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WO1996017947A9 - Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants - Google Patents

Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants

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Publication number
WO1996017947A9
WO1996017947A9 PCT/US1995/015892 US9515892W WO9617947A9 WO 1996017947 A9 WO1996017947 A9 WO 1996017947A9 US 9515892 W US9515892 W US 9515892W WO 9617947 A9 WO9617947 A9 WO 9617947A9
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WIPO (PCT)
Prior art keywords
aav
cell
packaging
vector
cells
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PCT/US1995/015892
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English (en)
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WO1996017947A1 (fr
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Priority to AU45963/96A priority Critical patent/AU707866B2/en
Priority to EP95944069A priority patent/EP0796339A1/fr
Priority to JP8517764A priority patent/JPH10511264A/ja
Publication of WO1996017947A1 publication Critical patent/WO1996017947A1/fr
Publication of WO1996017947A9 publication Critical patent/WO1996017947A9/fr
Priority to US09/731,941 priority patent/US6924128B2/en
Priority to US11/071,401 priority patent/US20050148076A1/en

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Definitions

  • This invention relates to gene therapy, and more specifically to materials and methods used for the generation of high titers of recombinant AAV vectors for use in gene therapy procedures.
  • AAV vectors may have utility for gene therapy but heretofore a significant obstacle has been the inability to generate sufficient quantities of such recombinant vectors in amounts that would be clinically useful for human gene therapy application. This is a particular problem for in vivo applications such as direct delivery to the lung.
  • Adeno-associated virus (AAV) vectors are among a small number of recombinant virus vector systems which have been shown to have utility as in vivo gene transfer agents (reviewed in Carter, 1992, Current Opinion in Biotechnology, 3:533-539; Muzcyzka, 1992, Curr. Top. Microbiol. Immunol. 158:97-129) and thus are potentially of great importance for human gene therapy.
  • AAV vectors are capable of high- frequency stable DNA integration and expression in a variety of cells including cystic fibrosis (CF) bronchial and nasal epithelial cells (see, e.g., Flotte et al., 1992a, Am. J. Respir. Cell Mol. Biol.
  • AAV may not require active cell division for stable expression which would be a clear advantage over retroviruses, especially in tissue such as the human airway epithelium where most cells are terminally differentiated and non-dividing.
  • AAV is a defective parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus (see Fig. l) .
  • a co-infecting helper virus see Fig. l.
  • co-infecting viruses that provide helper functions for AAV growth and replication are adenoviruses, herpesviruses and in some cases poxviruses such as vaccinia.
  • the nature of the helper function is not entirely known but appears to be some indirect effect of the helper virus which renders the cell permissive for AAV replication. This belief is supported by the observation that in certain cases AAV replication may occur at a low level of efficiency in the absence of helper virus co-infection if the cells are treated with agents that are either genotoxic or that disrupt the cell cycle.
  • AAV may replicate to a limited extent in the absence of helper virus in certain unusual conditions, as noted above, the more general result is that infection of cells with AAV in the absence of helper functions results in integration of AAV into the host cell genome.
  • the integrated AAV genome may be rescued and replicated to yield a burst of infectious progeny AAV particles if cells containing an integrated AAV provirus are superinfected with a helper virus such as adenovirus. Because the integration of AAV appears to be an efficient event, this suggested that AAV would be a useful vector for introducing genes into cells for stable expression for uses such as human gene therapy.
  • AAV has a very broad host range with neither any obvious species nor tissue specificity and will replicate in virtually any cell line of human, simian or rodent origin provided an appropriate helper is present.
  • AAV is ubiquitous and has been isolated from a wide variety of animal species including most mammalian and several avian species.
  • AAV has not been associated with the cause of any disease.
  • AAV is not a transforming or oncogenic virus.
  • AAV integration into chromosomes of human cell lines does not cause any significant alteration in the growth properties or morphological characteristics of the cells.
  • These properties of AAV also recommend it as a potentially useful human gene therapy vector because most of the other viral systems proposed for this application such as retroviruses, adenoviruses, herpesviruses, or poxviruses are disease-causing viruses.
