantially less than the amount that would be
produced in the host cell using an AAV helper function vector
expressing the long forms of Rep protein under the control of an
AAV promoter. In certain other embodiments, the amount of the long
forms of Rep protein produced are less than the quantities of the
short forms of Rep protein produced by the host cell. It will be
appreciated that both transcriptional and translational controls
may be used to limit the levels of the long forms of Rep protein
that are produced.
[0020] The AAV vector and AAV helper function vector can be
transfected into the host cell, either sequentially or
simultaneously, using well-known techniques. Accessory functions
may be expressed in any of several ways, including infecting the
host cell with a suitable helper virus (such as adenovirus,
herpesvirus, or vaccinia virus), or by transfecting one or more
accessory function vectors into the host cell. It is also well
known in the art that certain cell lines, e.g., 293 cells,
inherently express one or more accessory functions.
[0021] An AAV helper function vector of the present invention may
be a plasmid, bacteriophage, transposon, cosmid, chromosome,
artificial chromosome, virus, or other suitable genetic element,
and may include selectable genetic markers such as antibiotic
resistance genes. Such vectors may also include one or more
accessory function genes, such as the E1A, E1B, E2A, VA RNA, and E4
regions of adenovirus.
[0022] Also provided herein are host cells for producing rAAV
virions. In certain embodiments, a host cell of the present
invention comprises an AAV helper function vector that directs the
host cell to produce only small amounts of the long forms of Rep
protein. Upon introduction of an AAV vector and expression of
accessory functions in the host cell, rAAV virions are produced. In
certain preferred embodiments, a host cell of the present invention
also includes one or more accessory functions.
[0023] The rAAV virions produced using the present invention may be
used to introduce genetic material into animals, including humans,
or isolated animal cells for a variety of research and therapeutic
uses. For example, rAAV virions produced using the methods of the
present invention may be used to express a protein in animals to
gather preclinical data or to screen for potential drug candidates.
Alternatively, the rAAV virions may be used to transfer genetic
material into a human to cure a genetic defect or to effect a
desired treatment.
[0024] These and other objects and advantages of the present
invention will become apparent upon reference to the accompanying
drawing and upon reading the following detailed description and
appended claims.
4. BRIEF SUMMARY OF THE DRAWINGS
[0025] FIG. 1 depicts the promoter, rep, and cap configurations of
a number of AAV helper function vectors.
[0026] FIG. 2 illustrates a Western Blot of lysates from 293 cells
transfected with various AAV helper function vectors and probed
with anti-Rep antibody.
5. DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention is directed to novel systems and
methods for producing rAAV for introducing genetic material into
animals or animal cells for a variety of research and therapeutic
uses. A physician or researcher may wish to introduce DNA into an
organism (or cells isolated from an organism) for any of several
reasons. First, DNA may be introduced to correct a defective gene.
Second, DNA may be introduced to specifically delete or mutate a
given gene by, for example, homologous recombination. Third, DNA
may be introduced to express a protein. Such a protein may be
expressed to achieve a therapeutic benefit within the organism
treated with rAAV. Alternatively, a protein may be expressed in an
organism or in cells isolated from an organism with the goal of
isolating and purifying the protein product. Unlike previously
described methods for producing rAAV, however, the methods of the
present invention utilize AAV helper function vectors that effect
the production of only low levels of the long forms of Rep protein,
while generating high titer stocks of rAAV.
[0028] All publications, patents, and patent applications cited
herein are hereby incorporated by reference.
Definitions
[0029] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0030] "Gene transfer" or "gene delivery" refers to methods or
systems for reliably inserting foreign DNA into host cells. Gene
transfer can result in transient expression of non-integrated
transferred DNA, extrachromosomal replication and expression of
transferred replicons (e.g., episomes), or integration of
transferred genetic material into the genomic DNA of host cells.
Gene transfer provides a unique approach for the treatment of
acquired and inherited diseases. A number of systems have been
developed for gene transfer into mammalian cells. See, e.g., U.S.
Pat. No. 5,399,346.
[0031] By "vector" is meant any genetic element, such as a plasmid,
phage, transposon, cosmid, chromosome, artificial chromosome,
virus, virion, etc., which is capable of replication when
associated with the proper control elements and which can transfer
gene sequences between cells. Thus, the term includes cloning and
expression vehicles, as well as viral vectors.
[0032] By "adeno-associated virus inverted terminal repeats" or
"AAV ITRs" is meant the art-recognized regions found at each end of
the AAV genome which function together in cis as origins of DNA
replication and as packaging signals for the viral genome. AAV
ITRs, together with the AAV rep coding region, provide for the
efficient excision and rescue from, and integration of a nucleotide
sequence interposed between two flanking ITRs into a mammalian cell
genome.
[0033] The nucleotide sequences of AAV ITR regions are known. See,
e.g., Kotin, R. M. (1994) Human Gene Therapy 5, 793-801; Berns, K.
I. "Parvoviridae and their Replication" in Fundamental Virology, 2d
ed., (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence.
As used herein, an "AAV ITR" need not have the wild-type nucleotide
sequence depicted in these references, but may be altered, e.g., by
the insertion, deletion or substitution of nucleotides.
