WO2000072887A9 - A novel packaging cell line for the rescue, production and titration of high-capacity adenovirus amplicon vectors - Google Patents

Abstract

The present invention describes a method of producing adenovirus gutless amplicon viral vector substantially reduced in the content of helper virus. The invention also describes a system for the helper virus independent replication and packaging of adenovirus gutless vectors. A cell line for this system is also discussed.

Description

A NOVEL PACKAGING CELL LINE FOR THE RESCUE, PRODUCTION AND TITRATION OF HIGH-CAPACITY ADENOVIRUS AMPLICON

VECTORS

The United States Government has certain rights to this invention by virtue of funding received from Grant Nos. RO1-DK 51700 and R21-DK 53333.

This application claims priority under 35 U.S.C. § 119 from provisional patent application Serial No. 60/136,481, filed May 28, 1999 which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention pertains to methods for the efficient, large-scale, helper virus-free

production of recombinant adenovirus vectors that are free of most if not all viral genes

using a novel packaging cell line.

BACKGROUND OF THE INVENTION

Recombinant adenovirus vectors are a highly efficient means of transferring

genes into a wide variety of cell types in vivo. Although the adenovirus has a natural

tropism for lung epithelium, adenoviral vectors have quite a broad host cell range. In

addition to lung and other epithelial-derived tissues (Gilardi et al. 1990); (Stratford- Perricaudet et al. 1990); (Rosenfeld et al. 1991); (Quantin et al. 1992); (Rosenfeld et al.

1992); (Bajocchi et al. 1993); (Yang et al. 1993), recombinant adenovirus vectors have

been successfully used to transduce normally non-dividing cell types including

hepatocytes (Levrero et al. 1991); (Jaffe et al. 1992); (Herz and Gerard 1993); (Ishibashi

et al. 1993); (Li et al. 1993) and neuronal and glial cells of the central nervous system

(Akli et al. 1993); (Davidson et al. 1993); (Le Gal La Salle et al. 1993). Especially high

transduction efficiencies are observed in liver, where 100% of all mouse hepatocytes

could be transduced following intraportal infusion of recombinant adenovirus vectors (Li

et al. 1993).

At present, this vector system has two limitations that have prevented its

application in various forms of somatic gene therapy. The first is the transient expression

of genes delivered by this means. Apart from muscle, where low levels of persistent gene

expression have been reported (Rosenfeld et al. 1992); (Stratford-Perricaudet et al.

1992); (Vincent et al. 1993), expression of genes delivered by recombinant adenovirus

vectors is largely transient in other tissues. For example, although 100% of hepatocytes

were successfully transduced one week after intraportal infusion of a recombinant

adenovirus vector expressing β-galactosidase, only 0.5% to 10% of hepatocytes were still

positive for β-galactosidase activity by 14 to 16 weeks post-infusion (Li et al. 1993).

This decline in the proportion of transduced cells was correlated with a decrease in the

amount of vector DNA that was present, indicating that the decline in positive cells was

not due to inactivation of transcription from the vector.

More recent studies have used recombinant adenovirus vectors to express

potentially therapeutic genes in normal animals (Herz and Gerard 1993) or in animal models of various monogenic diseases (Ishibashi et al. 1993); (Fang et al. 1994); (Kay

and Woo 1994); (Li and Davidson 1995). In these latter studies, partial (Li and Davidson

1995) or complete (Ishibashi et al. 1993); (Fang et al. 1994); (Kay and Woo 1994)

correction of the disease phenotype was observed shortly after infusion of a recombinant

adenovirus vector expressing the deficient gene product. However, in studies where

persistence was examined, the phenotypic correction was greatly diminished or totally

reversed within 21 to 28 days of treatment (Fang et al. 1994); (Kay and Woo 1994); (Li

and Davidson 1995). Again, this lack of persistence was due to loss of the vector DNA

from the target cells (Fang et al. 1994); (Kay and Woo 1994); (Li and Davidson 1995).

Several studies have indicated that the transient expression of genes delivered by

El -deleted adenovirus vectors is primarily a consequence of a cytotoxic T cell-mediated

immune response mounted against the transduced cells (Engelhardt et al. 1994a);

(Engelhardt et al. 1994b); (Yang et al. 1994a); (Yang et al. 1994b). These observations

are supported by the fact that only slight decreases are observed in the level of transgene

expression for at least six months after adenovirus-mediated gene transfer in immune-

deficient nu/nu (Yang et al. 1994b) or SCID mice (Barr et al. 1995). Further support for

this hypothesis is provided by the observation that administration of the

immunosuppressive agent cyclosporine A can significantly prolong transgene expression

following the infusion of El -deleted adenovirus vectors (Engelhardt et al.

1994b;Engelhardt et al. 1994a); (Fang et al. 1995), as can the depletion of CD4⁺ cells by

the anti-CD4 antibody GK1.5 (Yang and Wilson 1995); (Yang et al. 1996); (Kolls et al.

1996). To date, however, none of these studies have shown the indefinite persistence of a complete phenotypic correction following adenovirus-mediated transfer of a therapeutic

gene in an animal model of human disease.

Because cells transduced with El -deleted recombinant adenovirus vectors appear

to be specifically targeted due to residual amounts of late viral gene expression (Yang et

al. 1994a); (Yang et al. 1994b), several studies have examined whether additional

modifications of these vectors to reduce late viral gene expression can significantly

increase their persistence in vivo (Engelhardt et al. 1994b;Engelhardt et al. 1994a);

(Yang et al. 1994c); (Armentano et al. 1995), (Krougliak and Graham 1995); (Lee et al.

1995); (Wang et al. 1995); (Zhou et al. 1996); (Yeh et al. 1996); (Gao et al. 1996);

(Gorziglia et al. 1996). As many of these modified vectors are unable to grow in 293

cells, several new cell lines also were developed, expressing in trans the adenoviral

genes deleted from the vectors, such as E4 (Krougliak and Graham 1995); (Wang et al.

1995); (Gao et al. 1996); (Yeh et al. 1996), E2a (Zhou et al. 1996); (Gorziglia et al.

1996), terminal protein precursor (Schaack et al. 1995); (Langer and Schaack 1996) or

Ad polymerase (Amalfitano et al. 1998). Although some studies indicated that these so-

called "second generation" adenovirus vectors are less toxic and less immunogenic

(Wang et al. 1995); (Gao et al. 1996); (Gorziglia et al. 1996); (Hu et al. 1999), there is as

yet no conclusive evidence that such vectors are capable of indefinite (or at least

prolonged) persistence in immunocompetent animals. Furthermore, in nearly every case,

the introduction of additional deletions into the vector genome has significantly

decreased the resulting titers, making the vectors more difficult to produce in quantities

sufficient to support preclinical and clinical studies. A second drawback of the present vector systems is that they are contaminated

with high levels of viral genes. The presence of significant levels of these viral genes

may lead to imrnunological responses in the host. Such responses may lead to a

compromise in the efficiency of the adenovirus vector.

