EP1222299A1 - Production of recombinant aav using adenovirus comprising aav rep/cap genes - Google Patents

Abstract

This invention relates to novel adenoviruses useful in the production of high titers of recombinant adeno-associated virus (rAAV) comprising a foreign DNA insert and methods of making these adenoviruses. The adenovirus comprises the AAV rep gene in which the p5 promoter of rep is replaced by a minimal promoter or by no promoter. The invention also provides methods of producing high levels of rAAV as a substantially homogeneous preparation and compositions of rAAV.

Description

PRODUCTION OF RECOMBINANT AAV USING ADENO VIRUS COMPRISING AAV REP/CAP GENES

TECHNICAL FIELD OF THE INVENTION

This invention relates to novel adenoviruses useful in the production

of high titers of recombinant adeno-associated virus (rAAN) comprising a foreign

DΝA insert and methods of making these adenoviruses. The adenovirus comprises

the AAN rep gene in which the p5 promoter of rep is replaced by a minimal

promoter or by no promoter. The invention also provides methods of producing

high levels of rAAN as a substantially homogeneous preparation and compositions

of rAAN.

BACKGROUND OF THE INVENTION

A recombinant virus carrying a foreign DNA insert may be used to

deliver genes to cells, where the gene may be expressed, if desired, to permit

production of recombinant proteins in vitro or in vivo, vaccination of human and

non-human mammals, or treatment or amelioration of diseases or genetic defects in

humans or in non-human mammals. One may treat or ameliorate diseases or genetic

defects by providing some effective level of normal gene products, increased levels of gene products or by blocking endogenous production of a gene, whose expression would be deleterious to the cell or organism.

Methods for delivering an exogenous gene to a mammalian cell

include the use of mammalian viral vectors, such as those that are derived from retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polio viruses, adeno-

associated viruses, hybrid viruses (e.g., hybrid adenovirus- AAV, see U.S. Pat. No. 5,856,152) and the like. Other methods include direct injection of DNA, biolistic

administration of DNA, electroporation, calcium phosphate precipitation, as well as

methods of administration which utilize ligand-DNA conjugates, liposome conjugates of DNA, polycation-DNA complexes or adenovirus-ligand-DNA

conjugates.

Adeno-associated virus (AAV) systems have many advantages that

can be exploited for delivery of transgenes. AAV is a helper-dependent DNA

parvovirus which belongs to the genus Dependovirus. AAV requires helper

function in order for a productive infection to occur. Helper functions may be

provided by a number of agents, but generally co-infection with an unrelated helper

virus, either adenovirus, herpesvirus or vaccinia, is used. In the absence of such

co-infection, AAV establishes a latent state by insertion of its genome into a host

cell chromosome. Subsequent infection by a helper virus rescues the integrated

copy which can then replicate to produce infectious viral progeny. AAV has a wide

host range and is able to replicate in cells from any species so long as there is also a successful co-infection of such cells with a suitable helper virus. 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 For a review of AAV, see,

e g , Berns and Bohenzky (1987) Advances in Virus Research (Academic Press,

Inc ) 32 243-307

AAV has a genome of about 4 7 kb in length, including inverted

terminal repeats (ITRs) that often, but not necessarily are 145 nucleotides in length

The AAV genome encodes two genes, rep and cap, each of which expresses a

family of related proteins from separate open reading frames and which are

produced by alternative mRNA splicing and different transcriptional and

translational start sites Rep polypeptides (Rep78, Rep68, Rep52, and Rep40) are

involved in replication, rescue and integration of the AAV genome Rep78 and

Rep 68 have the same amino-terminal sequence and share the same promoter, p5,

but Rep78 contains an exon that is alternatively spliced out in re ?68 Similarly, Rep 52 and Rep40 have the same amino-terminal sequence and share the pl9

promoter, which is downstream from the p5 promoter, but rep52 contains an exon

that is alternatively spliced out in rep6% Cap proteins (VPI, VP2, and VP3) form

the virion capsid Cap gene transcription is driven by the p40 promoter See Fig

2B for a schematic diagram of the rep and cap genes and promoters p5, pi 9 and p40 Flanking the rep and cap open reading frames at the 5' and 3' ends of the

AAV genome are the ITRs In certain AAV genomes, the ITRs are 145 nucleotides

in length, the first 125 bp of which are capable of forming Y- or T- shaped duplex

structures The entire nucleic acid encoding rep and cap can be excised and

replaced with a transgene [B. J. Carter, in "Handbook of Parvoviruses", ed. , P.

Tijsser, CRC Press, pp.155-168 (1990)]. The ITRs represent the minimal sequence required for replication, rescue, packaging, and integration of the AAV

genome if other sources of rep and cap are provided.

When AAV infects a human cell, the viral genome integrates into chromosome 19 resulting in latent infection of the cell. Upon introduction of helper

functions into the cell, such as by infection with a helper virus, the AAV provirus is

rescued and amplified. The rescued AAV genomes are packaged into preformed protein capsids (icosahedral symmetry approximately 20 nm in diameter) and

released as infectious virions that have packaged either + or - single stranded DNA genomes following cell lysis.

Replacing the rep and cap sequences with a desired transgene yields

a rAAV capable of delivering the transgene to target host cells. In current methods,

the deleted rep and cap sequences are supplied to the host cells by other viruses or

plasmids where they are transiently or stably expressed. There are also a number of

cell lines that stably express rep and cap. The host cells also require helper

functions in order for the rAAV to replicate and excise from the host cell genome.

The helper functions usually are provided by helper viruses (either wildtype or crippled viruses), plasmids containing the helper virus functions or physical

methods.

Although it is known that rep is required for replication and excision

of AAV, the amount of Rep proteins required for effective rAAV production is, as yet, unclear. U.S. Pat. No. 5,354,678 states that Rep proteins may be toxic to

certain cell lines, and WO 97/06272, WO 98/46728 and Li et al. suggest that

attenuation of Rep78/68 production results in higher levels of production of rAAV. In contrast, other art, such as U.S. Pat. No. 5,658,776, explicitly states that high

expression of Rep proteins — a result of replacing the native p5 promoter with a strong promoter, such as the human immunodeficiency virus long terminal repeat

(HIV LTR) - results in high level expression of rAAV. Similarly, U.S. Pat. No.

5,837,484 states that the p5 promoter should be replaced by a strong constitutive promoter or inducible promoter, such as the metallothionein promoter, in order to

overcome the strong feedback inhibition by Rep of its own transcription. Thus, U.S. Pat. Nos. 5,658,776 and 5,837,484 suggest that high expression of Reρ78/68

is required for efficient rAAV production.

One method that has been used to produce recombinant AAV

(rAAV) vectors comprises co-transfecting eukaryotic cells with a plasmid

containing rAAV sequences (the cis plasmid) and a plasmid containing rep and cap

(the trans plasmid), and infecting the cells with a helper virus (e.g., adenovirus or

herpes virus). See U.S. Pat. No. 5,753,500. Li et al. (J. Virol. 71 :5236-5243,

1997) have modified this method by altering the translation initiation codon of the

Rep78/68 proteins in the trans plasmid to decrease the translation of the Rep protein and increase production of rAAV. However, the disadvantage of the

methods taught by U.S. Pat. No. 5,753,500 and Li et al. is that co-transfection of

two plasmids along with infection by a helper virus is inefficient, may exhibit poor

reproducibility, may result in generation of pseudo-wildtype replication-competent

AAV (rcAAV), and cannot be easily scaled up for industrial production of rAAV.

rcAAV, comprising rep and cap flanked by ITRs, is produced when the rep and cap genes recombine with the ITRs flanking the transgene which results in deletion of

the transgene.

A second method that has been used to produce rAAV involves co-

transfection of three plasmids into eukaryotic cells. In this method, one plasmid

carries the transgene and ITRs (the cis plasmid), a second plasmid encodes the rep and cap genes (the trans plasmid), and the third plasmid encodes the helper virus

functions, i.e. adenoviral genes such as Ela, Elb, E2a and E4 (the helper plasmid).

The disadvantages of the first method are shared with this method.

A third method involves the use of a packaging cell line such as one

including AAV functions rep and cap. See U.S. Pat. Nos. 5,658,785 and 5,837,484

and PCT US98/19463. The packaging cell line may be transfected with a cis

plasmid comprising the transgene and ITRs, and infected by wild-type adenovirus

(Ad) helper. See U.S. Pat. No. 5,658,785. Alternatively, the packaging cell line

may be co-infected by a hybrid Ad/ AAV, in which a hybrid Ad vector carries the cis

plasmid in the El locus (see U.S. Pat. No. 5,856,152), and by a wild-type or mutant

Ad that supplies El . See, e.g., Reference 7. The disadvantage of this method is

that it requires making a cell line that expresses sufficient levels of rep and cap, and

requires multiple components — including the cell line, the rAAV genome, and an adenovirus ~ to produce rAAV, which do not lend themselves to easy and

convenient downstream manufacturing processes. In addition, some of these packaging cell lines do not produce high levels of rAAV.

A fourth method is provided by a prophetic example in U.S. Pat. No.

5,354,678. The method involves using a recombinant adenovirus in which the rep and cap genes of AAV replace a part of the adenovirus genome not essential for

helper virus functions In this method, an AAV/EBV plasmid vector comprising an

rAAV genome is introduced into a cell to produce an rAAV producer cell It is

presumed that the rep gene is driven by its native p5 promoter or by a strong inducible promoter The recombinant adenovirus comprising rep and cap is then

introduced into the cell and their production is induced such that rAAV is produced

by the cells U S Pat No 5,354,678 does not disclose the levels of rAAV, if any,

produced by this method

As described above, current rAAV production methods are not

amenable for production of sufficient rAAV for pharmaceutical applications in a

convenient manner However, the problem of reproducibly generating high levels

of substantially homogeneous replication-deficient rAAV by an efficient method

that is applicable to large-scale industrial production is solved by the present

invention

SUMMARY OF THE INVENTION

The instant invention provides an alternative production method that

results in high yields of rAAV vector and is amenable to large-scale industrial

applications The invention provides a novel adenovirus vector comprising rep and

cap genes, thus providing AAV rep and cap and adenovirus helper functions in one

component In the adenovirus vector of the instant invention, the native AAV p5 promoter upstream of rep is removed and replaced with a minimal promoter or with

no promoter This novel vector, when infected into cells containing a nucleic acid sequence comprising a transgene flanked by AAV ITRs, results in the production of high levels of rAAV The nucleic acid sequence comprising the transgene flanked

by AAV ITRs may be established in the host cell by stable integration into the host

cell chromosome, secondary infection with an adenovirus or other viral vector

carrying the transgene flanked by ITRs (see, e g, U S Pat No 5,856,152), infection with an rAAV comprising the transgene, or any other method known in

the art, such as transfection, lipofection or microinjection, of plasmid DNA

comprising the transgene flanked by ITRs

In one embodiment of the invention, rep, operably linked to a

minimal promoter or to no promoter, is inserted into either the El or E3 regions of

an adenovirus The adenovirus is deleted in El or E3 alone, or a combination of

both In another embodiment, the adenovirus vector is further deleted in E4 In

this embodiment, rep sequences may be inserted in E4, while upstream of these rep

sequences there may be no promoter or a minimal promoter In a preferred

embodiment, cap is inserted along with the rep gene into the adenoviral vector In

another aspect of the invention, the adenoviral vector comprising the minimal

promoter or promoterless rep is used in a method to produce rAAV The

advantage of this method is that it is easily scaled for industrial production of

rAAV

In the method of the invention, the host cell is supplied with an rAAV genome, and the adenovirus comprising the minimal promoter or

promoterless rep is infected into the cell In one embodiment of the invention, the

host cell is either simultaneously or sequentially co-infected with two adenoviruses, wherein one adenovirus comprises cap and rep driven from a minimal promoter or

no promoter, as described above, and the other adenovirus comprises an rAAV

genome In another preferred embodiment, an adenovirus comprising cap and rep

driven from a minimal promoter or no promoter is used to infect a host cell

comprising a stably expressed rAAV genome.