Additionally, the AAV ITR may be derived from any of several AAV
serotypes, including without limitation, AAV-1, AAV-2, AAV-3,
AAV-4, AAV-5, AAVX7, etc. Furthermore, 5' and 3' ITRs which flank a
selected nucleotide sequence in an AAV vector need not necessarily
be identical or derived from the same AAV serotype or isolate, so
long as they function as intended, i.e., to allow for excision and
rescue of the sequence of interest from a host cell genome or
vector, and to allow integration of the heterologous sequence into
the recipient cell genome when AAV Rep gene products are present in
the cell.
[0034] By "AAV rep coding region" is meant the art-recognized
region of the AAV genome which encodes the replication proteins of
the virus which are required to replicate the viral genome and to
insert the viral genome into a host genome during latent infection.
The term also includes functional homologues thereof such as the
human herpesvirus 6 (HHV-6) rep gene which is also known to mediate
AAV-2 DNA replication (Thomson et al. (1994) Virology 204,
304-311). For a further description of the AAV rep coding region,
see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol. and
Immunol. 158, 97-129; Kotin, .R. M. (1994) Human Gene Therapy 5,
793-801. The rep coding region, as used herein, can be derived from
any viral serotype, such as the AAV serotypes described above. The
region need not include all of the wild-type genes but may be
altered, e.g., by the insertion, deletion or substitution of
nucleotides, so long as the rep genes present provide for
sufficient integration functions when expressed in a suitable
recipient cell.
[0035] The terms "long forms of Rep protein" and "large Rep" refer
to the Rep 78 and Rep 68 gene products of the AAV rep coding
region, including functional homologues thereof. The long forms of
Rep are normally expressed under the direction of the AAV p5
promoter, which has been described and characterized.
[0036] The terms "short forms of Rep protein" and "small Rep" refer
to the Rep 52 and Rep 40 gene products of the AAV rep coding
region, including functional homologues thereof. The short forms of
Rep are expressed under the direction of the AAV p19 promoter,
which has been described and characterized.
[0037] By "AAV cap coding region" is meant the art-recognized
region of the AAV genome which encodes the coat proteins of the
virus which are required for packaging the viral genome. For a
further description of the cap coding region, see, e.g., Muzyczka,
N. (1992) Current Topics in Microbiol. and Immunol. 158, 97-129;
Kotin, R. M. (1994) Human Gene Therapy 5, 793-801. The AAV cap
coding region, as used herein, can be derived from any AAV
serotype, as described above. The region need not include all of
the wild-type cap genes but may be altered, e.g., by the insertion,
deletion or substitution of nucleotides, so long as the genes
provide for sufficient packaging functions when present in a host
cell along with an AAV vector.
[0038] The term "AAV coding region" refers to a nucleic acid
molecule that includes the two major AAV open reading frames
corresponding to the AAV rep and cap coding regions; e.g., a
nucleic acid molecule comprising a nucleotide sequence
substantially homologous to base pairs 310 through 4,440 of the
wild-type AAV genome. Thus, for purposes of the present invention,
an AAV coding region does not include those sequences corresponding
to the AAV p5 promoter region, and does not include the AAV
ITRs.
[0039] By an "AAV vector" is meant a vector derived from an
adeno-associated virus serotype, including without limitation,
AAV-1, AAV-2, AAV-3, AAV4, AAV-5, AAVX7, etc. AAV vectors can have
one or more of the AAV wild-type genes deleted in whole or part,
preferably the rep and/or cap genes, but retain functional flanking
ITR sequences. Functional ITR sequences are necessary for the
rescue, replication and packaging of the AAV virion. Thus, an AAV
vector is defined herein to include at least those sequences
required in cis for replication and packaging (e.g., functional
ITRs) of the virus. The ITRs need not be the wild-type nucleotide
sequences, and may be altered, e.g., by the insertion, deletion or
substitution of nucleotides, so long as the sequences provide for
functional rescue, replication and packaging.
[0040] "AAV helper functions" refer to AAV-derived coding sequences
that can be expressed to provide AAV gene products that, in turn,
function in trans for productive AAV replication. Thus, AAV helper
functions include the rep and cap regions. The rep expression
products have been shown to possess many functions, including,
among others: recognition, binding and nicking of the AAV origin of
DNA replication; DNA helicase activity; and modulation of
transcription from AAV (or other heterologous) promoters. The cap
expression products supply necessary packaging functions. AAV
helper functions are used herein to complement AAV functions in
trans that are missing from AAV vectors.
[0041] The term "AAV helper construct" refers generally to a
nucleic acid molecule that includes nucleotide sequences providing
AAV functions deleted from an AAV vector which is to be used to
produce a transducing vector for delivery of a nucleotide sequence
of interest. AAV helper constructs are commonly used to provide
transient expression of AAV rep and/or cap genes to complement
missing AAV functions that are necessary for lytic AAV replication;
however, helper constructs lack AAV ITRs and can neither replicate
nor package themselves. AAV helper constructs can be in the form of
a plasmid, phage, transposon, cosmid, virus, or virion. A number of
AAV helper constructs have been described, such as the commonly
used plasmids pAAV/Ad and pIM29+45 which encode both Rep and Cap
expression products. See, e.g., Samulski et al. (1989) J. Virology
63, 3822-3828; McCarty et al. (1991) J. Virology 65, 2936-2945. A
number of other vectors have been described which encode Rep and/or
Cap expression products. See, e.g., U.S. Pat. No. 5,139,941.