The ultimate form of adenovirus vector modification is the creation of a so-called

"gutless", "gutted" or amplicon vector. This is a vector containing only the cis elements

necessary for replication and packaging, but lacking most if not all adenovirus genes. All

of the amplicon vectors created thus far (Mitani et al. 1995); (Fisher et al. 1996);

(Kochanek et al. 1996); (Lieber et al. 1996); (Parks et al. 1996); (Alemany et al. 1997);

(Chen et al. 1997); (Parks and Graham 1997);(Morsy et al. 1998); (Schiedner et al.

1998); (Chen et al. 1999) share a number of disadvantages. Foremost among these is the

use of helper viruses or plasmid co-transfection to provide the necessary virus proteins in

trans. The use of helper viruses almost always results in significant contamination of the

amplicon vector by the helper virus. The use of plasmids results in much lower titers of

the vector. Both of these outcomes would significantly limit future clinical applications

of this highly promising vector system.

What is needed in the art is a system for the generation of adenovirus vectors

which overcomes the above-mentioned drawbacks of prior art systems.

SUMMARY OF THE INVENTION

A system has been developed with the potential to overcome the limitations of

both low yield and significant helper virus contamination that are associated with the

previous approaches described above. This system involves the creation of a true packaging cell line for the production of adenovirus amplicon vectors. All existing cell

lines that complement the function of adenoviral genes, including El (293 cells;

(Graham et al. 1977), El and E2 (C2; (Zhou et al. 1996) and El and E4 (Krougliak and

Graham 1995); (Wang et al. 1995); (Gao et al. 1996); (Yeh et al. 1996), resulted from

the incorporation of different adenovirus genes into chromosomes of the host cell.

Although this approach has worked well for the generation of El -complementing 293

cell lines, the addition of other adenoviral genes such as E2 or E4 has resulted in a

decrease of the yield of adenoviruses carrying mutations in the corresponding genes.

Further attempts to insert DNA fragments containing additional early and late adenovirus

genes into the chromosomes of host cells are unlikely to be successful because late viral

genes must be expressed at high level, some of the viral gene products are likely to be

cytotoxic, and the expression of the many different adenoviral genes must be highly

regulated.

In one embodiment, the present invention describes a method of producing

adenovirus gutless amplicon viral vector substatially reduced in the content of helper

virus, which comprises the steps of, transfecting a cell expressing EBV replicative

elements and a helper episome, comprising an adenovirus deficient genome, with an

amplicon vector expressing adenoviral pTP and E4 genes thereby causing replication of

the vector, packaging the vector into virions, and recovering the virions.

In one embodiment, the present invention describes a system for the helper virus

independent replication and packaging of adenovirus gutless vectors that comprises a cell

line and a vector, where the cell line is stably transformed by a helper episome, which

lacks the adenovirus DBP gene, and the vector comprises the adenovirus DBP and pTP genes, and where the packaged vectors are contain substantially reduced levels of helper

virus.

In one embodiment, the present invention describes a method of replicating and

packaging an adenovirus amplicon gutless vector substantially reduced in the level of

helper virus comprising the steps of amplifying and packaging the vector in a cell with a

helper virus capable of replication and providing helper function, where the helper virus

can not be packaged into virions.

In another embodiment, the present invention describes a cell line designated

VK128-3, which was deposited with the ATCC Accession No. PTA-154.

The invention will be better understood with reference to the following drawings

and detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic representation showing the construction of plasmid

pNK9-l, which is the initial skeleton of the helper episome.

Figure 2 is a schematic representation of the construction of plasmid pNK-A2

which contains the pTP gene with two internal deletions.

Figure 3 is a schematic representation of the construction of plasmid pNK-A3

which contains a portion of the adenovirus genome including the E2b genes, but does not

contain the El a, El b, and pIX genes.

Figure 4 is a schematic representation of the construction of plasmid pNK-A7 in

which portions of the adenovirus L4 and L5 genes as well as the promoter for the E2a

gene were inserted into plasmid pNK-A3. Intervening portions of the adenoviral genome found between the region of the adenovirus genome containing the E2b genes and the

region containing the L4 and L5 genes were also inserted into the plasmid to form pNK-

A7.

Figure 5 is a schematic representation of the construction of plasmid pVNK-A22-

1 in which the region of the adenoviral genome in pNK-A7 containing the pTP gene was

replaced with the homologous sequence derived from pNK-A2 containing two internal

deletions in pTP. Elements from the Epstein-Barr virus were also incorporated into the

plasmid to allow it to be maintained in human cells as an episome.

Figure 6 is a schematic representation of the construction of plasmid pAVec4-2

which contains the adenovirus cis elements, known as inverted terminal repeats or ITRs,

necessary for packaging and replication of the virus. A stuffer fragment of human

genomic DNA was also inserted into the plasmid to optimize the size of the resulting

adenovirus vector for packaging into mature virions.

Figure 7 is a schematic representation of the construction of plasmid pAVec5 in

which an expression cassette containing the bacterial lacZ gene driven by a human

promoter was inserted into pAVec4-2 to form the plasmid pAVec5.

Figure 8 is a schematic representation of the construction of plasmid pVK71

which contains the adenovirus E4 and pIX genes.

Figure 9 is a schematic representation of the construction of pAVecl2 which

contains the pTP gene and the E4 and pIX genes from pVK71.

Figure 10 is a schematic representation of the construction of the pVNK22-

1ΔDBP and pVNK24-lΔDBP helper episomes. Figure 11 is a schematic representation of the pAVecl2-l vector that is

linearized.

Figure 12 is a schematic representation of the construction of the pAVeclό and

pAVecl 7 vectors.

Figure 13 is a schematic representation of the construction of the pAVeclδ and

pAVec 19 vectors.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents, and literature references cited in this specification

are hereby incorporated by reference in their entirety.

The adenovirus vector is one of the most efficient vehicles for in vivo gene

delivery; however, the duration of adeno virus-delivered transgene expression is limited.

The expression of adenoviral genes from the vector backbone is thought to be

responsible for the rapid elimination of the adenovirus-transduced cells in

immunocompetent animals. Recently developed adenovirus vectors devoid of most or

all viral genes (so-called "gutless" or "gutted" vectors) offer a promising alternative to

previous adenovirus vectors because they have larger capacity, are less immunogenic,

and apparently more persistent. The major disadvantage of these new vectors is that they

require the presence of a helper virus for their rescue and propagation. This often leads

to significant contamination of the vectors with the helper virus. The contaminating

helper virus in the vector preparations may elicit significant host immune responses that

could impair the persistence of transgene expression in vivo. Substantial reduction in the level or complete removal of the helper virus from the final preparation is thus the most

critical step during production of gutless adenovirus vectors.

The term "substantial reduction" or "substantially reduced" refers to complete

absence of helper virus in the system or less than about 0.1% helper virus, based on the

total content of the helper- free system.