In a preferred embodiment of the invention, the method is one in

which a high titer of substantially homogeneous rAAV lysates and stocks is

achieved

In any of these embodiments, the host cell may stably express those

adenoviral sequences that are deleted from the adenovirus comprising rep and cap

For instance, a cell line such as 293 cells, which express El, 84-31 cells, which

express El and E4 (Ref 1), or 10-3 cells, which express El and E4ORF6 (Ref 11),

may be used Alternatively, a second helper virus is co-infected into the host cell

and expresses those adenoviral sequences deleted from the adenovirus comprising

rep and cap For instance, if a second adenovirus comprising a transgene cassette is

used to infect the host cell, this adenovirus could supply the deleted adenoviral

sequences

In another embodiment of this invention, the recombinant virus

carrying the rep gene may be any virus in which rep interferes with its replication

In this embodiment, the recombinant viral vector comprises a rep gene in which the

native p5 promoter of rep is removed and replaced with a minimal promoter or with no promoter BRTEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Construction of recombinant shuttle plasmids

pAV2cisEGFP, pAd-p5-RC and pAd-HSP-RC.

Fig. 2A. Genome of the parental E1/E3 deleted adenovirus.

Fig. 2B. Schematic diagrams of the recombinant adenoviruses Ad-

p5-RC and Ad-HSP-RC showing insertion of p5-rep-cap or HSP-rep-cap DNA

sequences into the El locus of the parental viral genome to generate Ad-p5-RC or Ad-HSP-RC, respectively.

Fig. 3A. Schematic diagram of the rep-cap insert in the El locus of

Ad-p5-RC or Ad-HSP-RC showing the location of PCR primers relative to the viral

genome and expected PCR DNA products I, II, III, and IV. PCR analysis is used

to determine the integrity of AAV rep-cap DNA sequences inserted into the

adenovirus genome.

Fig. 3B. Ethidium bromide stained agarose gel of PCR products

using primers whose locations are shown in Fig. 3A. Lanes 1, 3, 5, 7 are PCR

products from viral DNA of Ad-p5-RC. Lanes 2, 4, 6, 8 are PCR products from viral DNA of Ad-HSP-RC. M, 1 kb DNA ladder size marker (Gibco BRL).

Fig. 4. Production of rAAV after infection of 293-CG3 cells with

Ad-HSP-RC.

Fig. 5. Production of rAAV after co-infection of 293 cells with Ad-

HSP-RC and Ad-AAV-LacZ.

Fig. 6. Time-course study of rAAV production after co-infection of 293 cells with Ad-HSP-RC and Ad-AAV-LacZ. Fig. 7. Multiplicity of infection study of rAAV production after co-

infection of 293 cells with Ad-HSP-RC and Ad-AAV-LacZ.

Fig. 8. Ethidium bromide stained agarose gel of Hirt DNA

fractionated to detect replicating rAAV DNA in 293 cells (lanes 1-5) or in control

B50 cells (lane 6) Lane 1, Ad-p5-RC infection of 293 cells; lane 2, Ad-HSP-RC

infection of 293 cells, lane 3, Ad-AAVLacZ infection of 293 cells; lane 4, Ad-HSP- RC and Ad-AAV-LacZ co-infection of 293 cells; lane 5, Ad-p5-RC and Ad-AAV¬

LacZ co-infection of 293 cells, lane 6, sublOOr and Ad-AAV-LacZ stepwise

infection of B50 cells M, 1 kb DNA Ladder size marker (Gibco BRL)

Fig. 9 A. Ethidium bromide stained agarose gel of Hirt DNA samples

from 293 cells (lanes 2-6) or from control B-50 cells (lane 7) DNA samples.

Fig. 9B. Southern blot analysis of the gel shown in Fig. 9 A

hybridized to a lacZ DNA probe. Lane 1, lacZ DNA fragment as a positive

control; lane 2, Ad-HSP-RC infection of 293 cells; lane 3, Ad-p5-RC infection of

293 cells, lane 4, Ad-AAVLacZ infection of 293 cells; lane 5, Ad-HSP-RC and Ad-

AAV-LacZ co-infection of 293 cells; lane 6, Ad-p5-RC and Ad-AAV-LacZ co-

infection of 293 cells, lane 7, sublOOr and Ad-AAV-LacZ stepwise infection of B50 cells M, 1 kb DNA Ladder size marker (Gibco BRL)

Fig. 10. Western blot analysis of Rep and Cap protein expression in

293 cells infected with or Ad-p5-RC or Ad-HSP-RC viruses. Lane 1, Ad-p5-RC

alone, lane 2, Ad-p5-RC + Ad-AAV-LacZ; lane 3, Ad-HSP-RC alone, lane 4, Ad- AAV-LacZ alone, lane 5, Ad-HSP-RC + Ad/AAV-LacZ. DET AILED DESCRIPTION OF THE INVENTION

The instant invention relates to a novel adenoviral vector and a

method for producing high titer stocks of rAAV using this vector The adenoviral

vector comprises a rep gene in which the native AAV p5 promoter upstream of the

rep coding sequences has been deleted or effectively rendered inactive by mutation or partial deletion and replaced by a minimal promoter or no promoter

Although decreasing maximal production of Rep78 and Rep68 may

increase rAAV production (see WO 97/06272 and WO 98/46728), there has been

no evidence that one could obtain rAAV production if one replaced the p5

promoter with either a minimal promoter that promotes only basal expression of

Rep78/68 or with no promoter, l e , removing the promoter altogether and

incorporating rep into an adenovirus

In a preferred embodiment, Rep78 and Rep68 are produced at much

lower levels than Rep52 and Rep40 in 293 cells or other El -complementing cell

lines infected with an adenovirus containing a minimal promoter driving expression

of rep78 and rep68 See, e g , Example 9 and Fig 10 In another preferred

embodiment, host cells are infected with an adenovirus vector comprising a rep gene that lacks any promoter Although the exact amounts of Rep78 and Rep68

protein expressed in host cells infected by an adenovirus lacking any promoter

upstream of rep coding sequences are unknown, it is expected that Rep78 and

Rep68 protein levels would be expressed from this recombinant adenovirus at much lower levels than Rep52 and Rep40 or at levels much lower than that expressed by wildtype AAV during infection In one embodiment of the invention, the total amount of Rep78 and

Rep68 protein is less than 80%, more preferably less than 50%, of the total amount

of Rep52 and Rep40 produced in the infected cells. In a more preferred

embodiment, the total amount of Rep78 and Rep68 is less than 25% of the total

amount of Rep52 and Rep40 produced in the infected cells. In an even more

preferred embodiment, the total amount of Rep78 and Rep68 is less than 15%,

more preferably 10%, and more preferably is less than 5% of the total amount of

Rep52 and Rep40 produced in the infected cells. One may measure the amount of

Rep proteins by any method known in the art. These methods include, without

limitation, immunoprecipitation of metabolically labeled Rep proteins followed by

separation on SDS polyacrylamide gel electrophoresis and quantitation of the

labeled protein, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunofluorescence of infected cells, and quantitative Western blot analysis

using radioactive or enzymatic labeling of an anti-Rep antibodies.

Experiments performed using an adenovirus vector comprising a

promoterless rep gene demonstrate that the infected host cells also produce rAAV.

Without wishing to be bound by any theory, it is possible that in the absence of a promoter upstream of rep, low-level transcription of Rep78 and Rep68 may occur

from the upstream ITR of adenovirus or from sequences downstream of the ITR that are upstream of the rep gene.

The instant invention demonstrates that adenoviral vectors

comprising a rep gene with its native p5 promoter are unstable when infected into

host cells while adenoviral vectors comprising a rep gene with a minimal promoter or no promoter are stably propagated in host cells. See Example 4 and Figs. 3 A

and 3B. Example 4 demonstrates that Ad-p5-RC, which is an adenovirus

containing p5, rep and cap in the El site of the adenovirus vector, undergoes a rearrangement or deletion event in the rep-cap DNA sequences of the adenovirus

vector when passaged in 293 cells. In contrast, Ad-HSP-RC, which contains a

minimal heat shock protein promoter (HSP), rep and cap, is stable after insertion

into the El locus of the adenoviral genome and does not appear to undergo any

rearrangement when passaged in the same cells. In a preferred embodiment, a

minimal promoter or promoterless rep-containing adenovirus of the instant

invention is one which is stable upon propagation in a defined host cell system, such

as 293 cells.

The deletion or rearrangement of rep and cap is further borne out by

Examples 8-9 and Figs. 8-10. Southern blot analysis demonstrates that Hirt DNA

from 293 cells co-infected with Ad-HSP-RC and Ad-AAV-LacZ (an adenoviral

vector in which a transgene cassette comprising lacZ flanked by AAV ITRs is

inserted in El of adenovirus) contains LacZ DNA sequences in AAV replicating

form (RF) DNA, while Hirt DNA from 293 cells co-infected with Ad-p5-RC and

Ad-AAV-LacZ does not contain LacZ DNA sequences in AAV RF DNA. In

addition, 293 cells express Rep and Cap proteins when co-infected with Ad-HSP-

RC and Ad-AAV-LacZ (see lane 5 of Fig. 10), but do not express these proteins

when co-infected with Ad-p5-RC and Ad-AAV-LacZ (see lane 4 of Fig. 10).

The effect of the deletion or rearrangement in Ad-p5-RC is shown

by the levels of rAAV produced using this adenovirus vector. Little or no rep cating rAAV is produced in cells co-infected with Ad-p5-RC and Ad-AAV¬

LacZ, while replicating rAAV is observed in 293 cells co-infected with Ad-HSP-RC and Ad-AAV-LacZ See Example 8 and Figs 8, 9A and 9B Similarly, sufficient

amounts of replicating rAAV is produced in cells that have been infected with an

adenovirus vector comprising rep sequences downstream of no promoter

Definitions and General Techniques

Unless otherwise defined, all technical and scientific terms used

herein have the meaning as commonly understood by one of ordinary skill in the art

to which this invention belongs The practice of the present invention employs,

unless otherwise indicated, conventional techniques of chemistry, molecular

biology, microbiology, recombinant DNA, genetics, virology and immunology See, e g , Sambrook et al , 1989, Ausubel et al , 1992, Harlow et al 1989 (which

are incorporated herein by reference)

A "recombinant adeno-associated virus (rAAV) genome" comprises

all or a part of an AAV genome, wherein the viral genome may be wild type or may

contain point mutations or deletions, and optionally comprises a transgene operably linked to expression control sequences The transgene may be regulated in cis or in

trans In a preferred embodiment, the rAAV genome comprises a transgene flanked

by AAV inverted terminal repeats (ITRs) The rAAV genome of the invention may

be embedded in the genome of an adenovirus vector to form a hybrid Ad/ AAV

See U S Pat No 5,586,152, herein incorporated by reference Alternatively, the rAAV genome may be introduced into a host cell by any route known in the art The rAAV genome can be expressed transiently or stably in the host cell.

A "recombinant adeno-associated virus" or "rAAV" is the AAV

derived from the rAAV genome described above. The rAAV preferably comprises

a transgene. The rAAV comprising a transgene is capable of transducing

mammalian cells and delivering the transgene thereto.

A "flanking element" or "flanking nucleic acid" is a nucleic acid

sequence which, when located in positions flanking a transgene, permits the

packaging of the transgene into an rAAV. The flanking elements of AAV are

inverted terminal repeats (ITRs). Flanking elements may be the naturally-occurring

ITRs from any one of AAV serotypes 1-6 or may be artificial nucleic acid elements,

e.g. mutated sequences of ITRs, that have the same or equivalent packaging

function.

A "transgene" is a nucleic acid sequence that is to be delivered or

transferred to a mammalian cell. A transgene may encode a protein, peptide or

polypeptide that is useful as a marker, reporter or therapeutic molecule. The

transgene also may be a selection gene, such as one for antibiotic resistance. A

transgene may also encode a protein, polypeptide or peptide that is useful for

protein production, diagnostic assays or for any transient or stable gene transfer in

vitro or in vivo. Alternatively, a transgene may not encode a protein but rather be

used as a sense or antisense molecule, ribozyme or other regulatory nucleic acid to modulate replication, transcription or translation of a nucleic acid to which it is

complementary or to target a complementary mRNA for degradation.

"Expression control sequences" are nucleic acid sequences that regulate the expression of a gene by being operably linked to the gene of interest.

"Operably linked" sequences include both expression control

sequences that are contiguous with the gene of interest and expression control

sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation,

termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic

mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus

sequence); sequences that enhance protein stability; and when desired, sequences

that enhance protein secretion.