[0042] The term "accessory functions" refers to non-AAV derived
viral and/or cellular functions upon which AAV is dependent for its
replication. Thus, the term captures DNAs, RNAs and proteins that
are required for AAV replication, including those moieties involved
in activation of AAV gene transcription, stage specific AAV mRNA
splicing, AAV DNA replication, synthesis of Cap expression products
and AAV capsid assembly. Viral-based accessory functions can be
derived from any of the known helper viruses such as adenovirus,
herpesvirus (other than herpes simplex virus type-1) and vaccinia
virus.
[0043] For example, adenovirus-derived accessory functions have
been widely studied, and a number of adenovirus genes involved in
accessory functions have been identified and partially
characterized. See, e.g., Carter, B. J. (1990) "Adeno-Associated
Virus Helper Functions," in CRC Handbook of Parvoviruses, vol. I
(P. Tijssen, ed.); Muzyczka, N. (1992) Current Topics. Microbiol.
and Immun. 158, 97-129. Specifically, early adenoviral E1A, E2A,
E4, VA RNA and, possibly, E1B regions are thought to participate in
the accessory process. Janik et al. (1981) Proc. Natl. Acad. Sci.
USA 78, 1925-1929. Herpesvirus-derived accessory functions have
been described. See, e.g., Young et al. (1979) Prog. Med. Virol.
25, 113. Vaccinia virus-derived accessory functions have also been
described. See, e.g.,Carter, B. J. (1990), supra., Schlehofer et
al. (1986) Virology 152, 110-117.
[0044] The term "accessory function vector" refers generally to a
nucleic acid molecule that includes nucleotide sequences providing
accessory functions. An accessory function vector can be
transfected into a suitable host cell, wherein the vector is then
capable of supporting AAV virion production in the host cell.
Expressly excluded from the term are infectious viral particles as
they exist in nature, such as adenovirus, herpesvirus or vaccinia
virus particles. Thus, accessory function vectors can be in the
form of a plasmid, phage, transposon, cosmid or virus that has been
modified from its naturally occurring form.
[0045] By "recombinant virus" is meant a virus that has been
genetically altered, e.g., by the addition or insertion of a
heterologous nucleic acid construct into the particle.
[0046] By "AAV virion" is meant a complete virus particle, such as
a wild-type (wt) AAV virus particle (comprising a linear,
single-stranded AAV nucleic acid genome associated with an AAV
capsid protein coat). In this regard, single-stranded AAV nucleic
acid molecules of either complementary sense, i.e., "sense" or
"antisense" strands, can be packaged into any one AAV virion and
both strands are equally infectious.
[0047] A "recombinant AAV virion," or "rAAV virion" is defined
herein as an infectious, replication-defective virus composed of an
AAV protein shell encapsulating a heterologous nucleotide sequence
of interest that is flanked on both sides by AAV ITRs. A rAAV
virion is produced in a suitable host cell comprising an AAV
vector, AAV helper functions, and accessory functions. In this
manner, the host cell is rendered capable of encoding AAV
polypeptides that are required for packaging the AAV vector
(containing a recombinant nucleotide sequence of interest) into
infectious recombinant virion particles for subsequent gene
delivery.
[0048] The term "transfection" is used to refer to the uptake of
foreign DNA by a cell. A cell has been "transfected" when exogenous
DNA has been introduced inside the cell membrane. A number of
transfection techniques are generally known in the art. See, e.g.,
Graham et al. (1973) Virology, 52, 456; Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratories, New York; Davis et al. (1986) Basic Methods in
Molecular Biology, Elsevier; Chu et al. (1981) Gene 13, 197. Such
techniques can be used to introduce one or more exogenous DNA
moieties, such as a nucleotide integration vector and other nucleic
acid molecules, into suitable host cells.
[0049] The term "host cell" denotes, for example, microorganisms,
yeast cells, insect cells, and mammalian cells, that can be, or
have been, used as recipients of an AAV helper construct, an AAV
vector plasmid, an accessory function vector, or other transfer
DNA. The term includes the progeny of the original cell which has
been transfected. Thus, a "host cell" as used herein generally
refers to a cell which has been transfected with an exogenous DNA
sequence. It is understood that the progeny of a single parental
cell may not necessarily be completely identical in morphology or
in genomic or total DNA complement to the original parent, due to
natural, accidental, or deliberate mutation.
[0050] As used herein, the term "cell line" refers to a population
of cells capable of continuous or prolonged growth and division in
vitro. Often, cell lines are clonal populations derived from a
single progenitor cell. It is further known in the art that
spontaneous or induced changes can occur in karyotype during
storage or transfer of such clonal populations. Therefore, cells
derived from the cell line referred to may not be precisely
identical to the ancestral cells or cultures, and the cell line
referred to includes such variants.