The abbreviation "pTP" is the precursor of the terminal protein.

The abbreviation of "IRES" is internal ribosomal entry sites.

The abbreviation of "TRE" is tetracycline responsive promoter.

The abbreviation of "DBP" is DNA binding protein.

The abbreviation of "Cre" is Cre recombinase.

The term "EBV replicative elements" refers to the Epstein-Barr virus EBNA-1

and oriP sequences.

The term "antibiotic resistance gene" refers to DNA that confers cellular

resistance to antibiotics such as hygromycin or neomycin, for example.

The term "stuffer DNA" refers to DNA which does not encode for any protein or

has regulatory function. One example of stuffer DNA is intron DNA. The stuffer DNA

may be of any length to achieve optimal packaging size, which may range from 27.5to

37.5 kilobases.

The term "helper episome" refers to a circular episomal copy of a modified

packaging-deficient adenovirus genome that is deleted in a number of genes. These

deleted viral genes include El, E3, E4, pTP, and DBP.

A helper-independent vector system has been designed that allows for the

production of a gutless-like adenovirus vector. The system consists of two components: a packaging cell line and a complementing virus vector. The cell line, based on 293

cells, contains a circular episomal copy of the modified packaging-deficient adenovirus

genome that is deleted in a number of genes including El, E3, E4, pTP, and DBP.

Incorporation of EBV replicative elements along with the hygromycin resistance gene

allows for stable low-copy number (2-20 copies per cell) maintenance of this helper

episome. The helper episomes are designated as pVNK-A22-l, pVNK-A24-2, pVNK-

22-1ΔDBP and pVNK-24-2ΔDBP (Figures 5, 9, and 10) . The pVNK-A22-l and

pVNK-22-lΔDBP helper episomes can be stably transformed into 293-Tet-On cells,

which are 293 cell derivatives that constitutively express the tetracycline reverse

transactivator protein (Clontech). The pVNK-A24-2 and pVNK-24-2ΔDBP helper

episomes can be stably transformed into pTP40 cells, which are 293 cell derivatives that

express both pTP and tTA. The construction of these episomes is taught in Example 1.

The second component of the system, the vector, contains all necessary cis

elements and a transgene (LacZ) expression cassette. Two designed vectors, pAVeclό

and pAVecl7, are based on the pAVecl2-l vector (Figure 12). In the pAVeclό vector

(Figure 13), the pTP expression cassette has been replaced with a TRE-DBP-IRES-pTP

expression cassette. In the pAVecl7 vector (Figure 13), the pTp expression cassette is

replaced with a TRE-DBP-IRES-Cre expression cassette. The pAVeclό vector can be

used with the 293VK15 cells and the pAVecl7 vector can be used with the pTP40 cells.

When packaging cells are transfected with this vector, both the helper episome and the

vector co-replicate using adenovirus replication machinery. This results in a high copy

number (>1000 copies per cell) of both genomes, but only the vector genome can be packed into virions. The construction of pAVeclό and pAVecl7 are taught in Example 3.

The pAVeclό and pAVecl7 vectors can be further modified to produce vectors that

restrict expression of the virus genes to the packaging cell line and prevent their expression

in the target cell. To achieve this, the TRE-DBP-IRES-pTP and TRE-DBP-IRES-Cre

expression cassettes can be modified to replacing the TRE promoter element with a bi¬

directional promoter containing a tetracycline-responsive element (Figure 14).

The gutless vector produced using the materials and methods described below was

able to transduce 293 cells, but was not able to replicate in these cells. No helper genome

could be detected either by PCR or by serial passaging of the resulting virus through 293

cells. It is believed that this system will prove useful for generation, propagation, and

titration of helper virus-free gutless adenovirus vectors for gene therapy.

The novel packaging cell line of the present invention will now be more fully

described. The design for a true packaging cell line for adenovirus amplicon vectors does

not use the prior art existing approaches, but rather is based upon a unique binary concept in

which a positive interaction occurs between an adenoviral "helper" genome and an

adenovirus amplicon vector. The helper genome is designed such that it is constantly present

inside the cell at low copy number (<20 copies per cell). Furthermore, the helper genome is

transcriptionally silent in the absence of the vector, but, in the presence of the vector, can

replicate to a high copy number (>1000 copies per cell) and express all of the adenoviral

proteins required for the replication of the vector genome and its packaging into infectious

virus particles. Packaging of the helper genome is prevented through two independent means. The

first is by deletion of its packaging sequence. The second is through the incorporation of

additional sequences into the helper genome so that its size is greater than the packaging

capacity of the adenovirus particle. Both integrative and episomal strategies for

maintenance of the helper genome have been exploited in different variants of this system.

In the episomal approach, the helper genome, derived from an otherwise infectious

adenovirus plasmid lacking the adenovirus pTP gene, which is absolutely essential for

adenovirus replication and hence transcription of adenoviral genes, is maintained

episomally. Mechanisms derived from Epstein-Barr (EBV) virus (EBNA-1 protein

expression and the oriP sequence), allow the episome to be maintained at low copy number

(<20 copies per cell), non-toxic copy numbers in 293 cells in the absence of the vector when

it is not needed for vector production. When an adenovirus amplicon vector that expresses

pTP is introduced into the cell, expression of pTP from the vector will trigger replication of

the helper genome and expression of adenoviral genes, which in turn will permit replication

and packaging of the vector genome into infectious virus particles. The inclusion of a

dominant selective marker, such as the hygromycin resistance gene (Invitrogen Corporation,

Carlsbad CA) or neomycin resistance gene (Invitrogen Corporation, Carlsbad Ca) will

ensure maintenance of the helper genome through positive selection.

In the integrative method, the helper genome is integrated into the genome of the

host cell. The integration method improves the long-term stability of the system and allows

for selection of clones for integration of low copy numbers into chromosomal locations that are relatively quiescent. The fragment that can be integrated into the packaging cell line

would be identical to the helper genome used in the episomal method described above,

except that the helper episome would contain an bicistronic expression cassette containing

tTA-IRES-pTP driven by a RSV promoter. The major difference between the episomal and

integration methods is that in this system the helper genome does not replicate

autonomously but is maintained as part of the cell and is released and replicated only in the

presence of the vector.

The adenovirus vector that is produced in this packaging cell line consists of a

plasmid containing all of the cis elements necessary for replication and packaging (the

adenoviral ITRs and the packaging signal), an expression cassette containing the therapeutic

gene, inert "stuffer DNA" sequences to achieve a vector of appropriate size for optimal

packaging into adenovirus particles, and as few genes, viral or otherwise, as are necessary

for the induction of amplification and expression of the helper genome. Optimal packaging

size ranges from 27.5 to 37.5 kilobases. The only gene that is absolutely essential in the

vector in its initial embodiment is the pTP gene.