A "transgene cassette" is a nucleic acid sequence comprising a

transgene operably linked to expression control sequences in which the transgene

and expression control sequences are flanked by AAV flanking sequences. In a

preferred embodiment, the flanking sequences are AAV ITRs.

An "adenovirus genome" is the nucleic acid molecule backbone of an

adenovirus particle. The adenovirus genome may contain point mutations, deletions

or insertions of nucleotides. The adenovirus genome may further comprise a

foreign gene.

An "adenovirus" is an encapsidated adenovirus genome capable of binding to a mammalian cell and delivering the adenovirus genome to the cell's

nucleus. The term "adenovirus" encompasses both recombinant and non-

recombinant adenoviruses. The term "adenovirus" also encompasses both wildtype and mutant adenoviruses. A "recombinant adenovirus" is an adenovirus which contains one or

more genes that are foreign to a wildtype adenovirus. Recombinant adenoviruses

include, without limitation, those that include foreign genes such as rep and/or cap,

as well as adenoviruses that comprise an rAAV genome. An "adenovirus vector" is a recombinant adenovirus comprising one

or more foreign genes, wherein the adenovirus vector is capable of binding to a

mammalian cell and delivering the foreign gene to the cell's nucleus. The foreign

genes include, without limitation, genes such as rep and cap, rAAV genomes, such as transgenes and expression control sequences, or any foreign gene that is useful in

increasing production of rAAV.

A "locus" is a site within a virus wherein a particular gene normally

resides. For instance, the "adenovirus El locus" is the site at which El resides in

adenovirus. If a foreign gene or nucleic acid is inserted into a locus, it may either

replace the gene that resides there or it may be inserted at the site in addition to the

gene that resides there.

An "AAV p5 promoter" or "p5 promoter" is one that is derived

from any AAV serotype, including AAV Serotypes 1 to 6, as well as any AAV that infects non-human species, such as avian and bovine AAV. The p5 promoter of

AAV-2 directs the expression of rep78 and rep68, and is downregulated by the Rep protein, and is upregulated by certain adenoviral proteins, including El .

As used herein, the term "deleted p5 promoter" refers to a p5

promoter that has been completely deleted from the AAV genome or to a p5

promoter that has been effectively deleted or attenuated such that the p5 promoter is less active when compared to the wildtype p5 promoter in promoting

transcription in a cell into which it has been introduced. The p5 promoter may be

effectively deleted or attenuated by any method, including, without limitation,

removing or mutating a sufficient number of nucleotides to render the p5 promoter

less active or inactive or moving the promoter relative to the coding sequences of

rep such that the promoter is less active. In an alternative embodiment, one may

increase the distance between the p5 promoter and the start codon of the rep gene to decrease promoter activity. For instance, one may move the p5 promoter

downstream of the rep gene or insert nucleotide sequences between the p5

promoter and the downstream ATG start codon. One may measure whether a p5

promoter is effectively deleted by measuring the transcription of a gene operably linked to the mutated or partially deleted p5 promoter and comparing the gene's

transcription to the transcription of a gene in a promoterless construct.

In a preferred embodiment, a p5 promoter is effectively deleted

when it promotes less than 80%> of wildtype p5 promoter activity, more preferably

less than 50%o of wildtype p5 promoter activity. In a more preferred embodiment, a p5 promoter is effectively deleted when it promotes less than 25%> of wildtype p5 promoter activity. In an even more preferred embodiment, a p5 promoter is

effectively deleted when it promotes less than 15%>, more preferably promotes less

than 10%, and more preferably less than 5% of wildtype p5 promoter activity. In

another preferred embodiment, a p5 promoter is effectively deleted when the rep gene to which it is operably linked is not rearranged or deleted when an adenovirus

comprising the effectively deleted p5 promoter and rep gene is infected into a host cell, such as 293 cells. In another preferred embodiment, a p5 promoter is

effectively deleted when a host cell infected with an adenovirus comprising rep and

the deleted p5 promoter produces rAAV at a high titer. In a preferred embodiment,

the titer is at least 10² particles per cell; preferably at least 10³ particles per cell;

more preferably at least 10⁴ particles per cell; and, even more preferably, at least 10⁵ or 10⁶ particles per cell. In general, there are approximately 1 x 10³ to 3 x 10³

particles per transducing units (TU). The number of particles required to produce one TU varies based upon the transgene, purification method and assay method.

A "minimal promoter" is one that essentially comprises only a TATA

box and promotes only very low or basal levels of transcription of rep78 and rep68.

A promoter is a nucleotide sequence that promotes the initiation of transcription at

a particular site by the cell's transcriptional machinery.

In a preferred embodiment, a minimal promoter promotes

transcription that is less than 80%> of the wildtype p5 promoter, more preferably less

than 50%> of the wildtype p5 promoter, even more preferably less than 25% of the

wildtype p5 promoter. In a more preferred embodiment, a minimal promoter is one

that promotes transcription that is less than 20% of the wildtype p5 promoter, even

more preferably less than 15% of the wildtype p5 promoter. In an even more

preferred embodiment, a minimal promoter is one that promotes transcription that is less than 10% of the wildtype p5 promoter, even more preferably less than 5%o,

even more preferably less than 1% of the wildtype p5 promoter.

A minimal promoter also may be defined by functional measures. A

minimal promoter is one in which the rep gene to which it is operably linked is not rearranged or deleted when an adenovirus comprising the minimal promoter and rep

gene is infected into a host cell, such as 293 cells. A minimal promoter is one in

which a host cell infected with an adenovirus comprising a minimal promoter that

regulates transcription of rep78 and rep68 produces rAAV at a high titer. In a

preferred embodiment, the titer is at least 10² particles per cell; preferably at least

10³ particles per cell; more preferably at least 10⁴ particles per cell; and, even more

preferably, at least 10⁵ or 10⁶ particles per cell.

Many minimal promoters are known in the art. Alternatively, an

artificial minimal promoter may be constructed by using a sequence or a consensus

sequence of a TATA box and adding nucleotide sequences to the 5' and 3' ends of

the TATA box. The activity of the minimal promoter may be measured by measuring the transcription of the artificial minimal promoter and comparing it to an

natural minimal promoter, such as the Drosophila heat shock protein promoter.

Rep78/68 is "promoterless" or has "no promoter" when the p5

promoter has been deleted or effectively deleted, as defined supra, and no promoter

has been inserted in its place. Alternatively, rep78/68 is promoterless when the p5

promoter has been deleted and is replaced by a heterologous promoter that does not promote transcription in the host cell in which the adenovirus has been infected.

For instance, rep78/68 would be considered promoterless if p5 were substituted by

a promoter that was active in bacterial or insect cells, for example, but that was

inactive in a mammalian host cell. In another embodiment, rep78/68 is

promoterless when the p5 promoter has been deleted and replaced by an inducible promoter that permits low-level expression of rep78/68. The Adenoviral Vector

A large number of adenoviruses and adenoviral vectors are known,

including human adenoviruses types 1-46, chimpanzee adenoviruses, canine

adenoviruses, bovine adenoviruses [all available from the American Type Culture

Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209], and

ovine adenoviruses (Both et al , WO 97/06826 Al). Any of these adenoviruses may

be used in this invention, provided that the adenovirus is able to infect the target host cell For instance, a human adenovirus would generally be used to infect a

human cell, while a bovine adenovirus would be used to infect a bovine cell.

In one embodiment, the adenoviral vector comprises the AAV rep

gene downstream of a minimal promoter or no promoter (this is alternatively

referred to as a minimal promoter or promoterless AAV rep gene), and sufficient

helper virus functions for rAAV production in a host cell In a preferred

embodiment, the adenoviral vector further comprises the AAV cap gene The type

of adenoviral sequences required for replication and encapsidation of the rAAV

genome depends upon whether the host cell expresses any helper functions or

whether other vectors or viruses are introduced into the host cell which express

helper functions For instance, if the adenovirus is to be used to infect a cell line

that expresses El, e g , the 293 cell line, then the adenoviral vector could comprise rep and cap, and could also comprise those helper virus functions required in

addition to El (e g , E2a, E4ORF6 and VAI RNA) If the adenovirus is used to

infect a cell line such as 84-31, which expresses El and E4, then the adenoviral

vector could express rep, cap, E2a and VAI RNA If the adenovirus is used to infect a cell line that does not express any helper functions, then the adenovirus

vector could comprise, at least, El (both El a and Elb) and E2a, and, optionally,

may comprise E4ORF6 and VAI RNA. In an alternative embodiment, helper

functions may be supplied by chemical or physical methods or by other helper

viruses.

The recombinant adenovirus comprising the rep gene downstream of a minimal promoter or no promoter may be produced by any method known in the

art. In one preferred method, the recombinant adenovirus of the instant invention is

produced using homologous recombination. In another preferred method, the

recombinant adenovirus is produced using Cre-lox recombination (12).

In an alternative embodiment, some or all of the helper virus

functions may be provided by nucleic acid sequences that are introduced into the

host cell. For instance, the host cell may be co-infected with a second virus, such as

an adenovirus, that expresses some or all of the required helper functions. In a

preferred embodiment, a second adenoviral vector comprises a transgene cassette

and further comprises helper functions that are not expressed by either the host cell

or the adenovirus comprising the rep gene. See, e.g., Examples 6-8. Alternatively,

some or all of the helper virus functions may be provided by any method known in the art, such as by transfection or direct injection, as discussed above.

Based on this description, other embodiments of the adenoviral vector will be readily apparent to those of ordinary skill in the art. Other viral

vectors in which Rep interferes with viral replication also may be used. Rep and Cap Nucleic Acids

In AAV's life cycle, both rep and cap are required for excision,

replication and encapsidation of the recombinant viral genome into an infectious

recombinant vector or virus The Rep and Cap proteins may have a naturally

occurring sequence derived from any serotype of AAV, including serotypes 1 to 6

In one preferred embodiment, the rep and cap genes are derived from the same

AAV serotype In another preferred embodiment, the rep and cap genes are

derived from different AAV serotypes to permit the production a pseudotyped

rAAV Pseudotyped rAAV is desirable in cases in which the rAAV is to be administered to a patient as a gene therapy vector and there are existing neutralizing

antibodies in the patient's serum to the capsid proteins ofone AAV serotype and

not to another AAV serotype The cap gene from the serotype to which there is an

antibody response may be exchanged by the cap gene from a different serotype of

AAV to which there is no antibody response For example, the rep gene from

AAV-2 may be used with the cap gene from AAV-1 to produce a pseudotyped rAAV-2, or vice-versa

In an alternative embodiment, the Rep and/or Cap proteins may have

a mutated sequence, including insertions, deletions, fragments or point mutations of particular amino acid residues, so long as the mutated Rep and/or Cap proteins retain their respective excision, replication and encapsidation functions In a

preferred embodiment, the naturally-occurring Rep and Cap amino acid sequences

from AAV- 2 are used In one embodiment, a single adenovirus comprises the

nucleic acid sequences encoding the Rep and Cap proteins In another embodiment, the nucleic acid sequences encoding the Rep and Cap proteins are inserted at a

single site within one adenovirus, e g , both rep and cap are inserted at El, E3 or

E4 of adenovirus Alternatively, the nucleic acid sequences encoding Rep and Cap,

respectively, are each inserted at different loci in the adenovirus genome, e g , the

nucleic acid sequence encoding Rep may be in El, and the nucleic acid sequence encoding Cap may be in E4, and other combinations thereof Alternatively, a cell

line comprising the cap gene may be used, in which case only the rep gene would be

required on the adenoviral vector See, e.g , WO 98/27204

The minimal promoter that regulates expression of Rep78 or Rep68

may be any promoter that promotes only basal expression of the rep gene in a host

cell In general, the promoter is one that essentially contains a TATA box as its only regulatory element In a preferred embodiment, the minimal promoter is the

Drosophila heat shock promoter (HSP) In another preferred embodiment, the

minimal promoter is the minimal promoter derived from the adenovirus Elb gene

that provides only basal promoter activity In another preferred embodiment, the

minimal promoter is a 70 nucleotide DNA element derived from the promoter

region upstream of the adenovirus pIX gene The minimal pIX promoter comprises

a TATA box and an Spl box, and, in Ad5, corresponds to nucleotides 3511 to 3580 Many other adenovirus serotypes contain the pIX gene and its upstream

promoter as well, and the minimal promoters derived from these pIX promoters are

encompassed by this invention as well Other minimal promoters are well known in the art and may be used in the practice of this invention In another preferred

embodiment, the p5 promoter is deleted altogether and replaced by no promoter at all

In another embodiment of the invention, the activity of the Rep78/68

proteins are attenuated by mutating the rep78/68 genes to produce Rep78/68

proteins that are less active than wildtype Rep78/68. This may be done by altering

the coding sequence of the Rep78/68 proteins to make them less active

Alternatively, one may alter the DNA sequence of rep78/68 to destabilize the RNA encoded by the gene and thus decrease the amount of Rep78/68 proteins produced. One may determine whether a DNA sequence is appropriate for use

as a minimal promoter by inserting the DNA sequence upstream of the rep78/68

ATG codon in a plasmid construct or an adenoviral vector and transfecting or

infecting, respectively, a host cell that comprises an rAAV genome, incubating the host cell under conditions in which rAAV is produced, measuring the titer of rAAV

produced, and comparing the titer to that produced using a control plasmid or

adenovirus comprising a rep gene whose expression is regulated by a minimal

promoter The host cell may comprise the rAAV genome stably or transiently.