[0051] The term "heterologous" as it relates to nucleic acid
sequences such as coding sequences and control sequences, denotes
sequences that are not normally joined together, and/or are not
normally associated with a particular cell. Thus, a "heterologous"
region of a nucleic acid construct or a vector is a segment of
nucleic acid within or attached to another nucleic acid molecule
that is not found in association with the other molecule in nature.
For example, a heterologous region of a nucleic acid construct
could include a coding sequence flanked by sequences not found in
association with the coding sequence in nature. Another example of
a heterologous coding sequence is a construct where the coding
sequence itself is not found in nature (e.g., synthetic sequences
having codons different from the native gene). Similarly, a cell
transformed with a construct which is not normally present in the
cell would be considered heterologous for purposes of this
invention. Allelic variation or naturally occurring mutational
events do not give rise to heterologous DNA, as used herein.
[0052] A "coding sequence" or a sequence which "encodes" a
particular protein, is a nucleic acid sequence which is transcribed
(in the case of DNA) and translated (in the case of mRNA) into a
polypeptide in vitro or in vivo when placed under the control of
appropriate regulatory sequences. The boundaries of the coding
sequence are determined by a start codon at the 5' (amino) terminus
and a translation stop codon at the 3' (carboxy) terminus. A coding
sequence can include, but is not limited to, cDNA from prokaryotic
or eukaryotic mRNA, genomic DNA sequences from prokaryotic or
eukaryotic DNA, and even synthetic DNA sequences. A transcription
termination sequence will usually be located 3' to the coding
sequence.
[0053] A "nucleic acid" sequence refers to a DNA or RNA sequence.
The term captures sequences that include any of the known base
analogues of DNA and RNA such as, but not limited to,
4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,
pseudoisocytosine, 5- (carboxyhydroxylmethyl)uracil,
5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, 2-thiocytosine, and 2,6-diaminopurine.
[0054] The term DNA "control sequences" refers collectively to
promoter sequences, polyadenylation signals, transcription
termination sequences, upstream regulatory domains, origins of
replication, internal ribosome entry sites ("IRES"), enhancers, and
the like, which collectively provide for the replication,
transcription and translation of a coding sequence in a recipient
cell. Not all of these control sequences need always be present so
long as the selected coding sequence is capable of being
replicated, transcribed and translated in an appropriate host
cell.
[0055] The term "promoter region" is used herein in its ordinary
sense to refer to a DNA regulatory sequence to which RNA polymerase
binds, initiating transcription of a downstream (3' direction)
coding sequence.
[0056] An "AAV p5 promoter region" encompasses both promoter
sequences with identity to a p5 promoter region isolated from an
AAV serotype, including without limitation, AAV-1, AAV-2, AAV-3,
AAV-4, AAV-5, AAVX7, etc., as well as those that are substantially
homologous and functionally equivalent thereto. The AAV p5 promoter
directs the expression of the long forms of Rep, and has been
described and characterized.
[0057] The phrase "substantially less than an amount produced from
an AAV helper function vector expressing the long forms of Rep
protein under control of an AAV p5 promoter" refers to a comparison
between an AAV helper function vector of interest and an AAV helper
function vector in which the expression of the long forms of Rep
protein is directed by an AAV p5 promoter, e.g., the commonly used
plasmids pAAV/Ad and pIM29+45. The phrase implies a comparison
under equivalent conditions in equivalent host cells. Techniques
for conducting such a comparison are well known in the art, and
include RNA analytical methods, e.g., Northern blotting, and
protein analytical methods, e.g., Western blotting. An AAV helper
function vector of interest causes a host cell to produce an amount
of the long forms of Rep protein that is substantially less than an
amount produced from an AAV helper function vector expressing the
long forms of Rep protein under control of an AAV p5 promoter when
the amount of the long forms of Rep protein produced by the vector
of interest are less than 20%, more preferably less than 10%, and
most preferably less than 5%, of the amount produced by the vector
in which large Rep expression is driven by the p5 promoter.
[0058] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. Thus, control sequences operably linked to a
coding sequence are capable of effecting the expression of the
coding sequence. The control sequences need not be contiguous with
the coding sequence, so long as they function to direct the
expression thereof. Thus, for example, intervening untranslated yet
transcribed sequences can be present between a promoter sequence
and the coding sequence and the promoter sequence can still be
considered "operably linked" to the coding sequence.
[0059] By "isolated," when referring to a nucleotide sequence, is
meant that the indicated, molecule is present in the substantial
absence of other biological macromolecules of the same type. Thus,
an "isolated nucleic acid molecule which encodes a particular
polypeptide" refers to a nucleic acid molecule which is
substantially free of other nucleic acid molecules that do not
encode the subject polypeptide; however, the molecule may include
some additional bases or moieties which do not deleteriously affect
the basic characteristics of the composition.
[0060] For the purpose of describing the relative position of
nucleotide sequences in a particular nucleic acid molecule
throughout the instant application, such as when a particular
nucleotide sequence is described as being situated "upstream,"
"downstream," "3'," or "5'" relative to another sequence, it is to
be understood that it is the position of the sequences in the
"sense" or "coding" strand of a DNA molecule that is being referred
to, as is conventional in the art.