This novel system has three chief advantages over existing systems for the

production of high-capacity recombinant adenovirus vectors. The first is that the vectors

produced in this system contain a substantially reduced level of contaminating helper virus

compared to prior methods in the art. As significant levels of this helper virus could provoke

severe immunological or other responses in the host that could jeopardize both the efficacy

and safety of the adenovirus vector. The decreased helper virus levels are a significant benefit of the system of the present invention. Most (if not all) current production systems

lack this feature. A second advantage is that this production system can be easily expanded,

with a minimum of human labor, to quickly, easily and cheaply produce vectors on a scale

required for human clinical trials. This is certainly not the case with systems most

commonly employed (Parks et al., 1996; Morsy et al., 1998; Schiedner et al, 1998), which

requires highly time-consuming serial passaging of the vector in the presence of empirically-

determined amounts of helper virus. A third advantage of the system of the present

invention is that, because the cell line can fully support replication of the virus vector,

traditional methods of plaque formation can be used to accurately quantify the amount of

infectious virus that is present in a given production batch. Current vector production

systems can only estimate indirectly the amount of virus DNA that is in a given preparation,

but cannot accurately gauge the proportion of this vector DNA that is present in fully-

infectious virus particles.

The majority of human gene therapy trials currently in progress utilize recombinant

El -deleted adenovirus vectors. While some of the features of the adenovirus amplicon

vector system may not be necessary for some of these applications (long-term persistence

may not be an absolute requirement for certain trials where cancer or cardiovascular diseases

are the targets), other features of this vector system such as its increased capacity may result

in its increased use in these types of trials. Furthermore, in applications where highly-

efficient and persistent gene transfer is required, e.g. the treatment of genetic diseases,

diabetes or other acquired diseases, this vector system is currently without equal. The system disclosed herein has numerous uses, such as to prevent heart attacks, treat inoperable

cancers, reduce cholesterol, and reverse atherosclerosis among other possible applications.

The genes necessary for the treatment of most of these conditions are known, and

preliminary data are available showing that transient correction of these phenotypes can be

achieved through the delivery of these genes by transient, El-deleted adenovirus vectors. It

is expected that long-term corrections of these disease phenotypes will be achieved when

these same genes are delivered by adenovirus amplicon vectors, which have now been

shown to stably persist for periods exceeding one year in immunocompetent non-human

primates.

Cell line VK128-3 has been deposited with the American Type Culture Collection

(ATCC) Manassas, VA., on May 28, 1999 under ATCC Accession No. PTA-154.

The present invention is described below in specific examples which are intended to

further describe the invention without limiting the scope thereof.

In general, nucleic acid manipulations used in practicing the present invention

employ methods that are well known in the art, as disclosed in, e.g., Molecular Cloning, A

Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor) and

Current Protocols in Molecular Biology (Eds. Ausubel, Brent, Kingston, More, Feidman,

Smith and Stuhl, Greene Publ. Assoc, Wiley-Interscience, NY, NY, 1997). Preferred

disclosed vectors, restriction enzymes, ligases, nucleases, and the like are commercially

available from numerous sources, such as Sigma Chemical Co. (St. Louis, MO), New

England Biolabs, Inc. (Beverly, MA) and New England Nuclear (Boston, MA). Example 1 : Construction of the helper episome.

The initial steps in the construction of the helper episome involved the construction

of plasmid pNK9-l as shown in Figure 1. The commercially-available plasmid pREP7

(Invitrogen, Inc.) was opened between the Rous sarcoma virus LTR promoter and the SV40

polyadenylation signal by Nhel/Xhol digestion, and a humanized form of the green

fluorescent protein (EGFP) gene (Clontech Laboratories Inc., Palo Alto CA) was inserted

therein. This plasmid (pREP-EGFP) was then linearized by Xbal digestion and a fragment

of DNA containing functional fused inverted terminal repeats (ITRs) but NOT containing

the packaging signal sequence, obtained by Bsgl/SgrAI digestion of the plasmid pFG140

freely acquired from the laboratory of Dr. Frank Graham at McMaster University and

commercially available from Microbix Biosystems, Inc., Toronto, ON, was inserted to create

pNK9-l.

For the helper episome to function as intended, it must contain a significant portion

of the adenoviral genome in a form that cannot replicate as an adenovirus in the absence of

the complementing adenoviral amplicon vector. To prevent replication of the episome as an

adenovirus, the gene encoding the precursor of the terminal protein (pTP) was altered by the

introduction of two deletions, using the strategy shown in Figure 2. A BsaBI/Xbal fragment

of the Ad type 5 genome (ATCC# VR-5), containing the pTP gene, was subcloned into the

commercial plasmid pLithmus29 (New England Biolabs). The resulting plasmid, pNK-Al,

was digested with Bglll, and Bglll-Sall adaptors were added followed by Xbal digestion and the addition of an Xbal-Sall adapter. To this plasmid, a 379 bp Sail fragment containing an

internal portion of the pTP gene was added to create plasmid pNK-A2, in which the pTP

gene now had two internal deletions.

The reconstruction of the remainder of the adenovirus genome contained in the

helper episome is shown in Firgures 3 and 4. A portion of the adenovirus genome

containing the adenovirus E2b genes, but deleted of Ela, Elb and pIX was generated (Figure

3) by isolating a BsaBI restriction fragment from wild-type Ad 5 (ATCC# VR-5) and adding

to this fragment a synthetic oligonucleotide designed to restore the missing polyadenylation

sequence of the E2b gene. This fragment was then inserted into the commercially-available

plasmid pNEB193 (New England Biolabs, Inc., Beverly MA) to create pNK-A3.

PNK-A3 was further modified through the addition of a fragment of the adenoviral

genome containing portions of the L4 and L5 genes as well as promoter of the E2a gene, but

deleted of the adenoviral E3 and E4 regions. This fragment was isolated from the

commercially-available plasmid pBHGlO by Mfel digestion. The result of this insertion is

the plasmid pNK-A5 (Figure 4). The intervening portions of the adenoviral genome

contained between the two adenoviral genome fragments now present in pNK-A5 was

obtained by digesting the Ad 5 genome (ATCC# VR-5) with Bstl 107 and Spel, and this

fragment was then inserted into the same sites contained in pNK-A5 to create pNK-A7.

While now containing a large fragment of the adenoviral genome, several additional

modifications of the adenoviral sequences contained in pNK-A7 were required to be made in

order to obtain the desired helper episome. These changes are shown in Figure 5. First, the region of the adenoviral genome in pNK-A7 containing the pTP gene was replaced with

homologous sequences derived from pNK-A2. The swapping of these fragments introduced

the two deletions previously engineered into the pTP gene contained in pNK-A2 into the

pTP gene contained in pNK-A7. The resulting plasmid was designated pNK-A8-ΔpTP. To

permit this plasmid to replicate either to low copy numbers as an EBV-based episome or to

high copy numbers using adenovirus replication machinery, pNK-A8-ΔpTP was fused with

pNK9-l (Figure 1) to form pVNK-A22-l. This plasmid now contains all of the elements

derived from Epstein-Barr virus (e.g. EBNA-1 expression cassette and oriP) to allow it to be

maintained in human cells as an episome, and also all of the information necessary for

replication as an adenovirus when transcomplemented by the products of the adenovirus El,

pTP and E4 genes.