Alternatively, one may measure the level of Rep78 and Rep68 produced in the host cell after infection to determine if sufficiently low levels of Rep78 and Rep68 are

produced.

Helper Functions

As discussed above, AAV requires helper functions for excision, replication and encapsidation of AAV. AAV helper functions can be provided by

adenovirus, herpesvirus [including herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2), cytomegalovirus (CMV) and pseudorabies virus (PRV)] or by exposure

of the cells to different chemical or physical agents. Alternatively, one of skill in the

art may determine which helper functions are required by producing rAAV using

the compositions and methods disclosed in the instant specification.

To identify which helper functions are required for high levels of

rAAV production, one may transfect a host cell containing an rAAV genome with a plasmid comprising rep and cap and then transfect with one or nucleic acids

encoding various potential helper functions to determine which potential helper

functions are required for rAAV production. The rAAV genome may be stably

integrated into the host cell or may be transfected or infected into the host cell by methods known in the art. After transfecting the host cell with the nucleic acid

encoding the potential helper function, one may then measure the titer of the rAAV

that is produced to determine if the nucleic acid encodes a helper function.

In a preferred embodiment, the helper functions are nucleic acids

derived from a virus. In a more preferred embodiment, the helper functions are

derived from adenovirus types 2 or 5, HSV-1, HSV-2, CMV or PRV. In an even

more preferred embodiment, the helper functions are El a, Elb, E2a, E4ORF6

proteins and VAI RNA from adenovirus. In another preferred embodiment, the

nucleic acid encodes the helper functions from the helicase-primase complex of

HSV (UL5, UL8 and UL52) and the major single-stranded DNA binding protein of HSV (UL29). Alternatively, helper functions for recombinant AAV may be

provided by chemical or physical agents, including ultraviolet light, cycloheximide,

hydroxyurea and various carcinogens. The required helper functions for production of a rAAV may be

delivered to the host cell by any method known in art The helper functions may be

delivered by transfection with a vector, such as a plasmid, by infection with a viral

vector comprising the helper functions, or by any other method known in the art,

including those discussed above (e g , biolistic injection of DNA, use of DNA

conjugates, etc ) The transfection or infection may be stable or transient

Alternatively, the cell line may stably express (either on an extrachromosomal episome or through integration in the cell's genome) the helper functions In

addition, some of the helper functions may be expressed by the mammalian cell line

while other helper functions are introduced by a vector Thus, for production of

rAAV in 293 cells (ATCC CRL-1573), which constitutively produce adenoviral

Ela and Elb proteins, only E2a, E4ORF6 and VAI must be introduced into the

host cell by transfection or infection of a vector

In a preferred embodiment, the helper functions are transduced into

the host cells by an adenovirus In a more preferred embodiment, some or all of the

helper functions are transduced into the host cell by the adenovirus that comprises

the rep and/or cap genes In a preferred embodiment, the native helper function

sequences are used However, mutated helper function sequences may be used so long as they retain their helper function activity The helper function nucleic acids

may be supplied with its native promoter or may be under the regulatory control of a variety of promoters, constitutive or inducible, such as the CMV immediate-early

promoter/enhancer or the zinc-inducible metallothionein promoter, respectively, as known in the art or as described above The rAAV Transgene Cassette

In order to manufacture a rAAV containing a transgene, the method

of the present invention begins with a desired transgene, then associates the

transgene with appropriate expression regulatory sequences (ERS), e.g., promoter, enhancer, polyadenylation site, then inserts this ERS-transgene construct between

AAV flanking sequences, e.g., the ITRs, in place of rep and cap genes normally

found therein. Where the length of the ERS-transgene cassette is shorter than the AAV rep and cap sequences, and that shorter length would pose an obstacle to

proper packaging, an optional spacer or "sniffer" sequence may be inserted in order

to maintain the proper length for packaging. The transgene cassette comprised of

the ERS-transgene bordered by the AAV flanking sequences may then be embedded

in an adenovirus vector separate from that which carries the rep gene.

Alternatively, the transgene cassette may be inserted into a plasmid vector and

transfected into a host cell. The transgene cassette may be maintained in the host

cell stably, either by integration into the host cell genome or as an episome, or may

be introduced transiently, such as by infection with a hybrid Ad/ AAV virus. See,

e.g., Examples 3 and 6-8. Each element of the transgene cassette is further

described below:

The Transgene

A transgene is a nucleic acid encoding a protein of interest; it may be

a gene to allow for genetic or drug selection, e.g., a gene conferring resistance to

antibiotics, or a reporter gene allowing detection, e.g., by color in the case of the use of green fluorescent protein. Alternatively, the transgene may be one that is

useful for corrective applications. For instance, a transgene may be a normal gene

that replaces or augments the function of a patient's defective gene. The transgene may be one that counteracts the effects of a disease, such as introduction and

expression of a gene that is distinct from the one that it replaces or augments, but

which has the same function or compensates for the defective gene's function. The

transgene may be a gene which blocks or represses the expression of a malfunctioning, mutated, or viral gene in the patient, thereby giving rise to a

corrective effect. A transgene may also be a protective gene, such as one that

prevents cellular apoptosis, injury, toxicity or death. A transgene may also be used

for immunization against various agents, by provoking an immunogenic response in

an animal. Delivery of therapeutic transgenes to a patient thus effects some level of

correction of a defect or is beneficial for prevention of disease. The transgene also

may be a gene which would confer sensitivity to a reagent that results in cellular

toxicity, e.g., introduction of HSV thymidine kinase, which confers sensitivity to

gancyclovir. The transgene also may be one which is useful for production of

proteins in vitro, such as for large-scale production of therapeutic proteins.

Many gene therapy methods involve supplying an exogenous gene to overcome a deficiency in the expression of a gene in a patient. Some of these

deficiencies are congenital and are due to a mutation in a particular gene in all the

cells of the patient. For instance, in cystic fibrosis, there are one or more mutations

in the gene encoding the cystic fibrosis transmembrane conductance regulator

(CFTR) which prevents the CFTR protein from functioning properly. In other cases, a deficiency in gene expression is due to an accident or disease that occurs

duπng the patient's life For instance, in Type I diabetes mellitus, the β pancreatic

islet cells, which produce insulin, are destroyed, such that patients with this disease

can no longer synthesize insulin. In other cases, the endogenous gene may be

structurally normal but is not transcribed and/or translated in high enough quantities

due to disease, medical treatment or other environmental conditions, or mutations in the regulatory elements of the endogenous gene For example, there are a number

of blood disorders, such as anemia, in which there is insufficient production of red

blood cells, which may be treated with erythropoietin (EPO) or with a transgene encoding EPO Conversely, gene therapy methods may be used where

overexpression of a particular gene results in a disease state. For instance, overexpression of z-myc by the immunoglobulin heavy chain promoter results in

leukemia Transgenes may also be used for genetic immunization, i e , to elicit an

immune response to a pathogen in an animal, including humans For instance, a

transgene may include a sequence from a viral, bacterial or fungal pathogen, such as

influenza virus, human immunodeficiency virus (HIV), or mycobacterium

tuberculosis

Appropriate genes for expression in the cell include, without

limitation, those genes which are normally expressed in cells but whose products are

produced in abnormal amounts due to over- or under-expression Alternatively, the appropriate gene for expression is one which expresses a normal gene product

which replaces a defective gene product, encodes ribozymes or antisense molecules

which repair or destroy mutant cellular RNAs expressed from mutated genes, or modifies or destroys viral RNAs. Transgenes used for production of proteins in

vitro include proteins such as secreted factors, including hormones, growth factors

and enzymes.

The composition of the transgene sequence depends upon the

intended use for the resulting rAAV. For example, one type of transgene sequence

comprises a reporter or marker sequence, which upon expression produces a detectable signal. Such reporter or marker sequences include, without limitation,

DNA sequences encoding E. coli β-lactamase, β-galactosidase (LacZ), alkaline

phosphatase, HSV thymidine kinase, green fluorescent protein (GFP), bacterial

chloramphenicol acetyltransferase (CAT), firefly luciferase, eukaryotic membrane

bound proteins including, for example, CD2, CD4, CD8, the influenza

hemagglutinin protein, and others well known in the art, to which high affinity

antibodies directed to them exist or can be made routinely, and fusion proteins

comprising a membrane bound protein appropriately fused to an antigen tag domain

from, among others, hemagglutinin or myc.

These sequences, when associated with regulatory elements which

drive their expression, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectroscopic assays,

fluorescent activated cell sorting assay and immunological assays, including ELISA,

RIA and immunohistochemistry. For example, where the transgene is the LacZ

gene, the presence of a rAAV is detected by assays for β-galactosidase activity.

Similarly, where the transgene is luciferase, the rAAV gene expression may be measured by light production in a luminometer. However, desirably, the transgene is a non-marker gene which can

be delivered to a cell or an animal via the rAAV produced by this method The

transgene may be selected from a wide variety of gene products useful in biology

and medicine, such as proteins, sense or antisense nucleic acids (e.g , RNAs), or catalytic RNAs. The invention may be used to correct or ameliorate gene

deficiencies, wherein normal genes are expressed but at greater than normal or less

than normal levels, and may also be used to correct or ameliorate genetic defects

wherein a functional gene product is not expressed. A preferred type of transgene

sequence is a therapeutic gene which expresses a desired corrective gene product in

a host cell at a level sufficient to ameliorate the disease, including partial

amelioration. These therapeutic nucleic acid sequences typically encode products

which, upon expression, are able to correct, complement or compensate an inherited

or non-inherited genetic defect, or treat an epigenetic disorder or disease.

However, the selected transgene may encode any product desirable for study The

selection of the transgene sequence is not a limitation of this invention Choice of a

transgene sequence is within the skill of the artisan in accordance with the teachings

of this application.

The invention also includes methods of producing rAAV and

compositions thereof which can be used to correct or ameliorate a gene defect

caused by a multi-subunit protein. In certain situations, a different transgene may be used to encode each subunit of the protein. This may be desirable when the size

of the DNA encoding the protein subunit is large, e.g., for an immunoglobulin or

the platelet-derived growth factor receptor. In order for the cell to produce the multi-subunit protein, a cell would be infected with rAAV expressing each of the

different subunits

Alternatively and more preferably, different subunits of a protein

may be encoded by the same transgene. In this case, a single transgene would

include the DNA encoding each of the subunits, with the DNA for each subunit

separated by an internal ribosome entry site (IRES) The use of IRES permits the creation of multigene or polycistronic mRNAs IRES elements are able to bypass

the ribosome scanning model of 5' methylated cap-dependent translation and begin

translation at internal sites (Pelletier and Sonenberg, 1988) For example, IRES

elements from hepatitis C and members of the picornavirus family (e g , polio and encephalomyocarditis) have been described, as well an IRES from a mammalian

mRNA (Macejak and Sarnow, 1991) IRES elements can be linked to heterologous

open reading frames By virtue of the IRES element, each open reading frame is

accessible to ribosomes for efficient translation Thus, multiple genes can be

efficiently expressed using a single promoter/enhancer to transcribe a single

message This is preferred when the size of the DNA encoding each of the subunits

is sufficiently small that the total of the DNA encoding the subunits and the IRES is

no greater than the maximum size of the DNA insert that the virus can encompass

For instance, for rAAV, the insert size can be no greater than approximately 4 8

kilobases, however, for an adenovirus which lacks all of its helper functions, the insert size is approximately 28 kilobases

Useful gene products include hormones and growth and

differentiation factors including, without limitation, insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), calcitonin, growth hormone releasing

factor (GRF), thyroid stimulating hormone (TSH), adrenocorticotropic hormone

(ACTH), prolactin, melatonin, vasopressin, β-endorphin, met-enkephalin, leu-

enkephalin, prolactin-releasing factor, prolactin-inhibiting factor, corticotropin-

releasing hormone, thyrotropin-releasing hormone (TRH), follicle stimulating

hormone (FSH), luteinizing hormone (LH), chorionic gonadotropin (CG), vascular

endothelial growth factor (VEGF), angiopoietins, angiostatin, endostatin,

granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), bFGF2, acidic

fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming

growth factor α (TGFα), platelet-derived growth factor (PDGF), insulin-like

growth factors I and II (IGF-I and IGF-II), any one of the transforming growth

factor β (TGFβ) superfamily comprising TGFβ, activins, inhibins, or any of the bone

morphogenic proteins (BMP) BMPs 1-15, any one of the

heregulin/neuregulin/ARIA/neu differentiation factor (NDF) family of growth

factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),

neurotrophins NT-3, NT-4/5 and NT-6, ciliary neurotrophic factor (CNTF), glial

cell line derived neurotrophic factor (GDNF), neurtuin, persephin, agrin, any one of

the family of semaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.