  • AAV particles are comprised of a protein capsid having three capsid proteins, VPl, VP2, and VP3, and enclosing a DNA genome.
  • the AAV DNA genome is a linear single-stranded DNA molecule having a molecular weight of about 1.5 x 10 6 daltons or approximately 4680 nucleotides long. Strands of either complementary sense, "plus” or “minus” strands, are packaged into individual particles but each particle has only one DNA molecule. Equal numbers of AAV particles contain either a plus or minus strand.
  • Either strand is equally infectious and replication occurs by conversion of the parental infecting single strand to a duplex form and subsequent amplification of a large pool of duplex molecules from which progeny single strands are displaced and packaged into capsids.
  • Duplex or single-strand copies of AAV genomes inserted into bacterial plasmids or phagemids are infectious when transfected into adenovirus-infected cells, and this has allowed the study of AAV genetics and the development of AAV vectors.
  • the AAV2 genome has two copies of a 145-nucleotide-long
  • ITR inverted terminal repeat
  • the unique region contains three transcription promoters p5, pl9, and p40 (Laughlin et al., 1979, Proc. Natl. Acad. Sci. USA. 76:5567-5571) that are used to express the rep and cap genes.
  • the ITR sequences are required in cis and are sufficient to provide a functional origin of replication (ori ) and also are sufficient to provide signals required for integration into the cell genome as well as for efficient excision and rescue from host cell chromosomes or from recombinant plasmids.
  • the ITR can function directly as a transcription promoter in an AAV vector (Flotte et al. , 1993, vide supra) .
  • the rep and cap genes are required in trans to provide functions for replication and encapsidation of viral genome respectively.
  • the rep gene is expressed from two promoters, p5 and pl9. Transcription from p5 yields an unspliced 4.2 kb mRNA which encodes a protein, Rep78, and a spliced 3.9 kb mRNA which encodes a protein, Rep68. Transcription from pl9 yields an unspliced mRNA which encodes Rep52 and a spliced 3.3 kb mRNA which encodes Rep40.
  • the four Rep proteins all comprise a common internal region sequence but differ with respect to their amino and carboxyl terminal regions.
  • Rep78 and Rep68 are required for AAV duplex DNA replication, but Rep52 and Rep40 appear to be needed for progeny,- single- strand DNA accumulation. Mutations in Rep78 and Rep68 are phenotypically Rep- whereas mutations affecting only Rep52 and Rep40 are Rep+ but Ssd-. Rep68 and Rep78 bind specifically to the hairpin conformation of the AAV ITR and possess several enzyme activities required for resolving replication at the AAV termini. Rep52 and Rep40 have none of these properties.
  • the Rep proteins, primarily Rep78 and Rep68 exhibit several pleiotropic regulatory activities including positive and negative regulation of AAV genes and expression from some heterologous promoters, as well as inhibitory effects on cell growth (Tratschin et al. , 1986, Mol. Cell.
  • Virology. 166:154-165) reported a very low level expression of some Rep proteins in certain cell lines after stable integration of AAV genomes.
  • the proteins VPl, VP2, and VP3 all share a common overlapping sequence but differ in that VPl and VP2 contain additional amino terminal sequence. All three are coded from the same cap gene reading frame expressed from a spliced 2.3 kb mRNA transcribed from the p40 promoter. VP2 and VP3 are generated from the same mRNA by use of alternate initiation codons. VPl is coded from a minor mRNA using 3' donor site that is 30 nucleotides upstream from the 3' donor used for the major mRNA that encodes VP2 and VP3. VPl, VP2, and VP3 are all required for capsid production. Mutations which eliminate all three proteins (Cap-) prevent accumulation of single- strand progeny AAV DNA whereas mutations in the VPl a ino- terminus (Lip-, Inf-) permit single-strand production but prevent assembly of stable infectious particles.
  • AAV infectious genomes of AAV were constructed by insertion of double-strand molecules of AAV into plasmids by procedures such as GC tailing (Sa ulski et al. , 1982, Proc. Natl. Acad. Sci. USA. 79:2077-2081), addition of synthetic linkers containing restriction endonuclease (Laughlin et al., 1983, Gene. 23:65-73) or by direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem..