[0061] "Homology" refers to the percent of identity between two
polynucleotide or two polypeptide moieties. The correspondence
between the sequence from one moiety to another can be determined
by techniques known in the art. For example, homology can be
determined by a direct comparison of the sequence information
between two polypeptide molecules by aligning the sequence
information and using readily available computer programs.
Alternatively, homology can be determined by hybridization of
polynucleotides under conditions which allow for the formation of
stable duplexes between homologous regions, followed by digestion
with singlestranded-specific nuclease(s), and size determination of
the digested fragments. Two DNA or two polypeptide sequences are
"substantially homologous" to each other when at least about 80%,
preferably at least about 90%, and most preferably at least about
95% of the nucleotides or amino acids match over a defined length
of the molecules, as determined using the methods above.
[0062] A "functional homologue" or a "functional equivalent" of a
given polypeptide includes molecules derived from the native
polypeptide sequence, as well as recombinantly produced or
chemically synthesized polypeptides which function in a manner
similar to the reference molecule to achieve a desired result.
Thus, a functional homologue of AAV Rep68 or Rep78 encompasses
derivatives and analogues of those polypeptides, including any
single or multiple amino acid additions, substitutions and/or
deletions occurring internally or at the amino or carboxy termini
thereof--so long as integration activity remains.
[0063] A "functional homologue" or a "functional equivalent" of a
given adenoviral nucleotide region includes similar regions derived
from a heterologous adenovirus serotype, nucleotide regions derived
from another virus or from a cellular source, and recombinantly
produced or chemically synthesized polynucleotides which function
in a manner similar to the reference nucleotide region to achieve a
desired result. Thus, a functional homologue of an adenoviral VA
RNA gene region or an adenoviral E2A gene region encompasses
derivatives and analogues of such gene regions--including any
single or multiple nucleotide base additions, substitutions and/or
deletions occurring within the regions, so long as the homologue
retains the ability to provide its inherent accessory function to
support AAV virion production at levels detectable above
background.
General Methods
[0064] It is a primary object of the present invention to provide
improved AAV helper function systems, host cells, and methods of
their use for the production of rAAV virions. More particularly, it
is an object of the present invention to provide AAV helper
function systems, host cells, and methods that support
high-efficiency rAAV production by producing only small amounts of
the long forms of Rep protein.
[0065] In certain embodiments, a method of the present invention
includes the steps of (1) introducing an AAV vector into a suitable
host cell; (2) introducing an AAV helper function vector into the
host cell such that the host cell produces only small amounts of
the long forms of Rep protein; (3) expressing accessory functions
in the host cell; and (4) culturing the host cell to produce rAAV
virions. In certain preferred embodiments, the amount of the long
forms of Rep protein produced is substantially less than the amount
that would be produced in an equivalent host cell using an AAV
helper function vector expressing the long forms of Rep protein
under the control of an AAV promoter. The relative amounts of Rep
proteins produced may be compared using techniques that are well
known in the art, including immunochemical techniques such as
Western blotting, immunoprecipitation, immunoaffinity purification,
and immunofluorescence. These immunochemical techniques utilize
anti-Rep antibodies, which are commercially available, e.g.,
Mab226.7 from American Research Products (Belmont, Mass.).
[0066] In certain other embodiments, the amount of the long forms
of Rep protein produced are less than the quantities of the short
forms of Rep protein produced by the host cell. The relative
amounts of the long and short forms of Rep proteins produced may be
compared by the techniques listed above.
[0067] It will be appreciated that both transcriptional and
translational controls may be used to limit the levels of the long
forms of Rep protein that are produced. Transcriptional controls
include the use of heterologous promoter and enhancer sequences
such as inducible promoters that attenuate the expression of the
long forms of Rep protein. The effects of transcriptional
regulation may be assayed by techniques that are well known in the
art, including Northern blotting of host cell total RNA or mRNA.
The effects of transcriptional control may also be assayed by
determining the effect on Rep protein concentration using the
techniques listed above.
[0068] An AAV helper function vector that contains rep and cap
sequences with wild-type promoter and gene configurations may be
used to determine baseline levels of the production of Rep isoforms
and expression of rAAV. A modified vector that effects decreased
expression of the long forms of Rep protein and increased
expression of the short forms of Rep protein shows increases in
rAAV virion production relative to such a wild-type construct. In
fact, we show that contrary to conventional wisdom, expression of
the long forms of Rep protein is inversely proportional to rAAV
virion production. By contrast, expression of the short forms of
Rep protein was directly proportional to rAAV virion
production.
[0069] Relocation of the p5 promoter to the 3' end of the cap gene
(pW1909) resulted in the lowest expression of the long forms of Rep
protein, the highest expression of the short forms of Rep protein,
and the highest production of recombinant virions. Introduction of
an efficient polyadenylation site (pRCM.globinpolyA) between the
transcriptional start site and the first codon in the coding
sequence of the long forms of Rep protein also decreased expression
of the long forms of Rep and increased expression of the short
forms of Rep protein, but less so. Consequently, recombinant virion
production was improved relative to the wild-type constructs but
was less than pW1909. Modification of the Kozak sequence so as to
reduce translation initiation efficiency (pRCM.kozak) and
introduction of a sub-optimal polyadenylation site had little
effect on Rep protein expression and resulted in little improvement
in recombinant virion production.