Other useful gene products include proteins that regulate the

immune system including, without limitation, cytokines and lymphokines such as

thrombopoietin (TPO), interleukins (IL) IL-lα, IL-lβ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, and IL-17,

monocyte chemoattractant protein (MCP-1), leukemia inhibitory factor (LIF),

granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony

stimulating factor (G-CSF), monocyte colony stimulating factor (M-CSF), Fas

ligand, tumor necrosis factors α and β (TNFα and TNFβ), interferons (IFN) IFN-α,

IFN-β and IFN-γ, stem cell factor, flk-2/flt3 ligand. Gene products produced by the

immune system are also encompassed by this invention. These include, without limitations, immunglobulins IgG, IgM, IgA, IgD and IgE, chimeric

immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors,

chimeric T cell receptors, single chain T cell receptors, class I and class II MHC

molecules, as well as engineered MHC molecules including single chain MHC

molecules. Useful gene products also include complement regulatory proteins such as membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CR2

and CD59.

Still other useful gene products include any one of the receptors for

the hormones, growth factors, cytokines, lymphokines, regulatory proteins and

immune system proteins. Examples of such receptors include flt-1, flk-1, TIE-2; the trk family of receptors such as TrkA, MuSK, Eph, PDGF receptor, EGF

receptor, HER2, insulin receptor, IGF-1 receptor, the FGF family of receptors, the

TGFβ receptors, the interleukin receptors, the interferon receptors, serotonin

receptors, α-adrenergic receptors, β-adrenergic receptors, the GDNF receptor, p75 neurotrophin receptor, among others. The invention encompasses receptors for

extracellular matrix proteins, such as integrins, counter-receptors for transmembrane-bound proteins, such as intercellular adhesion molecules (ICAM-1, ICAM-2, ICAM-3 and ICAM-4), vascular cell adhesion molecules (VCAM), and

selectins E-selectin, P-selectin and L-selectin. The invention encompasses receptors

for cholesterol regulation, including the LDL receptor, HDL receptor, VLDL

receptor, and the scavenger receptor. The inventions encompasses the

apolipoprotein ligands for these receptors, including ApoAI, ApoAIV and ApoE.

The invention also encompasses gene products such as steroid hormone receptor

superfamily including glucocorticoid receptors and estrogen receptors, Vitamin D

receptors and other nuclear receptors. In addition, useful gene products include

antimicrobial peptides such as defensins and maginins, transcription factors such as

jun,fos, max, mad, serum response factor (SRF), AP-1, AP-2, myb, MRG1,

CREM, Alx4, FREACl, NF-κB, members of the leucine zipper family, C2H4 zinc

finger proteins, including Zif268, EGR1, EGR2, C6 zinc finger proteins, including

the glucocorticoid and estrogen receptors, POU domain proteins, exemplified by

Pit 1 , homeodomain proteins, including HOX-1 , basic helix-loop-helix proteins,

including myc, MyoD and myogenin, ETS-box containing proteins, TFE3, E2F,

ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-

box binding proteins, interferon regulation factor 1 (IRF-1), Wilms tumor protein,

ETS-binding protein, STAT, GATA-box binding proteins, e.g., GATA-3, and the forkhead family of winged helix proteins.

Other useful gene products include carbamoyl synthetase I, ornithine

transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase,

fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha- 1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor VII, factor VIII, factor

IX, factor II, factor V, factor X, factor XII, factor XI, von Willebrand factor,

superoxide dismutase, glutathione peroxidase and reductase, heme oxygenase,

angiotensin converting enzyme, endothelin-1, atrial natriuetic peptide, pro-

urokinase, urokinase, plasminogen activator, heparin cofactor II, activated protein

C (Factor V Leiden), Protein C, antithrombin, cystathione beta- synthase, branched

chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl

CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase,

insulin, beta-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase (also referred to as P-protein), H-

protein, T-protein, Menkes disease protein, tumor suppressors (e.g., p53), cystic

fibrosis transmembrane regulator (CFTR), the product of Wilson's disease gene

PWD, Cu/Zn superoxide dismutase, aromatic aminoacid decarboxylase, tyrosine

hydroxylase, acetylcholine synthetase, prohormone convertases, protease inhibitors,

lactase, lipase, trypsin, gastrointestinal enzymes including chyromotrypsin, and

pepsin, adenosine deaminase, αl anti-trypsin, tissue inhibitor of metalloproteinases

(TIMP), GLUT-1, GLUT-2, trehalose phosphate synthase, hexokinases I, II and

III, glucokinase, any one or more of the individual chains or types of collagen,

elastin, fibronectin, thrombospondin, vitronectin and tenascin, and suicide genes such as thymidine kinase and cytosine deaminase. Other useful proteins include

those involved in lysosomal storage disorders, including acid β-glucosidase, α-

galactosidase a, α-1-iduronidase, iduroate sulfatase, lysosomal acid α-glucosidase,

sphingomyelinase, hexosamina\idase A, hexominidases A and B, arylsulfatase A, acid lipase, acid ceramidase, galactosylceramidase, α-fucosidase, α-, β-

mannosidosis, aspartylglucosaminidase, neuramidase, galactosylceramidase,

heparan-N-sulfatase, N-acetyl-α-glucosaminidase, Acetyl-CoA: α-glucosaminide N-

acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, N-acetylgalactosamine-

6-sulfate sulfatase, arylsulfatase B, β-glucuoronidase and hexosaminidases A and B.

Other useful transgenes include non-naturally occurring

polypeptides, such as chimeric or hybrid polypeptides or polypeptides having a non-

naturally occurring amino acid sequence containing insertions, deletions or amino

acid substitutions. For example, single-chain engineered immunoglobulins could be useful in certain immunocompromised patients. Other useful proteins include

truncated receptors which lack their transmembrane and cytoplasmic domain.

These truncated receptors can be used to antagonize the function of their respective

ligands by binding to them without concomitant signaling by the receptor. Other

types of non-naturally occurring gene sequences include sense and antisense

molecules and catalytic nucleic acids, such as ribozymes, which could be used to

modulate expression of a gene.

Other useful transgenes include those that encode antigenic peptides

capable of generating an immune response. Recombinant vectors comprising these

transgenes can be used for genetic immunization. Useful transgenes include those that encode peptides specific for Epstein Barr virus; HIV; simian immunodeficiency

virus (SIV); human T-cell leukemia viruses I and II (HTLV-I and HTLV-II);

hepatitis A, B, C, D, E and SEN; pseudorabies virus; rabies virus; cytomegalovirus;

respiratory syncytial virus; parainfluenza virus types 1-4; mumps virus; rubella virus; polio virus; measles virus; influenza virus types A, B and C; rotavirus; heφes

simplex viruses types 1 and 2; varicella-zoster virus; human herpes virus type 6, 7

and 8; hantavirus; denguevirus, sindbisvirus, adenoviruses; chlamydia pneumoniae;

chlamydia trachomatis; mycoplasma pneumoniae; mycobacterium tuberculosis;

atypical mycobacteria; feline leukemia virus; feline immunodeficiency virus; bovine

immunodeficiency virus; equine infectious anemia virus; caprine arthritis

encephalitis virus; visna virus; Staphlococcus species and Streptococcus species.

The transgenes may also be directed against peptides from tumor antigens to provide immunization for tumors and cancers.

Expression Control Sequences

A great number of expression control sequences ~ native,

constitutive, inducible and/or tissue-specific ~ are known in the art and may be

utilized to drive expression of the transgene and the nucleic acid sequences

encoding the replication and encapsidation functions of the rAAV, the helper

functions and the ligand. The choice of expression control sequence depends upon

the type of expression desired. For eukaryotic cells, expression control sequences typically include a promoter, an enhancer, such as one derived from an

immunoglobulin gene, SV40, cytomegalovirus, etc., and a polyadenylation

sequence. The polyadenylation sequence generally is inserted following the

transgene sequences and before the 3' flanking sequence of the transgene. A transgene-carrying molecule useful in the present invention may also contain an

intron, desirably located between the promoter/enhancer sequence and the transgene One possible intron sequence is also derived from SV40 and is referred

to as the latel6S/19S intron Another vector element that may be used is an

internal ribosome entry site (IRES), as described above An IRES element is used

to produce more than one polypeptide from a single transcript An IRES element

can be used for the transgene or for any of the other nucleic acid sequences

encoding the replication and encapsidation polypeptides, the helper functions or the

ligand Selection of these and other common vector elements are conventional and many such sequences are available [see, e g , Sambrook et al, and references cited

therein at, for example, pages 3 18-3 26 and 16 17-16 27 and Ausubel et al ,

Current Protocols in Molecular Biology. John Wiley & Sons, New York, 1989]

In one embodiment, high-level constitutive expression will be

desired Examples of such promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter/enhancer, the cytomegalovirus (CMV)

immediate early promoter/enhancer [see, e g , Boshart et al, Cell. 41 521-530

(1985)], the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic

β-actin promoter and the phosphoglycerol kinase (PGK) promoter

In another embodiment, inducible promoters may be desired

Inducible promoters are those which are regulated by exogenously supplied

compounds, either in cis or in trans, including without limitation, the zinc-inducible metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse

mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system

[WO 98/10088], the ecdysone insect promoter [No et al, Proc Natl Acad Sci

USA, 93 3346-3351 (1996)], the tetracycline-repressible system [Gossen et al, Proc Natl Acad Sci USA. 89 5547-5551 (1992)], the tetracycline-inducible

system [Gossen et al , Science. 268 1766- 1769 (1995), see also Harvey et al , Curr

Opin Chem Biol . 2 512-518 (1998)], the RU486-inducible system [Wang et al ,

Nat Biotech . 15 239-243 (1997) and Wang et al , Gene Ther . 4 432-441 (1997)],

and the rapamycin-inducible system [Magari et al , J Clin. Invest . 100 2865-2872

(1997), Rivera et al . Nat Medicine. 2.1028-1032 (1996)] Other types of inducible

promoters which may be useful in this context are those which are regulated by a

specific physiological state, e g , temperature, acute phase, or in replicating cells

only In another embodiment, the native promoter for the transgene or

nucleic acid sequence of interest will be used. The native promoter may be

preferred when it is desired that expression of the transgene or the nucleic acid

sequence should mimic the native expression The native promoter may be used

when expression of the transgene or other nucleic acid sequence must be regulated

temporally or developmentally, or in a tissue-specific manner, or in response to

specific transcriptional stimuli In a further embodiment, other native expression

control elements, such as enhancer elements, polyadenylation sites or Kozak

consensus sequences may also be used to mimic the native expression

In one embodiment, the recombinant viral genome comprises a

transgene operably linked to a tissue-specific promoter. For instance, if expression in skeletal muscle is desired, a promoter active in muscle may be used These include the promoters from genes encoding skeletal α-actin, myosin light chain 2A,

dystrophin, muscle creatine kinase, as well as synthetic muscle promoters with activities higher than naturally-occurring promoters [see Li et al , Nat Biotech ,