For the production of pVNK22-lΔDBP and pVNK24-2ΔDBP episomes, an Ascl

fragment corresponding to nucleotides 15627 to 25292 in the Ad5 genome was excised. This

fragment was subcloned into the unique Hindlll site of pZero2 (Invitrogen) using an

Ascl/Hindlll adaptor consisting of the following two oligonucleotides: 5'-

AGCTGGACCTAGGAGG-3' and 5'-CGCGCCTCCTAGGTCC-3'. This procedure

generated the pE2Al plasmid. The tTA encoding sequences between unique Xbal and

BamHI sites from plasmid pUHD15-l (Clontech) was then excised and the BamHI site was

blunted by Klenow reaction. An Xbal/Aatll adaptor consisting of the following two

oligonucleotides: 5'-CTAGAGGGGGGACGT-3' and 5'-CCCCCCT-3' was constructed and

ligated to the Xbal site. The pE2Al plasmid was partially digested with Dral and completely digest with Aatll to remove nucleotides 22445 through 23871 in the Ad5 genome, to remove

the DBP genes in the Ad5 genome. The tTA-encoding fragment, described above, was

subcloned into the linearized pE2Al plasmid to generate the pE2A2 plasmid. In this

plasmid, the E2 region from the Ad5 genome had been modified so that the E2 promoter led

to transcription of the tTA gene. The protein expressed from this gene also contains an

additional 24 amino acids from the DBP protein at its N-terminus. pE2A2 was digested with

Ascl to release the DBP-deleted Ad5 fragment. This fragment then was substituted for the

corresponding fragment from pVNK22-l and pVNK24-2 to create pVNK22-lΔDBP and

pVNK24-2ΔDBP (see Figure 10).

pVNK22-lΔDBP and pVNK24-2ΔDBP are identical except for that in the pVNK24-

2ΔDBP there is a strong transcriptional stop signal flanked by two loxP sites that was

inserted into an Xbal site that had been generated during the course of the deletion of pTP

from the Ad5 genome, as previously described above. The transcriptional stop signal

consisted of three polyadenylation signals aligned in series, and ensured complete inhibition

of transcription from the Ad5 major late promoter.

Example 2: Method for the stable transformation of the helper episome into cultured

mammalian 293 cells.

Once constructed, pVNK-A22-l was introduced into commercially-available 293

cells (Microbix Biosystems, Inc., Toronto, ON) by standard calcium phosphate transfection procedures. Because this episome contains a hygromycin resistance gene, stable

transformants in which the episome is being maintained by replication can be positively

selected on the basis of their growth in the presence of the antibiotic hygromycin. Moreover,

because the episome also expresses EGFP, the presence of green fluorescence in the stably

transformed cells further confirms the presence of the episome.

Example 3: Method for the construction of an adenovirus amplicon vector that can be

rescued, propagated and titered using the novel adenovirus packaging cell line.

The adenovirus cis elements necessary for virus replication and packaging are

located at the termini of the adenoviral genome. These regions are referred to as inverted

terminal repeats or ITRs. Plasmid pNC-1.2, which was constructed by inserting into

pNEB193 (New England Biolabs, Inc., Beverly MA) two copies of the left ITR and

packaging signal amplified from human Ad 5 (ATCC# VR-5) in a head-to-head

configuration, was digested with EcoRl, and a 19kb piece of DNA was inserted to produce

the plasmid pAVec4-2 (Figure 6). The DNA can be and was of no specific source or

sequence composition. This insertion was performed to optimize the size of the resulting

adenovirus vector for packaging into mature virions. PAVec4-2 was then digested with Sail

and an expression cassette consisting of the bacterial lacZ gene driven by the human

elongation factor lα (EFla) promoter, excised from plasmid pAVec2, which was

constructed by inserting the EF- la promoter (Invitrogen Corporation, Carlsbad CA), the lacZ gene (Invitrogen Corporation, Carlsbad CA) and the SV40 polyadenylation sequence

(Invitrogen Corporation, Carlsbad CA) into pNEB 193 (New England Biolabs, Inc., Beverly

MA). This produced plasmid pAVec5 (Figure 7).

In order for this vector to transcomplement the helper episome pVNK-A22-l, it must

express the adenovirus pIX, E4 and pTP genes. These genes were introduced as shown in

Figures 8 and 9. To recreate the adenovirus E4 gene, the E4 region of Ad 5 (ATCC# VR-5)

as amplified by PCR, using the primer sequences shown in Figure 8, was blunt-end cloned

into the EcoRV site of the commercially-available vector pZERO-2 (Invitrogen) to create

pVK70-l (Figure 8). To this plasmid was added a 613 bp fragment containing the

adenovirus pIX gene derived in part from the commercially-available plasmid pdEl splB

(Microbix, Canada), to create pVK71 (Figure 8). This plasmid served as a source of the E4

and pIX genes to be introduced into the adenoviral amplicon vector.

To introduce the pTP gene, pAVec5 was digested with Nhel and an Spel fragment,

containing the pTP gene excised from pL-TK-pTP, was inserted into the site (Figure 9). The

resulting plasmid, pAVec9-2, was digested with Avrll and the Spel/Xbal fragment of

pVK71, containing the E4 and pIX genes, was inserted to create the final vector construct,

pAVecl2.

To produce the pAVeclόvector, the 1.6 kb Ad5 DBP gene was cloned by PCR, using

TGCCTATAGGAGAAGGAAATGGCCAGTCGGG and

TCTCGGGTGATTATTTACCCCCACCC primers.. The blunt-ended amplified product was

subcloned into a unique EcoRV site contained within the multiple cloning region of pZero2. The fidelity of the DBP gene sequence in the resulting plasmid pZero-DBP was confirmed

by sequence analysis of this plasmid. The 1.6 kb Spel-Xbal fragment pZero-DBP containing

the DBP gene was then inserted into the Nhel site an pIRES (Clontech) plasmid in front of

the IRES sequence to generate plasmid pDBP/IRES. The pTP coding sequence from

pAVecl2-l was subcloned into the Xbal site downstream of the IRES sequence in

pDBP/IRES to generate pDBP/IRES/pTP. The CMV promter contained in pDBP/IRES/pTP

was excised by digestion with Bglll and EcoRl and replaced by a tet-inducible promoter

obtained by digestion of pTRE (Clontech) with Xhol and EcoRl. This fragment was

modified by addition of a Bglll-Xhol adapter, consisting of the following two

oligonucleotides: 5'-TCGAGGGATCGATGGA-3' and 5'-GATCTCCATCGATCCC-3'.