[0070] In the methods of the present invention, an AAV vector and
AAV helper function vector can be transfected into the host cell,
either sequentially or simultaneously, using well-known techniques.
These vectors can be readily introduced into a suitable host cell
using transfection techniques that are known in the art. Such
transfection methods have been described, including calcium
phosphate co-precipitation (Graham et al. (1973) Virol.
52:456-467), direct micro-injection into cultured cells (M. R.
Capecchi (1980) Cell 22:479-488), electroporation (Shigekawa et al.
(1988) BioTechniques 6:742-751), liposome-mediated gene transfer
(Mannino et al. (1988) BioTechniques 6:682-690), lipid-mediated
transfection (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA
84:7413-7417), and nucleic acid delivery using high-velocity
microprojectiles (Klein et al. (1987) Nature 327:70-73).
[0071] AAV helper function vectors can be engineered using
conventional recombinant techniques. Particularly, nucleic acid
molecules can be readily assembled in any desired order by
inserting one or more accessory function nucleotide sequences into
a construct, such as by ligating restriction fragments or
PCR-generated products into a cloning vector using polylinker
oligonucleotides or the like. The newly formed nucleic acid
molecule can then be excised from the vector and placed in an
appropriate expression construct using restriction enzymes or other
techniques that are well known in the art.
[0072] More particularly, selected AAV nucleotide sequences or
functional homologues thereof can be excised either from a viral
genome or from a vector containing the same. Alternatively,
selected AAV nucleotide sequences may be generated as PCR products
using as a template either viral DNA or a vector containing such
DNA. The nucleotide sequences are then inserted into a suitable
vector either individually or linked together to provide a helper
function construct using standard ligation techniques such as those
described in Sambrook et al., supra.
[0073] Nucleic acid molecules comprising one or more helper
functions can also be synthetically derived using a combination of
solid phase direct oligonucleotide synthesis chemistry and
enzymatic ligation methods that are conventional in the art.
Synthetic sequences may be constructed having features such as
restriction enzyme sites, and can be prepared in commercially
available oligonucleotide synthesis devices such as those devices
available from Applied Biosystems, Inc. (Foster City, Calif.) using
the phosphoramidite method. See, e.g., Beaucage et al. (1981)
Tetrahedron Lett. 22:1859-1862. Preferred codons for expression of
the synthetic molecule in mammalian cells can also be readily
synthesized. Complete nucleic acid molecules are then assembled
from overlapping oligonucleotides prepared by the above methods.
See, e.g., Edge, Nature 292, 756 (1981); Nambair et al., Science
223, 1299 (1984); Jay et al., J. Biol. Chem. 259, 6311 (1984).
[0074] An AAV helper function vector utilized in the present
invention may be a plasmid, bacteriophage, transposon, cosmid,
chromosome, artificial chromosome, virus, or other suitable genetic
element, and may include selectable genetic markers. Suitable
markers include genes which confer antibiotic resistance or
sensitivity, or impart color, or change the antigenic
characteristics when cells which have been transfected with the
nucleic acid constructs are grown in an appropriate selective
medium. Particular selectable marker genes useful in the practice
of the invention include the Neomycin resistance gene (encoding
Aminoglycoside phosphotranferase (APH)) that allows selection in
mammalian cells by conferring resistance to G418 (available from
Sigma, St. Louis, Mo.) and the Hygromycin-B resistance gene
(encoding Hygromycin-B-phosphotransferase (HPH)) that confers
resistance to Hygrogmycin-B. Other suitable markers are known to
those of skill in the art.
[0075] Such vectors can further include elements that control the
replication and expression of the nucleic acid sequences that code
for one or more AAV helper functions.
[0076] Accessory functions may be expressed in any of several ways,
including infecting the host cell with a suitable helper virus
(such as adenovirus, herpesvirus, or vaccinia virus), or by
transfecting one or more accessory function vectors into the host
cell. An AAV helper function vector may also include one or more
accessory function genes, such as those derived from adenovirus,
and may include one or more of the adenoviral E1A, E1B, E2A, VA
RNA, and E4 regions.
[0077] Also provided herein are host cells for producing rAAV
virions. In certain embodiments, a host cell of the present
invention comprises an AAV helper function vector that directs the
host cell to produce only small amounts of the long forms of Rep
protein. Upon introduction of an AAV vector and expression of
accessory functions in the host cell, rAAV virions are produced. In
certain preferred embodiments, a host cell of the present invention
also includes one or more accessory functions.
[0078] An AAV vector, AAV helper function vector and accessory
function vector(s) can be introduced into the host cell, either
simultaneously or serially, using transfection techniques described
above.