17 241-245 (1999)] Examples of promoters that are tissue-specific are known for liver [albumin, Miyatake et al J Virol , 71 5124-32 (1997), hepatitis B virus core

promoter, Sandig et al , Gene Ther . 3 1002-9 (1996), alpha-fetoprotein (AFP),

Arbuthnot et al , Hum Gene Ther . 7 1503-14 (1996)], bone [osteocalcm, Stein et

al , Mol Biol Rep . 24 185-96 (1997), bone sialoprotein, Chen et al , J Bone

Miner Res . 11 654-64 (1996)], lymphocytes [CD2, Hansal et al , J Immunol . 161 1063-8 (1998), lmmunoglobulin heavy chain, T cell receptor α chain], neuronal

[neuron-specific enolase (NSE) promoter, Andersen et al Cell Mol Neurobiol ,

3 503-15 (1993), neurofilament light-chain gene, Piccioli et al , Proc Natl Acad

Sci USA 88 5611-5 (1991), the neuron-specific vgf gene, Piccioli et al , Neuron,

15 373-84 (1995)], among others

Of course, not all vectors and expression control sequences will

function equally well to express all of the transgenes or other nucleic acid sequences

of this invention However, one of skill in the art may make a selection among

these expression control sequences without departing from the scope of this

invention Suitable promoter/enhancer sequences which function in the appropriate

host cell of choice may be selected by one of skill in the art using the guidance

provided by this application Such selection is a routine matter and is not a limitation of the molecule or construct

One may identify a suitable expression control sequence for a desired transgene by selecting one or more expression control sequences and operably

linking the expression control sequence to the nucleic acid sequence to be regulated Then, one may insert these operably linked sequences comprising the

expression control sequence and regulated sequence into the genome of the

adenovirus vector. In one embodiment, one may insert a recombinant viral genome

comprising the expression control sequence and the transgene into a vector of the

instant invention. After following one of the methods for producing and packaging

the rAAV as taught in this specification one may infect suitable cells in vitro or in

vivo The number of copies of the transgene in the cell may be monitored by

Southern blotting or quantitative PCR; the level of RNA expression may be

monitored by Northern blotting or quantitative RT-PCR; and the level of protein

expression may be monitored by Western blotting, immunohistochemistry, ELISA,

RIA, tests of the transgene' s gene product's biological activity, either in vitro or in vivo, or tests for correction or amelioration of a genetic defect.

Flanking Elements

The naturally-occurring AAV ITRs consist of repeated sequences,

usually but not necessarily approximately 145 nucleotides in length, at the 5' and 3'

ends of the AAV genome. The AAV ITRs are required for replication, excision and encapsidation of both wild type and recombinant AAV virions. The ITRs flank the

transgene when the AAV DNA integrates into a host cell chromosome. When

rAAV is rescued from the host chromosome, the ITRs excise along with the transgene and remain in flanking positions surrounding the rescued DNA, in a form

suitable for packaging into virions. The ITRs may be derived from any one of the

adeno-associated viruses known, including AAV serotypes 1 to 6. In a preferred embodiment of the invention, the rAAV comprises a selected transgene operably linked to expression regulatory sequences and AAV flanking elements

Host Cells

Any type of mammalian cell that can be adapted to cell culture may

be used as a host cell to produce the recombinant viral genome In general, a host

cell used in this invention is one that may be infected by the adenovirus vector of

the instant mvention Another preferred characteristic of the host cell is that it is

able to replicate the rAAV at high levels

Appropoate host cells include, without limitation, CHO, BHK,

MDCK and vaoous muone cells, e g , 10T1/2 and WEHI cells, African green

monkey cells such as VERO, COS 1, COS 7, BSC 1, BSC 40, and BMT 10, and

human cells such as WI38, MRC5, A549, human embryonic retinoblast (HER),

human embryonic kidney (HEK), human embryonic lung (HEL) and HT1080 cells

In a preferred embodiment, appropoate cells include 293 cells (human embryonic

kidney cells that express adenoviral Ela and Elb proteins), 911 or PER C6 cells

(human embryonic retinoblast cells that express adenoviral El, see WO 97/00326),

B50 cells (HeLa cells that express AAV rep and cap, see PCT US98/19463), 84-31

cells (293-based cells that express adenovirus Ela, Elb and E4, Ref 4), 10-3 cells

(293 -based cells that express adenovirus Ela, Elb and E4ORF6, Ref 1 1), 3T3 cells (mouse embryonic fibroblast cell line), NIH3T3 cells (subline of 3T3 cells), HepG2

cells (human liver carcinoma cell line), Saos-2 cells (human osteogenic sarcoma cell line), HuH7 cells or HeLa cells (human carcinoma cell line) In addition to the host cells listed above, other host cells may be

used. One may determine whether a cell line would be suited for use as a

mammalian host cell by infecting the cell line with an adenovirus of the instant

invention in the presence of rep, cap and all required helper functions, culturing the

cells under conditions in which rAAV is produced, and then measuring the titer of

infectious rAAV. One may then compare the titer of infectious rAAV produced in

the potential host cell with the titers produced by other host cells to determine

whether the cell line is good for rAAV production.

Methods of Producing Recombinant Adeno-Associated Virus from Adenovirus

Another aspect of the instant invention is a method of producing

rAAV from the adenovirus of the instant invention. The method comprises the

steps of:

1. Infecting host cells comprising an rAAV genome with an

adenovirus comprising a rep gene under the regulatory control of a minimal promoter or no promoter;

2. growing the infected host cells under conditions in which the

rAAV genome is excised, replicated and encapsidated; and

3. collecting the rAAV from the mammalian host cells.

The host cells may be any mammalian cell known in the art or as

described herein. The host cell, prior to infection by the adenovirus comprising the

rep gene, may be one that expresses one or more of the following genetic elements:

1 ) the cap gene, 2) some or all necessary helper functions (e.g., 293 cells), and/or 3) an rAAV genome Alternatively, the host cell may comprise none of these genetic elements prior to infection by the adenovirus. In this case, the genetic elements are

supplied by the adenovirus comprising the rep gene, other viral vectors, and/or

plasmids The host cells may be infected by the adenovirus by any method

known in the art or as described herein Methods for infecting host cells with adenoviruses are well known in the art and are also described herein. Once the host

cell has been infected, the helper functions are activated and rep and cap are

produced Low levels of Rep78 and Rep68 are produced because the p5 promoter

has been replaced by a minimal promoter or by no promoter. This, combined with

levels of Rep52 and Rep40, both expressed from the adenoviral pl9 promoter, and the capsid proteins, expressed from the AAV p40 promoter, are sufficient to

produce high titers of rAAV

Methods of producing rAAV using other viral vectors in which Rep

interferes with viral replication also may be performed following the teachings of

the instant specification.

The rAAV may be purified from the supernatant produced by the host cells or from cell lysates by any method known in the art or as described

herein. A method of collecting and purifying rAAV is described in Examples 5 and

6

The method is easily scaled to industrial production because it does

not require transfection of a large number of host cells to produce rAAV In a

preferred embodiment, only a single infection of host cells by an adenovirus is required to produce rAAV in large amounts at high titers See, e g , Example 5 In

another preferred embodiment, host cells are co-infected with two different adenoviruses, one comprising rep downstream of a minimal promoter or no

promoter and cap, and the other adenovirus comprising the rAAV genome

Infection of host cells by adenovirus is highly efficient and may be easily scaled to a

large number of cells

The instantly described method produces rAAV at a high titer In a

preferred embodiment, the titer is at least 10² particles per cell, preferably at least

10³ particles per cell, more preferably at least 10⁴ particles per cell, and, even more

preferably, at least 10⁵ or 10⁶ particles per cell The instant invention also encompasses lysates and supernatants of host cells comprising rAAV These lysates

and supernatants differ from those produced by prior art methods because of the higher level of rAAV contained therein without concentration

rAAV Compositions

The rAAV produced by the method of this invention may be

formulated as a pharmaceutical or pharmacological composition for use for any

form of transient and stable gene transfer in vivo and in vitro The composition

comprises at least the rAAV and a pharmaceutically acceptable carrier The rAAV

may be used for in vivo and ex vivo gene therapy, genetic immunization, in vitro protein production and diagnostic assays

For gene therapy, the rAAV may be introduced into cells ex vivo or

in vivo Where the virus is introduced into a cell ex vivo, the rAAV may be used to infect a cell in vitro, and then the cell may subsequently be introduced into a

mammal (e.g., into the portal vein or into the spleen), if desired. Alternatively, the

rAAV may be administered to a mammal directly, e.g., intravenously or

intraperitoneally. A slow-release device, such as an implantable pump, may be used

to facilitate delivery of the virus to a cell. Where the virus is administered to a

mammal, the specific cells to be infected may be targeted by controlling the method

of delivery. For example, intravascular administration of rAAV to the portal vein or

to the hepatic artery may be used to facilitate targeting rAAV to a liver cell.

The rAAV produced by the above-described method may be

administered to a patient, preferably suspended in a biologically compatible solution

or pharmaceutically acceptable delivery vehicle. A suitable vehicle includes sterile saline. Other aqueous and non-aqueous sterile suspensions known to be

pharmaceutically acceptable carrier and well known to those of skill in the art may

be employed for this purpose.

The rAAV is administered in sufficient amounts to infect the desired

cells and provide sufficient levels of transduction and expression of the selected

transgene (or viral gene products in the case of a vaccine) to provide some level of

a corrective effect without undue adverse or with medically acceptable physiological effects, which can be determined by those skilled in the medical arts.

Conventional and pharmaceutically acceptable routes of administration include

direct administration to the target organ, tissue or site; intranasal; intravenous;

intramuscular; subcutaneous; intradermal; oral and other parenteral routes of

administration. Routes of administration may be combined, if desired. Dosages of rAAV will depend primarily on factors such as the

condition being treated and the selected gene. The dosage may also vary depending

upon the age, weight and health of the patient. For example, an effective human

dosage of rAAV is generally in the range of from about 0.5 ml to 50 ml of saline

solution containing rAAV at concentrations of 1 x 10⁷ or 1 x 10⁸ or 1 x 10⁹ or 1 x

10¹⁰ or 1 x 10¹¹ or 1 x 10¹² or 1 x 10¹³ or 1 x 10¹⁴ or 1 x 10¹⁵ or 1 x 10¹⁶ particles

per dose administered. The dosage will be adjusted to balance the corrective

benefits against any adverse side effects. The levels of expression of the selected gene may be monitored to determine the type and frequency of dosage

administration.

The following examples of the present inventions are illustrative

only, and are not intended to limit the scope of the invention.

EXAMPLE 1 Cell Lines and Viruses and Maintenance and Propagation Thereof

All cell lines are maintained in Dulbecco's Modified Eagle's Medium

(DMEM; Gibco BRL) supplemented with 10% FBS (Hyclone) and 50 μg/ml of

penicillin, 50 μg/ml of streptomycin, and 10 μg/ml of neomycin (Gibco BRL).

Human embryonic kidney cell line 293 is obtained from ATCC (CRL 1573). 293-

derived 84-31 cells (1) which express adenovirus El/E4orf6 proteins, and HeLa- derived B50 cells (7) which express AAV-2 Rep and Cap proteins from the native p5 promoter, are obtained from Dr. Guangping Gao, Institute for Human Gene

Therapy, University of Pennsylvania. 293-CG3 is a 293 -derived cell line carrying stably integrated copies of AAV ITRs flanking GFP as marker gene (Chen et al., unpublished data). Human adenovirus type 5 (ATCC VR-5) and derived

recombinant adenoviruses are propagated on 293 cells and purified through CsCl

gradient centrifugation according to the method of Jones and Shenk with

modification (2).