This generated plasmid pTRE-DBP/IRES/pTP. The expression cassette consisting of the tet-

inducible promoter, the DBP gene, the IRES sequence, the pTP gene and the SV40

polyadenylation signal was excised from pTRE-DBP/IRES/pTP by digestion with Clal. The

isolated fragment was subcloned into pAVecl2-l from which the TK-pTP expression

cassette had been removed by digestion with BstBI and Pmel, and the Pmel site was

transformed into BstBI ^'by the addition of a BstB linker 5'-CGTTCGAACG3'. This insertion

generated plasmid pAVeclό, which could be rescued as an adenovirus vector if transfected

into 293 cells stably transformed with pVNK22-lΔDBP.

The plasmid pAVecl7, which could be rescued as an adenovirus vector if transfected

into 293 cells stably transformed with pVNK24-2ΔDBP, was constructed in essentially the

same manner as the pAveclό vector with the following exceptions: instead of inserting the pTP gene downstream of the IRES sequence, a commercially-obtained Cre recombinase

gene was inserted into a unique cloning site located at this position in pDBP/IRES.

Example 4: DNA transfection. rescue, propagation, and titration of the gutless adenoviral

vector.

Mammalian VK- 128-3 cells (293 cells stably transformed with the helper episome

pVNK-A22-l) were grown at 37 °C in 60 mm dishes to approximately 70-80% confluence.

The growth media (90 % MEM + 10% Fetal Bovine Serum + 1%

Penicillin/Streptomycin/Fungizone; all obtained from Gibco) was removed and the cells

were washed with phosphate-buffered saline (PBS) or fresh serum-free MEM (Gibco).

Purified DNA of the pAVecl2 plasmid was linearized by digestion with the restriction

enzyme Swal and purified using the QIAquick Gel Extraction Kit (Qiagen Inc.). This

digestion released the adenoviral ITRs, making them functional for initiating adenovirus

replication. The purified plasmid DNA was transfected into VK- 128-3 cells using a standard

calcium phosphate procedure. At 24 hrs after transfection, the transfection media was

removed and replaced by fresh media. At 7 days after the infection/transfection procedure,

the cells were harvested and a crude lysate of the transfected cells was prepared.

Subconfluent (80%) VK- 128-3 cells grown in 60-mm dishes were infected by 0.1 ml

of the crude lysate containing the adenoviral vector rescued from the cells transfected as

described immediately above. At 5 days postinfection, a crude lysate of the infected cells was made. These lysates were stored at -20 or -70 °C. The multiplicity of infection (moi) of

the vector was less than 1 "blue-forming unit" (bfu)/cell at the initial stages of vector

propagation, but maximum vector yields were obtained when cells were infected with the

vector at mois ranging from 2-5.

The crude lysates from the propagation procedure described above were clarified by

centrifugation at 700 rpm for 10 min at 4 °C and filtered through 0.8 micron filter units. 0.1

ml of the crude extract was used for infection of 293 cells. As the vector expressed E. coli

β-galactosidase, cells infected by serial dilutions of the vector could be identified by staining

with 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal) dissolved in PBS (0.5

mg/ml). In this procedure, the cells were fixed by a 5 min exposure to 0. 5% glutaraldehyde

diluted in PBS at 20 hrs after infection, rinsed several times with PBS, and then stained by

exposure to the X-gal solution for 5 hrs at 37°C. The number of positively-stained cells

multiplied by the dilution factor corresponded to the infectious titer of the recombinant

adenoviral vector produced by this method.

Reference List

Akli, S., Caillaud, C, Vigne, E., Stratford-Perricaudet, L. D., Poenaru, L., Perricaudet, M.,

Kahn, A., and Peschanski, M. R. (1993). Transfer of a foreign gene into the brain using

adenovirus vectors [see comments]. Nat. Genet. 3, 224-228.

Alemany, R., Dai, Y., Lou, Y. C, Sethi, E., Prokopenko, E., Josephs, S. F., and Zhang, W.

W. (1997). Complementation of helper-dependent adenoviral vectors: size effects and titer

fluctuations. J. Virol.Methods 68, 147-159.

Amalfitano, A., Hauser, M. A., Hu, H., Serra, D., Begy, C. R., and Chamberlain, J. S.

(1998). Production and characterization of improved adenovirus vectors with the El, E2b,

and E3 genes deleted. J. Virol. 72, 926-33 Other Formats.

Armentano, D., Sookdeo, C. C, Hehir, K. M., Gregory, R. J., St.George, J. A., Prince, G. A.,

Wadsworth, S. C, and Smith, A. E. (1995). Characterization of an adenovirus gene transfer

vector containing an E4 deletion. Hum.Gene Ther. 6, 1343-1353.

Bajocchi, G., Feldman, S. H., Crystal, R. G., and Mastrangeli, A. (1993). Direct in vivo gene

transfer to ependymal cells in the central nervous system using recombinant adenovirus

vectors [see comments]. Nat. Genet. 3, 229-234.

Barr, D., Tubb, J., Ferguson, D., Scaria, A., Lieber, A., Wilson, C, Perkins, J., and Kay, M.

A. (1995). Strain related variations in adenovirally mediated transgene expression from mouse hepatocytes in vivo: comparisons between immunocompetent and immunodeficient

inbred strains. Gene Ther. 2, 151-155.

Chen, H. H., Mack, L. M., Choi, S. Y., Ontell, M., Kochanek, S., and Clemens, P. R. (1999).

DNA from both high-capacity and first-generation adenoviral vectors remains intact in

skeletal muscle [In Process Citation]. Hum.Gene Ther. 10, 365-373.

Chen, H. H., Mack, L. M., Kelly, R., Ontell, M., Kochanek, S., and Clemens, P. R. (1997).

Persistence in muscle of an adenoviral vector that lacks all viral genes.

Proc.Natl.Acad.Sci. U.S.A. 94, 1645-1650.

Davidson, B. L., Allen, E. D., Kozarsky, K. F., Wilson, J. M., and Roessler, B. J. (1993). A

model system for in vivo gene transfer into the central nervous system using an adenoviral

vector [see comments]. Nat. Genet. 3, 219-223.

Engelhardt, J. F., Litzky, L., and Wilson, J. M. (1994b). Prolonged transgene expression in

cotton rat lung with recombinant adenoviruses defective in E2a. Hum.Gene Ther. 5, 1217-

1229.

Engelhardt, J. F., Ye, X., Doranz, B., and Wilson, J. M. (1994a). Ablation of E2A in

recombinant adenoviruses improves transgene persistence and decreases inflammatory

response in mouse liver. Proc.Natl.Acad.Sci. U.S.A. 91, 6196-6200.

Fang, B., Eisensmith, R. C, Li, X. H., Finegold, M. J., Shedlovsky, A., Dove, W., and Woo,

S. L. (1994). Gene therapy for phenylketonuria: phenotypic correction in a genetically deficient mouse model by adeno virus-mediated hepatic gene transfer. Gene Ther. 1, 247-

254.