[0079] AAV vectors used to produce rAAV virions for delivery of a
nucleotide sequence of interest can be constructed to include one
or more heterologous nucleotide sequences flanked on both ends (5'
and 3') with functional AAV ITRs. In the practice of the invention,
an AAV vector generally includes at least one AAV ITR and an
appropriate promoter sequence suitably positioned relative to a
heterologous nucleotide sequence, and at least one AAV ITR
positioned downstream of the heterologous sequence. The 5' and 3'
ITRs need not necessarily be identical to, or derived from, the
same AAV isolate, so long as they function as intended.
[0080] Suitable heterologous nucleotide sequences for use in AAV
vectors include any functionally relevant nucleotide sequence.
Thus, AAV vectors for use in the practice of the invention can
include any desired gene that encodes a protein that is defective
or missing from a recipient cell genome or that encodes a
non-native protein having a desired biological or therapeutic
effect (e.g., an antiviral function), or the sequence can
correspond to a molecule having an antisense or ribozyme
function.
[0081] AAV vectors can also include control sequences, such as
promoter and polyadenylation sites, as well as selectable markers
or reporter genes, enhancer sequences, and other control elements
which allow for the induction of transcription. Such AAV vectors
can be constructed using techniques well known in the art.
[0082] In the methods of the invention, accessory functions are
used for rAAV production. Accessory functions may be provided by
infecting the host cell with a suitable helper virus, such as
adenovirus, herpesvirus, or vaccinia virus, or by transfecting the
host cell with one or more accessory function vectors.
Materials and Methods
[0083] Plasmid pW1909 has previously been described. See U.S.
patent application Ser. No. 08/688,648. Briefly, as shown in FIG.
1, pW1909 comprises rep and cap coding regions and a downstream p5
promoter sandwiched between two Flip Recombination Target (FRT)
sites.
[0084] Plasmid pRCM is composed of a DNA fragment encoding the
entire AAV genome without the ITR sequences (base pairs 146-4536)
cloned into the SmaI site of pUC119. Plasmids pRCM.kozak,
pRCM.polyA and pRCM.globinpolyA are modified versions of pRCM in
which the 5' untranslated region between the transcriptional start
site and the first codon of large Rep has been modified by the
introduction of Kozak sequence alterations and polyadenylation
sites. In pRCM.kozak, the five base pair sequence immediately
preceding the first codon was changed from 5'-CCGCC-3' (SEQ ID
NO:1) to 5'-AAAAA-3' (SEQ ID NO:2). This was done by
oligonucleotide-directed mutagenesis using the following
oligonucleotide: 5'-GAACGCGCAGAAAAAATGCCGGG-3' (SEQ ID NO:3).
Plasmid pRCM.polyA was made by changing base pairs -10 through -5,
relative to the first codon of large Rep, from 5'-CGCACG-3' (SEQ ID
NO:4) to 5'-AATAAA-3' (SEQ ID NO:5) using the following
oligonucleotide: 5'-GGTTTGAACGAATAAACGCCATGC-3' (SEQ ID NO:6).
Plasmid pRCM.globinpolyA was constructed by inserting a 48 bp
fragment encoding an optimized polyadenylation site (based on the
rabbit beta-globin polyadenylation site, Levitt et al. (1989),
Genes and Development 3,1019-1025) between base pairs -8 and -7
relative to the start codon of large Rep. To do this, an Eco47III
site was introduced in the 5' untranslated region using the primer
5'-GGTTTGAACGAGCGCTCGCCATGC-3' (SEQ ID NO:7). The resulting 7559 bp
plasmid was called pUCRCM47III. Synthetic DNA encoding the
polyadenylation site
(5'-AATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGTTTTTTGTGT-3' (SEQ ID
NO:8)) was inserted at this site.
EXAMPLES
[0085] The following examples are given to illustrate various
embodiments which have been made within the scope of the present
invention. It is to be understood that the following examples are
neither comprehensive nor exhaustive of the many types of
embodiments which can be prepared in accordance with the present
invention.
Example 1
Western Blot
[0086] 293 cells (available from ATCC, catalog number CRL-1573) in
10 cm dishes were transfected with 10 .mu.g each of pVlacZ,
pladeno5, and various AAV helper function constructs, which are
depicted schematically in FIG. 1. Rep isoforms were analyzed by
western blot analysis (shown in FIG. 2) using an anti-Rep
monoclonal antibody.
[0087] AAVlacZ vector was produced and titer as previously
described. Western blot analysis of the freeze/thaw lysates from
vector production was carried out by standard methods using an 8%
SDS polyacrylamide gel, electrotransfer to a nitrocellulose
membrane, a monoclonal antibody to the AAV Rep protein (American
Research Products, Belmont, Mass., Mab226.7) and chemiluminescent
detection.
[0088] FIG. 2 illustrates a Western Blot of lysates prepared from
293 cells transfected with various AAV helper function vectors and
probed with anti-Rep antibody. Lane 1 contains lysate from a cell
transfected with the pAAVAd vector. Lane 2 contains lysate from a
cell transfected with the pRCM vector. Lane 3 contains lysate from
a cell transfected with the pW1909 vector. Lane 4 contains lysate
from a cell transfected with the pRCM.kozak vector. Lane 5 contains
lysate from a cell transfected with the pRCM.polyA vector. Lane 6
contains lysate from a cell transfected with the pRCM.globinpolyA.