EXAMPLE 2 Construction of Plasmids and Generation of Recombinant Adenoviruses

Standard recombinant DNA techniques are employed to create

recombinant plasmids (3). DNA containing the rep and cap sequences of pAV2

(ATCC 37216) between Dralll site (nucleotide 241, upstream of the AAV-2 p5

promoter) and Ncol site (nucleotide 4489, downstream of the polyA signal) is removed and replaced through multiple cloning steps with a DNA cassette

containing GFP under the transcriptional control of elongation factor 1 alpha

(EFlα) promoter and upstream of the SV40 polyA signal to create pAV2cisEFGFP

(Fig.1). The AAV-2 rep and cap genes located between a Dra III site (nucleotide

241, upstream of the p5 promoter) and a Bsal site (nucleotide 4464, downstream of

the polyA signal) are further subcloned to obtain pAd-p5-RC (Fig.1). A small DNA

fragment between nucleotides 241 and 287 of pAd-p5-RC containing the p5

promoter is removed and replaced with a Drosophila melanogaster minimal heat shock protein (HSP) promoter from pLND (Invitrogen) to create pAd-HSP-RC

(Fig.1). Recombinant adenoviruses Ad-p5-RC and Ad-HSP-RC (Fig. 2) are generated according to standard protocols known in the art (see, e.g., Refs. 4 and 12). The recombinant adenoviruses are passaged five to six times on appropriate

mammalian cells to generate a stock of recombinant adenovirus that is used for

production of rAAV.

EXAMPLE 3 Transfection of 293 Cells and Selection of the 293-CG3 Stable Cell Line

293 cells are grown to -70% confluency in 6-cm tissue culture

dishes and co-transfected overnight with 1 μg pIRESlneo and 10 μg

pAV2cisEFGFP by the calcium phosphate transfection method. The monolayer is

replenished with fresh medium containing 10%> FBS and cultured for 24 hours.

Following trypsinization, cells are seeded at a 1 :20 dilution in fresh medium

containing 10%) FBS. After incubation for another 24 hours, fresh medium

containing 1,250 μg/ml of G418 (Gibco BRL) is added to the cell monolayer for

genetic selection of G418-resistant cells. The medium containing G418 is replaced

every 3-4 days to allow formation of G418-resistant cell colonies. A total of fifty

colonies are picked, six of which demonstrate constitutive GFP expression. These

six clones are expanded and tested for their ability to rescue functional rAAV by

transfection with pBV-EiOV-RC, a plasmid that carries adenovirus E2A, E4ORF6,

and VAI genes as well as AAV rep-cap genes. One cell clone, 293-CG3, shows

high efficiency of rAAV rescue and is expanded and used for further experiments. EXAMPLE 4 PCR Analysis of AAV-2 rep and cap Genes Inserted into the Adenovirus Genome

In order to determine the integrity of AAV-2 rep and cap genes

recombined into the adenovirus genome, a polymerase chain reaction (PCR) assay

is employed Four sets of primer pairs overlapping the entire DNA sequence

encoding AAV-2 rep and cap genes, as well as the junctions between adenoviral and rep-cap sequences, are synthesized These primers are

HC#30 (5'-CGTAACCGAGTAAGATTTGG-3', SEQ ID NO 1),

HC#31 (5'-ATGTTGGTGTTGGAGGTGAC-3', SEQ ID NO 2),

HC#32 (5 '-TGGACCAGAAATGCAAGTCC-3', SEQ ID NO 3),

HC#33 (5 '-AGCCTTGACTGCGTGGTGGT-3', SEQ ID NO 4),

HC#34 (5 '-GTACCTGT ATT ACTTGAGCA-3', SEQ ID NO 5), HC#35 (5 -ACGAGTCAG GTATCTGGTGC-3', SEQ ID NO 6),

HC#36 (5 '-GGACTTTACTGTGGACACTA-3', SEQ ID NO 7), and HC#37 (5' -GACCC AGACT ACGCTGACGA-3 ' , SEQ ID NO 8)

To obtain adenoviral DNA for the PCR assay, a miniprep method is

employed Briefly, 293 cells are grown in 10-cm dishes until nearly confluent and then infected with either Ad-HSP-RC or Ad-p5-RC for 3 days Infected cells are

harvested, pelleted and lysed in DOC lysis buffer (100 mM Tris-HCl, pH9 0, 20%

ethanol, 0 4%> sodium deoxycholate) Cellular nucleic acids are precipitated by

spermine-HCl and supernatant is collected The supernatant is then treated with

RNase A and pronase, followed by extraction with phenol-chloroform Adenoviral DNA in the supernatant is precipitated with isopropanol and dissolved in TE/RNase buffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA, 20 μg/ml RNase).

The PCR assay is performed using the Robocycler Gradient 96

thermal cycler (Stratagene), PCR products are separated on a 1% agarose gel and DNA is stained with ethidium bromide. As shown in Fig. 3, all four expected DNA

PCR products are obtained when using Ad-HSP-RC DNA as template, indicating

that the full-length AAV-2 rep-cap DNA is present in the genome of this recombinant. However, the 880 bp PCR product III is not amplified from Ad-p5-

RC DNA, suggesting a rearrangement or deletion event in rep-cap DNA sequences

of this recombinant. These results indicate the integrity and stability of HSP-rep-

cap DNA after insertion into the El locus of the adenoviral genome, and point out

that p5-rep-cap DNA sequences inserted into the same locus are unstable.

EXAMPLE 5

Production of rAAV through Infection of 293-CG3 Cells with Ad-HSP-RC

Since Ad-HSP-RC is shown to contain full length AAV-2 rep-cap

DNA sequences, its utility to produce rAAV is analyzed. 293-CG3 cells are seeded

in 6-well plates at a density of l.OxlO⁶ cells/well. Twelve to fifteen hours later, the

culture media is removed from the cell monolayer and dilutions of Ad-HSP-RC

virus in 0.5 ml of serum-free DMEM are added to the cells. Following a 30 min.

incubation, an additional 2.5 ml of DMEM containing 10% FBS is added and the

infection is allowed to proceed for a total of 3 days. Infected cells are harvested and pelleted. Cell pellets are lysed in 1 ml of lysis buffer (50 mM Tris-HCl, pH7.4,

1.0 mM MgCl₂, 0.5% DOC) with sonication for 3x1 min. Cell debris is removed by centrifugation at 3,000 rpm using a Beckman GS-6R centrifuge for 10 min. The

lysate supernatant is collected for titration of rAAV.

To titrate the rAAV in the lysate, 84-31 cells (1) are plated 3-4

hours before use on 24-well plates at a density of 2x10⁵ cells/well. The rAAV

lysate is diluted in DMEM containing 10%> FBS and heated at 56°C for 60 min to

inactivate contaminating Ad-HSP-RC. The rAAV lysate is added to the monolayer,

cells are incubated for approximately 24 hours, and GFP-expressing cells are scored

as transducing units (TU).

As shown in Fig.4, the results demonstrate that rAAV is successfully

produced through infection of 293-CG3 cells with the recombinant Ad-HSP-RC. The rAAV titer increases by increasing the multiplicity of infection (MOI) of Ad-

HSP-RC. Using an MOI of 250 particles/cell, as much as 70 TU/cell of rAAV is

produced. However, further increasing the MOI of the Ad-HSP-RC virus does not

increase the yield of rAAV. Instead, a slight decrease in rAAV yield is observed,

probably due to the increased cytotoxicity associated with higher MOI of Ad-HSP-

RC virus.

EXAMPLE 6

Production of rAAV through Co-Infection of 293 Cells with Ad-HSP-RC and Ad-AAV-LacZ

In the previous example, AAV vector sequences are stably

integrated into the host cell chromosome while rep-cap functions necessary for their

rescue and packaging into rAAV are provided by Ad-HSP-RC. As a separate

measure to determine the functionality of the Ad-HSP-RC recombinant, AAV vector sequences are delivered exogenously into 293 cells to determine whether

they could be rescued and packaged into rAAV particles following infection with

Ad-HSP-RC To perform this experiment, 293 cells are seeded on 6-well plates at a density of lxlO⁶ cells/well 12-15 hours later, the cells are co-infected with Ad-

HSP-RC and Ad-AAV-LacZ, an El -deleted adenovirus containing the E. coli lacZ

gene flanked by AAV-2 ITR's inserted into the El locus (4)

To further investigate the effects of varying MOI's of either virus on

rAAV yield, the following experiment is performed Different quantities of the two

viruses (50 to 450 particles/cell) are mixed together, keeping the total inoculum at a

constant 500 particles/cell (Fig 5) The virus mixture is diluted in 0 5 ml of serum-

free DMEM at various particle/cell ratios (50 450, 100 400, 200 300, 300 200,

400 100, and 450 50) and is added to the cell monolayer Thirty minutes later, an

additional 2 5 ml of DMEM containing 10%> FBS is added and the cells are incubated in the presence of virus inoculum for 3 additional days

Infected cells are harvested, lysed, and rAAV titrated as described in

Example 5 except that rAAV transduction is scored cells by X-gal staining

according to standard protocols (5) Cell stain is possible because the rAAV in this

experiment carries lacZ as a transgene Briefly, the rAAV transduced cells are first fixed with 0 5 ml of 0 05% glutaraldehyde for 10 min and then rinsed with 3x0 5 ml

of PBS Fixed cells are stained with 0 5 ml of X-gal solution at room temperature

overnight The X-gal solution is removed, 0 5 ml of 70%> ethanol is added to

terminate the reaction, and blue-staining cells are scored as transducing units

As shown in Fig 5, the results clearly demonstrate that co-infection of 293 cells by the two recombinant adenoviruses, Ad-HSP-RC and Ad-AAVLacZ, can produce high titers of rAAV in 293 cells. The data indicate that with

decreasing MOI of Ad-HSP-RC and increasing MOI of Ad-AAV-LacZ, the yield of

rAAV produced remains relatively steady until the Ad-HSP-RC MOI reaches 100

particles/cell or less. Further decrease of the MOI of Ad-HSP-RC dramatically

decreases the rAAV yield, presumably due low levels of Rep and Cap proteins that are produced at the lower MOFs. On the other hand, increasing the MOI of Ad-

AAV-LacZ does not increase the rAAV yield. While the conventional method for

rAAV production using plasmid co-transfection is limited in its yield of rAAV, the

current invention provides the means to easily and efficiently produce high yields of

rAAV.

EXAMPLE 7

Time-Course and Particle Ratio Studies of rAAV Production through Co-Infection of 293 Cells with Ad-HSP-RC and Ad-AAV-LacZ

To further study the conditions required for optimal production of

rAAV through co- infection of 293 cells with Ad-HSP-RC and Ad-AAV-LacZ,

separate time-course and MOI studies are performed. 293 cells are seeded in 6-well

plates as described in Example 6 and are infected with both viruses for different

time intervals or at different particles/cell ratios. Infected cells are harvested and

lysed, and rAAV is titrated as described in Example 6. For the time-course study,

100 particles/cell each of Ad-HSP-RC and Ad-AAV-LacZ are used to infect 293 cells for various times.

As shown in Fig.6, the results demonstrate that rAAV is detected as early as 24 hours post-infection by the two viruses, but its levels peaks at 72 hours

after infection. More than 200 TU/cell of rAAV is produced between 48 and 96 hours after infection by the two viruses. To determine the optimal MOI of the two

viruses required for high level rAAV production, 293 cells are infected by the two

viruses at equal MOI's for 72 hours and harvested and rAAV titers are determined.

As shown in Fig. 7, it is apparent that with increasing input of Ad-HSP-RC and Ad-

AAV-LacZ viruses, the yield of rAAV increases up to an MOI of 125 particles/cell of each virus. Further increasing the input adenovirus MOI does not increase the

rAAV yield and instead results in a slight decrease of rAAV yield. This may be due

to higher cytopathic effects resulting from higher MOI's of input adenovirus which

in turn may affect rAAV yields from the infected cell.

EXAMPLE 8

Analysis of rAAV DNA Replication Following Co-Infection of 293 Cells with Ad-HSP-RC and Ad-AAV-LacZ

To analyze the excision and replication of rAAV DNA from the Ad-

AAV-LacZ hybrid genome, extrachromosomal DNA is analyzed in 293 cells co-

infected by Ad-HSP-RC and Ad-AAV-LacZ using the method of Hirt (6). As a

positive control for rAAV DNA rescue and replication, a separate system

previously shown to produce rAAV at high titers is similarly assayed in parallel.

The control system is based on the use of the B50 cell line, that was previously created by stably transfecting into HeLa cells a rep/cαp-containing plasmid utilizing

endogenous AAV-2 promoters (7). rAAV production in this cell line occurs in a

two-step process: B50 cells are initially infected with sublOOr, an adenovirus temperature-sensitive mutant in the E2b gene, to induce rep and cap expression and provide helper functions. 24 h later, cell are infected by a hybrid El -deleted

adenovirus in which the AAV vector sequence is cloned in the El region of a

replication-defective adenovirus. In the presence of Rep and Cap proteins

expressed by the cell, as well as adenoviral helper functions expressed from

sublOOr, the rAAV genome delivered by the hybrid vector is rescued, replicated and encapsidated into rAAV particles (7).