Fang, B., Eisensmith, R. C, Wang, H., Kay, M. A., Cross, R. E., Landen, C. N., Gordon, G.,

Bellinger, D. A., Read, M. S., and Hu, P. C. (1995). Gene therapy for hemophilia B: host

immunosuppression prolongs the therapeutic effect of adenovirus-mediated factor IX

expression. Hum.Gene Ther. 6, 1039-1044.

Fisher, K. J., Choi, H., Burda, J., Chen, S. J., and Wilson, J. M. (1996). Recombinant

adenovirus deleted of all viral genes for gene therapy of cystic fibrosis. Virology 217, 11-22.

Gao, G. P., Yang, Y., and Wilson, J. M. (1996). Biology of adenovirus vectors with El and

E4 deletions for liver- directed gene therapy. J. Virol. 70, 8934-8943.

Gilardi, P., Courtney, M., Pavirani, A., and Perricaudet, M. (1990). Expression of human

alpha 1-antitrypsin using a recombinant adenovirus vector. FEBS Lett. 267, 60-62.

Gorziglia, M. I., Kadan, M. J., Yei, S., Lim, J., Lee, G. M., Luthra, R., and Trapnell, B. C.

(1996). Elimination of both El and E2 from adenovirus vectors further improves prospects

for in vivo human gene therapy. J. Virol. 70, 4173-4178.

Graham, F. L., Smiley, J., Russell, W. C, and Nairn, R. (1977). Characteristics of a human

cell line transformed by DNA from human adenovirus type 5. J.Gen. Virol. 36, 59-74. Herz, J. and Gerard, R. D. (1993). Adenovirus-mediated transfer of low density lipoprotein

receptor gene acutely accelerates cholesterol clearance in normal mice.

Proc.Natl.Acad.Sci. U.S.A. 90, 2812-2816.

Hu, H., Serra, D., and Amalfitano, A. (1999). Persistence of an [E1-, polymerase-]

adenovirus vector despite transduction of a neoantigen into immune-competent mice [In

Process Citation]. Hum.Gene Ther. 10, 355-364.

Ishibashi, S., Brown, M. S., Goldstein, J. L., Gerard, R. D., Hammer, R. E., and Herz, J.

(1993). Hypercholesterolemia in low density lipoprotein receptor knockout mice and its

reversal by adenovirus-mediated gene delivery [see comments]. J.Clin.Invest. 92, 883-893.

Jaffe, H. A., Danel, C, Longenecker, G., Metzger, M., Setoguchi, Y., Rosenfeld, M. A.,

Gant, T. W., Thorgeirsson, S. S., Stratford-Perricaudet, L. D., and Perricaudet, M. (1992).

Adenovirus-mediated in vivo gene transfer and expression in normal rat liver. Nat. Genet. 1,

372-378.

Kay, M. A. and Woo, S. L. (1994). Gene therapy for metabolic disorders. Trends. Genet. 10,

253-257.

Kochanek, S., Clemens, P. R., Mitani, K., Chen, H. H., Chan, S., and Caskey, C. T. (1996).

A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA

independently expressing both full- length dystrophin and beta-galactosidase.

Proc.Natl.Acad.Sci. U.S.A. 93, 5731-5736. Kolls, J. K., Lei, D., Odom, G., Nelson, S., Summer, W. R., Gerber, M. A., and Shellito, J.

E. (1996). Use of transient CD4 lymphocyte depletion to prolong transgene expression of

El -deleted adenoviral vectors. Hum.Gene Ther. 1, 489-497.

Krougliak, V. and Graham, F. L. (1995). Development of cell lines capable of

complementing El, E4, and protein IX defective adenovirus type 5 mutants. Hum.Gene

Ther. 6, 1575-1586.

Langer, S. J. and Schaack, J. (1996). 293 cell lines that inducibly express high levels of

adenovirus type 5 precursor terminal protein. Virology 221, 172-179.

Le Gal La Salle, G., Robert, J. J., Berrard, S., Ridoux, V., Stratford-Perricaudet, L. D.,

Perricaudet, M., and Mallet, J. (1993). An adenovirus vector for gene transfer into neurons

and glia in the brain. Science 259, 988-990.

Lee, M. G., Kremer, E. J., and Perricaudet, M. (1995). Adenoviral vectors. Moi. Cell

Biol.Hum.Dis.Ser. 5:20-32Other Formats, 20-32Other Formats.

Levrero, M., Barban, V., Manteca, S., Ballay, A., Balsamo, C, Avantaggiati, M. L., Natoli,

G., Skellekens, H., Tiollais, P., and Perricaudet, M. (1991). Defective and nondefective

adenovirus vectors for expressing foreign genes in vitro and in vivo. Gene 101, 195-202.

Li, Q., Kay, M. A., Finegold, M., Stratford-Perricaudet, L. D., and Woo, S. L. (1993).

Assessment of recombinant adenoviral vectors for hepatic gene therapy. Hum.Gene Ther. 4,

403-409. Li, T. and Davidson, B. L. (1995). Phenotype correction in retinal pigment epithelium in

murine mucopolysaccharidosis VII by adenovirus-mediated gene transfer.

Proc.Natl.Acad.Sci. U.S.A. 92, 7700-7704.

Lieber, A., He, C. Y., Kirillova, I., and Kay, M. A. (1996). Recombinant adenoviruses with

large deletions generated by Cre- mediated excision exhibit different biological properties

compared with first- generation vectors in vitro and in vivo. J. Virol. 70, 8944-8960.

Mitani, K., Graham, F. L., Caskey, C. T., and Kochanek, S. (1995). Rescue, propagation,

and partial purification of a helper virus- dependent adenovirus vector.

Proc.Natl.Acad.Sci. U.S.A. 92, 3854-3858.

Morsy, M. A., Gu, M., Motzel, S., Zhao, J., Lin, j., Su, Q., Allen, H., Franlin, L., Parks, R.

j., Graham, F. L., Kochanek, S., Bett, A. J., and Caskey, C. T. (1998). An adenoviral vector

deleted for all viral coding sequences results in enhanced safety and extended expression of

a leptin transgene. Proc.Natl.Acad.Sci. U.S.A. 95, 7866-7871.

Parks, R. J., Chen, L., Anton, M., Sankar, U., Rudnicki, M. A., and Graham, F. L. (1996). A

helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated

excision of the viral packaging signal. Proc.Natl.Acad.Sci. U.S.A. 93, 13565-13570.

Parks, R. J. and Graham, F. L. (1997). A helper-dependent system for adenovirus vector

production helps define a lower limit for efficient DNA packaging. J. Virol. 71, 3293-3298. Quantin, B., Perricaudet, L. D., Tajbakhsh, S., and Mandel, J. L. (1992). Adenovirus as an

expression vector in muscle cells in vivo. Proc.Natl.Acad.Sci. U.S.A. 89, 2581-2584.