Arrow A indicates the position of the long forms of Rep protein,
while Arrow B indicates the position of the short forms of Rep
protein.
[0089] Lanes 1 and 2 (pAAVAd and pRCM, respectively) expressed
similar amounts of both the long and short forms of Rep protein. By
contrast, the cells transfected with pW1909 (lane 3) produced very
little of the long forms of Rep. The cells transfected with pW1909
also produced more of the short forms of Rep than were produced by
cells transfected with the other AAV helper function vectors.
Modification of the Kozak sequence so as to reduce translation
initiation efficiency (pRCM.kozak; lane 4) and introduction of a
sub-optimal polyadenylation site between the transcriptional start
site and the first codon in the coding sequence of the long forms
of Rep protein (pRCM.polyA; lane 5) had little effect on Rep
protein expression. Introduction of an efficient polyadenylation
site (pRCM.globinpolyA; lane 6) between the transcriptional start
site and the first codon in the coding sequence of the long forms
of Rep protein also decreased expression of the long forms of Rep
and increased expression of the short forms of Rep protein, but
less so.
Example 2
rAAV Titers
[0090] AAVlacZ vector was produced by transfecting 293 cells in 10
cm dishes with 10 .mu.g each of pVlacZ, pladeno5, and various rep
and cap constructs, as described in Example 1, above. AAV lacZ
virion preparations were titered on 293 cells in the presence of
adenovirus. The results of the vector production are shown in the
table below. TABLE-US-00001 TABLE 1 AAVlacZ Titer/ Relative Helper
Function Vector 10 cm Plate Production pAAVAd 7.3 .times. 10.sup.8
0.85 pRCM 8.6 .times. 10.sup.8 1.0 pW1909 3.0 .times. 10.sup.9 3.5
pRCM.kozak 9.0 .times. 10.sup.8 1.05 pRCM.polyA 8.5 .times.
10.sup.8 0.98 pRCM.globinpolyA 2.0 .times. 10.sup.9 2.32
[0091] pAAVAd and pRCM both contain rep and cap sequences with
wild-type promoter and gene configurations. These plasmids produced
similar amounts of AAVlacZ and expressed similar amounts of both
the long and short forms of Rep protein. With the modified
constructs, alterations that decreased expression of the long forms
of Rep protein and increased expression of the short forms of Rep
protein, relative to the wild-type constructs (PRCM and pAAVAd),
also showed increases in AAVlacZ virion production relative to the
wild-type constructs. In this series, expression of the long forms
of Rep protein is inversely proportional to AAVlacZ virion
production. By contrast, expression of the short forms of Rep
protein was directly proportional to AAVlacZ virion production.
Relocation of the p5 promoter to the 3' end of the cap gene
(pW1909) resulted in the lowest expression of the long forms of Rep
protein, the highest expression of the short forms of Rep protein,
and the highest production of recombinant virions. Introduction of
an efficient polyadenylation site (pRCM.globinpolyA) between the
transcriptional start site and the first codon in the coding
sequence of the long forms of Rep protein also decreased expression
of the long forms of Rep and increased expression of the short
forms of Rep protein, but less so. Consequently, recombinant virion
production was improved relative to the pRCM but was less than
pW1909. Modification of the Kozak sequence so as to reduce
translation initiation efficiency (pRCM.kozak) and introduction of
a sub-optimal polyadenylation site had little effect on Rep protein
expression and resulted in little improvement in recombinant virion
production.
[0092] In summary, the AAV helper function systems, host cells, and
methods of the present invention allow for high-efficiency
production of rAAV virions by reducing the amount of the long forms
of Rep proteins produced. Here, we show that AAV helper function
vectors that produce only small amounts of the long forms of Rep
protein provide for higher titer rAAV virion production.
[0093] The invention may be embodied in other specific forms
without departing from its essential characteristics. The described
embodiments are to be considered in all respects only as
illustrative and not restrictive. The scope of the invention is,
therefore, indicated by the appended claims rather than by the
foregoing description. All changes that come within the meaning and
range of equivalency of the claims are to be embraced within their
scope.
Sequence CWU 1
1
8 1 5 DNA adeno-associated virus 2 1 ccgcc 5 2 5 DNA Artificial
Sequence Description of Artificial Sequenceartificial sequence 2
aaaaa 5 3 23 DNA Artificial Sequence Description of Artificial
Sequence oligonucleotide 3 gaacgcgcag aaaaaatgcc ggg 23 4 6 DNA
adeno-associated virus 2 4 cgcacg 6 5 6 DNA Artificial Sequence
Description of Artificial Sequenceartificial sequence 5 aataaa 6 6
24 DNA Artificial Sequence Description of Artificial Sequence
oligonucleotide 6 ggtttgaacg aataaacgcc atgc 24 7 24 DNA Artificial
Sequence Description of Artificial Sequence oligonucleotide 7
ggtttgaacg agcgctcgcc atgc 24 8 48 DNA Artificial Sequence
Description of Artificial Sequence oligonucleotide 8 aataaaggaa
atttattttc attgcaatag tgtgttggtt ttttgtgt 48
* * * * *