293 cells are grown in 10-cm dishes to subconfluency and are either

co-infected with Ad-HSP-RC plus Ad-AAV-LacZ or with Ad-p5-RC plus Ad- AAV-LacZ at 200 particles/cell of each virus. Positive control B50/subl00r

experiments are carried out in a similar fashion except that Ad-AAV-LacZ is added

to B50 cells 24 hours after addition of sublOOr, each at 1,000 particles/cell.

Negative controls include single virus infections of 293 cells with either Ad-p5-RC,

Ad-HSP-RC or Ad-AAV-LacZ alone. Infected cells are harvested 72 hours post-

infection and lysed in 0.85 ml of Hirt solution (10 mM Tris-HCl, pH 7.4, 100 mM

EDTA, 0.6% SDS). The lysate is mixed with 0.25 ml of 5 M NaCl and incubated at

4"C overnight. After centrifugation at 14,000 RPM for 40 min. in a Sorvall

centrifuge, supernatant is collected and extracted three times with phenol-

chloroform. The low molecular weight DNA is precipitated by isopropanol,

dissolved in TE/RNase buffer, fractionated by electrophoresis on a 0.8%> agarose gel and stained with ethidium bromide.

The results of this experiment are presented in Fig. 8. Co-infection

of 293 cells by Ad-HSP-RC and Ad-AAV-LacZ results in the generation of DNA bands of 4 8 and 9 6 kb which correspond to monomeric and dimeric forms of

replicating, double-stranded rAAV DNA (Fig 8, lane 4) The positive control also shows the same two DNA species following infection of B-50 cells with sublOOr

and Ad-AAV-LacZ (Fig 8, lane 6) Neither DNA band is observed in negative

controls in which 293 cells are infected singly with either Ad-p5-RC (Fig 8, lane 1),

Ad-HSP-RC (Fig 8, lane 2), or Ad-AAV-LacZ (Fig 8, lane 3) Furthermore, co-

fection of 293 cells by Ad-p5-RC and Ad-AAV-LacZ also does not result in the formation of either rAAV DNA species (Fig 8, lane 5), suggesting that Ad-p5-RC

is defective in its rep/cap functions for rescue or replication of rAAV DNA

To confirm that the 4 8 and 9 6 kb extrachromosomal DNA species

detected in Fig 8 does indeed contain the lacZ transgene, and to confirm that low

quantities of such DNA species are indeed absent in negative control lanes, a

Southern blot analysis is performed Hirt-extracted DNA, isolated from either 293

cells co-infected by Ad-HSP-RC and Ad-AAV-LacZ or B50 cells infected by

sub 1 OOr and Ad-AAV-LacZ, is separated on a 0 8%> agarose gel (Fig 9A) The gel

is transferred to a nitrocellulose membrane and hybridized with a digoxigenin

labeled, lacZ DNA probe using the DIG High Prime DNA Labeling Detection

Starter Kit II from Boehringer Mannheim (Fig 9B) A lacZ DNA fragment,

isolated from a bacterial plasmid, is used as positive control for the Southern blot (Fig 9 A and 9B, lane 1)

Both monomeric and dimeric forms of replicating rAAV DNA,

isolated from either 293 cells co-infected by Ad-HSP-RC and Ad-AAV-LacZ (Fig 9A and 9B, lane 5) or B50 cells infected by sublOOr and Ad-AAV-LacZ (Fig 9A and 9B, lane 7), hybridize to the lacZ probe Southern blotting does not detect any replicated rAAV DNA species following co-infection of 293 cells by Ad-p5-RC and

Ad-AAV-LacZ (Fig 9A and 9B, lane 6), or from the negative controls which

include Hirt DNA from 293 cells infected singly by either Ad-HSP-RC (Fig 9A and

9B, lane 2), Ad-p5-RC (Fig 9A and 9B, lane 3), or Ad-AAV LacZ (Fig 9A and

9B, lane 4) Taken together, these results indicate that co-infection of 293 cells by

Ad-HSP-RC and Ad-AAV-LacZ results in rescue and replication of rAAV DNA

from the Ad-AAV-LacZ genome, and that replicating rAAV DNA indeed contains

the lacZ transgene Moreover, the lack of replicating rAAV DNA after co-infection

of 293 cells by Ad-p5-RC and Ad-AAV-LacZ supports earlier observations that

Ad-p5-RC is indeed defective in its rep/cap functions for rescue or replication of

rAAV DNA

EXAMPLE 9 Analysis of Rep/Cap Protein Expression in 293 Cells Infected with Ad-HSP-RC Virus

To analyze the expression of Rep and Cap proteins in cells infected

by Ad-HSP-RC virus, Western blotting is performed 293 cells are grown in 10-cm

plates to subconfluency and are either infected with Ad-HSP-RC, Ad-AAV-LacZ,

or Ad-p5-RC using 200 particles/cell of each virus 72 hours post-infection, cells

are harvested, pelleted, and processed as described by Xiao et al (8) Briefly, the infected cell pellet from one 10-cm dish is lysed in 0 5 ml lysis buffer containing 10 mM Tris-HCl, pH 8 2, 1% Triton X-100, 1% SDS and 150 mM NaCl by

somcation Samples are separated by SDS-polyacrylamide gel electrophoresis (PAGE) on a 4%>~20%> gradient polyacrylamide gel and transferred to a

nitrocellulose membrane. The Rep proteins are detected using monoclonal antibody

303.9 and Cap proteins are detected using monoclonal antibody Bl (American

Research Products, Inc.), both at a 1 :20 dilution. Protein-antibody complexes are

visualized using the ECL™ Western Blotting Analysis System (Amersham).

As shown in Fig.10, Rep and Cap proteins are expressed in 293 cells

infected with Ad-HSP-RC alone (Lane 3), or co-infected with Ad-HSP-RC and Ad-

AAV-LacZ (Lane 5), but not in 293 cells infected with Ad-p5-RC alone (Lane 1),

Ad-p5-RC plus Ad-AAV-LacZ (Lane 2), or Ad-AAV-LacZ (Lane 4). Levels of

Rep52 and Rep40 proteins expressed following Ad-HSP-RC infection are much

higher than those of Rep78 and Rep68, a phenomenon that has been previously

reported to contribute to higher levels of rAAV produced (9). The low basal transcriptional activity of the HSP promoter may indeed play an important role in

the success of creating the Ad-HSP-RC recombinant adenovirus since it is known

that expression of AAV Rep proteins may interfere with adenovirus replication

(10). Low level expression of these proteins may minimize the interference but

provide enough Rep function for excision and replication of the rAAV genome.

All publications and patent applications cited in this specification are

herein incorporated by reference as if each individual publication or patent

application were specifically and individually indicated to be incorporated by

reference. Although the foregoing invention has been described in some detail by

way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this

invention that certain changes and modifications may be made thereto without

departing from the spirit or scope of the appended claims.

References

Fisher KJ, Gao GP, Weitzman MD, DeMatteo R, Burda JF, Wilson JM

1996 Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis J Virol 70(1) 520-532

Jones N, Shenk T 1978 Isolation of deletion and substitution mutants of adenovirus type 5 Cell 13(1) 181-188

Sambrook J , Fritsch E F , and Maniatis T 1989 "Molecular Cloning — A Laboratory Manual" Cold Spring Harbor Laboratory Press

Fisher KJ, Kelley WM, Burda JF, and Wilson JM 1996 A novel adenovirus-adeno-associated virus hybrid vector that displays efficient rescue and delivery of the AAV genome Hum Gene Ther 7(17) 2079- 2087

Ausubel FM et al 1994 Current Protocols In Molecular Biology John Wiley & Sons, Inc

Hirt B 1967 Selective extraction of polyoma DNA from infected mouse cell cultures J Mol Biol 26(2) 365-369 Gao GP, Qu G, Faust LZ, Engdahl RK, Xiao W, Hughes JV, Zoltick PW, and Wilson JM 1998 High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus Hum Gene Ther 9 2254-2362

Xiao X, Li J, and Samulski RJ 1998 Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus J Virol 1998 72(3) 2224-2232

Li J, Samulski RJ, and Xiao X. 1997 Role for highly regulated rep gene expression in adeno-associated virus vector production J Virol 71(7) 5236- 5243

Weitzman MD, Fisher KJ, and Wilson JM 1996 Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers J Virol 70(3) 1845-1854

Gao GP, Yang Y, and Wilson JM 1996 Biology of adenovirus vectors with El and E4 deletions for liver-directed gene therapy J Virol

70(12) 8934-43 2. Hardy S, Kitamura M, Harris- Stansil T, Dai Y, and Phipps L. 1997.

Construction of Adenovirus Vectors through Cre-/øx Recombination. J. Virol. 71(3), 1842-1849.

Claims

1 An adenovirus vector for the manufacture of a rAAV, wherein

the adenovirus vector comprises an AAV rep gene, and wherein the AAV p5

promoter is deleted upstream of the AAV rep gene

2 The adenovirus vector of claim 1, wherein the AAV rep gene

comprises a minimal promoter in place of the p5 promoter

3 The adenovirus vector of claim 1 , wherein the AAV rep gene

contains no promoter in place of the p5 promoter

4 The adenovirus vector of any one of claims 1 to 3, wherein the

adenovirus vector further comprises an AAV cap gene

5 The adenovirus vector of claim 2, wherein the minimal promoter

contains a TATA box

6 The adenovirus vector of claim 2, wherein the minimal promoter is a minimal Drosophila heat shock promoter

7 The adenovirus vector of claim 2, wherein the minimal promoter is a minimal adenoviral Elb promoter

8 The adenovirus vector of claim 4, wherein the rep gene and the

cap gene are inserted in place of at least a portion of one or more of the El, E3 or E4 genes of adenovirus in a locus of the adenovirus vector

9 The adenovirus vector of claim 8, wherein both the rep gene and

the cap gene are inserted within the same locus of the adenovirus vector

10 The adenovirus vector of claim 8, wherein the rep gene and the

cap gene are inserted within different loci of the adenovirus vector

11 The adenovirus vector of claim 4, wherein the rep and cap

genes are derived from different AAV serotypes from each other

12 A method for producing recombinant rAAV, comprising the

steps of

a) infecting the adenovirus vector according to any one of claims 1

to 1 1 into a host cell containing an rAAV genome,

b) growing the host cells under conditions in which rAAV is

produced, and c) collecting the rAAV from the host cells

13 The method according to claim 12, wherein the rAAV genome is stably integrated in a chromosome of the host cells

14 The method according to claim 12, wherein an adenovirus

vector comprises the rAAV genome and is co-infected with the adenovirus vector

comprising the rep gene into the host cell.

15 The method according to claim 12, wherein the adenovirus

vector provides necessary helper functions for rAAV production

16 The method according to claim 12, wherein the host cell is 293

cells

17 The method according to any one of claims 12 to 16, further

comprising the step of purifying the rAAV.

18 The method according to claim 12, wherein the host cells are

selected from CHO, BHK, MDCK, 10T1/2, WEHI cells, COS, BSC 1, BSC 40, BMT 10, VERO, WI38, MRC5, A549, HT1080, 293, B-50, 3T3, NIH3T3,

HepG2, Saos-2, Huh7, HER, HEK, HEL, or HeLa cells.

19 A lysate or supernatant comprising the rAAV produced by the

method according to any one of claims 12 to 16 or 18.

20 A purified rAAV produced by the method according to claim 19

21. A pharmaceutical composition comprising the rAAV according to claim 20 and further comprising a pharmaceutically acceptable carrier.

22. A method for transient or stable gene transfer of a desired

transgene to a mammalian cell, comprising the step of infecting the mammalian cell

with the rAAV according to claim 20.

23. The method according to claim 22, wherein said transient or

stable gene transfer is for genetic immunization, correction of genetic defects or production of proteins in vitro, in vivo, or ex vivo.

24. The vector according to claim 15, wherein said helper functions

are encoded by at least one gene product selected from the group consisting of

adenoviral genes El A, EIB, E2A, E4orf6 and VAI, or at least one gene product

selected from the group consisting of HSV type 1 genes UL5, UL8, UL52, and UL29.