Rosenfeld, M. A., Siegfried, W., Yoshimura, K., Yoneyama, K., Fukayama, M., Stier, L. E.,

Paakko, P. K., Gilardi, P., Stratford-Perricaudet, L. D., and Perricaudet, M. (1991).

Adenovirus-mediated transfer of a recombinant alpha 1- antitrypsin gene to the lung

epithelium in vivo [see comments]. Science 252, 431-434.

Rosenfeld, M. A., Yoshimura, K., Trapnell, B. C, Yoneyama, K., Rosenthal, E. R.,

Dalemans, W., Fukayama, M., Bargon, J., Stier, L. E., and Stratford-Perricaudet, L. (1992).

In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to

the airway epithelium. Cell 68, 143-155.

Schaack, J., Guo, X., Ho, W. Y., Karlok, M., Chen, C, and Ornelles, D. (1995). Adenovirus

type 5 precursor terminal protein-expressing 293 and HeLa cell lines. J. Virol. 69, 4079-

4085.

Schiedner, G., Morral, N., Parks, R. J., Wu, Y., Koopmans, S. C, Langston, C, Graham, F.

L., Beaudet, A. L., and Kochanek, S. (1998). Genomic DNA transfer with a high-capacity

adenovirus vector results in improved in vivo gene expression and decreased toxicity

[published erratum appears in Nat Genet 1998 Mar;18(3): 298]. Nat.Genet. 18, 180-183. Stratford-Perricaudet, L. D., Briand, P., and Perricaudet, M. (1992). Feasibility of

adenovirus-mediated gene transfer in vivo. Bone Marrow Transplant. 9 Suppl 1:151-2, 151-152.

Stratford-Perricaudet, L. D., Levrero, M., Chasse, J. F., Perricaudet, M., and Briand, P.

(1990). Evaluation of the transfer and expression in mice of an enzyme- encoding gene using

a human adenovirus vector. Hum.Gene Ther. 1, 241-256.

Vincent, N., Ragot, T., Gilgenkrantz, H., Couton, D., Chafey, P., Gregoire, A., Briand, P.,

Kaplan, J. C, Kahn, A., and Perricaudet, M. (1993). Long-term correction of mouse

dystrophic degeneration by adenovirus- mediated transfer of a minidystrophin gene.

Nat.Genet. 5, 130-134.

Wang, Q., Jia, X. C, and Finer, M. H. (1995). A packaging cell line for propagation of

recombinant adenovirus vectors containing two lethal gene-region deletions. Gene Ther. 2,

775-783.

Yang, Y., Ertl, H. C, and Wilson, J. M. (1994b). MHC class I-restricted cytotoxic T

lymphocytes to viral antigens destroy hepatocytes in mice infected with El -deleted

recombinant adenoviruses. Immunity. 1, 433-442.

Yang, Y., Greenough, K., and Wilson, J. M. (1996). Transient immune blockade prevents

formation of neutralizing antibody to recombinant adenovirus and allows repeated gene

transfer to mouse liver. Gene Ther. 3, 412-420. Yang, Y., Janich, S., Cohn, J. A., and Wilson, J. M. (1993). The common variant of cystic

fibrosis transmembrane conductance regulator is recognized by hsp70 and degraded in a pre-

Golgi nonlysosomal compartment. Proc.Natl.Acad.Sci.U.S.A. 90, 9480-9484.

Yang, Y., Nunes, F. A., Berencsi, K., Furth, E. E., Gonczol, E., and Wilson, J. M. (1994a).

Cellular immunity to viral antigens limits El -deleted adenoviruses for gene therapy.

Proc.Natl.Acad.Sci. U.S.A. 91, 4407-4411.

Yang, Y., Nunes, F. A., Berencsi, K., Gonczol, E., Engelhardt, J. F., and Wilson, J. M.

(1994c). Inactivation of E2a in recombinant adenoviruses improves the prospect for gene

therapy in cystic fibrosis. Nat.Genet. 7, 362-369.

Yang, Y. and Wilson, J. M. (1995). Clearance of adeno virus-infected hepatocytes by MHC

class I- restricted CD4+ CTLs in vivo. J.Immunol. 155, 2564-2570.

Yeh, P., Dedieu, J. F., Orsini, C, Vigne, E., Denefle, P., and Perricaudet, M. (1996).

Efficient dual transcomplementation of adenovirus El and E4 regions from a 293-derived

cell line expressing a minimal E4 functional unit. J. Virol. 70, 559-565.

Zhou, H., O'Neal, W., Morral, N., and Beaudet, A. L. (1996). Development of a

complementing cell line and a system for construction of adenovirus vectors with El and

E2a deleted. J. Virol. 70, 7030-7038.

Claims

WHAT IS CLAIMED IS:

1. A method of producing adenovirus gutless amplicon viral vector substatially reduced in the content of helper virus, comprising the steps of,

transfecting a cell expressing EBV replicative elements and a helper episome, comprising an adenovirus deficient genome, with an amplicon vector expressing adenoviral pTP and DBP genes thereby causing replication of said vector,

packaging of said vector into virions,

and recovering said virions.

2. The method of claim 1, wherein the EBV replicative elements are EBNA-1 and oriP sequences.

3. The method of claim 1, wherein the helper episome is deficient in at least one adenovirus gene selected from the group consisting of El, E3, E4, pTP, and DBP

4. The method of claim 1, wherein the episome further comprises an antibiotic resistance gene.

5. A system for the helper virus independent replication and packaging of adenovirus gutless vectors comprising a cell line and a vector, wherein said cell line is stably transformed by a helper episome comprising an adenovirus deficient genome, lacking the adenovirus DBP gene, and said vector comprises the adenovirus DBP and pTP genes, wherein the packaged vectors are contain substantially reduced levels of helper virus.

6. The system of claim 5, wherein said helper episome has the structure of pAveclό.

7. The system of claim 5, wherein said helper episome has the structure of pAvecl7.

8. The system of claim 5, wherein said helper episome has the structure of pAveclδ.

9. The system of claim 5, wherein said helper episome has the structure of pAvecl9.

10. The system of claim 5, wherein said vector further comprises TRE and IRES genes.

11. The system of claim 5, wherein said vector further comprises TRE and Cre genes.

12. The system of claim 5, wherein said cell line is in a 293 cell line.

13. The system of claim 5, wherein said vector has the structure of pVNK22-

1ΔDBP.

14. The system of claim 5, wherein said vector has the structure of pVNK24- 2ΔDBP.

15. A cell line designated VK128-3, deposited with the ATCC on May 28, 1999 and having the Accession No. PTA-154.

16. A method of replicating and packaging an adenovirus amplicon gutless vector substantially reduced in the level of helper virus comprising the steps of amplifying and packaging said vector in a cell with a helper virus capable of replication and providing helper function, wherein said helper virus can not be packaged into virions.

17. The method of claim 16, wherein the helper virus lacks the packaging sequence.

18. The method of claim 16, wherein the helper virus is larger that the optimal packaging size.

M \2420\OF615\NYC0201 WPD