  • AAV vector construction were defined as reviewed recently (Carter, 1992, Current Opinions in Biotechnology. 3:533-539; Muzyczka, 1992, Current Topics in Microbiology and Immunology. 158:97-129).
  • AAV vectors are constructed in AAV recombinant plasmids by substituting portions of the AAV coding sequence with foreign DNA to generate a vector plasmid.
  • the terminal (ITR) portions of the AAV sequence must be retained intact because these regions are required in cis for several functions including excision from the plasmid after transfection, replication of the vector genome and integration and rescue from a host cell genome.
  • the vector can then be packaged into an AAV particle to generate an AAV transducing virus by transfection of the vector plasmid into cells that are infected by an appropriate helper virus such as adenovirus or herpesvirus.
  • an appropriate helper virus such as adenovirus or herpesvirus.
  • the vector plasmid In order to achieve replication and encapsidation of the vector genome into AAV particles, the vector plasmid must be complemented for any AAV functions required in trans , namely rep and cap, that were deleted in construction of the vector plasmid.
  • the transducing vector must be generated at sufficiently high titers that it is practicable as a delivery system. This is especially important for gene therapy stratagems aimed at in vivo delivery of the vector.
  • the required dose of transducing vector may be in excess of 10 10 .
  • the vector preparations must be free of wild-type AA V virus. The attainment of high titers of AAV vectors has b een difficult for several reasons including preferential - 7 -
  • the first AAV vectors that were described contained foreign reporter genes such as neo or cat or dhfr that were expressed from AAV transcription promoters or an SV40 promoter (Tratschin et al., 1984b, Mol. Cell. Biol. 4:2072-2081; Hermonat & Muzyczka, 1984, Proc. Natl. Acad. Sci. USA. 81:6466-6470; Tratschin et al. , 1985, Mol. Cell. Biol. 5:3251- 3260; McLaughlin et al., 1988, J. Virol.. 62:1963-1973; Lebkowski et al., 1988 Mol. Cell. Biol.. 7:349-356).
  • foreign reporter genes such as neo or cat or dhfr that were expressed from AAV transcription promoters or an SV40 promoter
  • the packaging plasmid had deleted the ITR regions of AAV in order that it could not be excised and replicated and thus could not be packaged. All of these approaches failed to prevent generation of particles containing wild-type AAV DNA and also failed to generate effective high titers of AAV transducing particles. Indeed titers of not more than 10 4 ml were cited by Hermonat & Muzyczka, 1984. The production of wild-type AAV particles in these studies was probably due to the presence of overlapping homology between AAV sequences present in the vector and packaging plasmids. It was shown by Senapathy and Carter (1984, J. Biol. Chem.
  • AAV rep or cap gene still met with generation of wild-type AAV and still produced very low transduction frequencies on human cell lines.
  • McLaughlin et al., 1988 reported that AAV rep- cap- vectors containing the neo gene packaged with the same packaging plasmid used earlier by Hermonat & Muzyczka
  • Lebkowski et al., 1988 packaged AAV vectors which did not contain either a rep or cap gene and used an ori- packaging plasmid pBalA identical to that used earlier by Tratschin et al., (1984b, 1985) and reported transduction frequencies that were similarly low, in that for several human cell lines not more than 1% of the cells could be transduced to geneticin resistance even with their most concentrated vector stocks.
  • Lebkowski et al., (1988) did not report the actual vector titers in a meaningful way but the biological assays showing not more than 1% transduction frequency when 5 x 10* cells were exposed to three ml of vector preparation indicates that the titer was less than 2 x 10 4 .
  • the pBal packaging plasmid contains overlapping homology with the ITR sequence in the vector and leads to generation by recombination of wild-type AAV.
  • Laface et al., (1988) used the same vector as that used by Hermonat & Muzyczka (1984) prepared in the same way and obtained a transduction frequency of 1.5% in murine bone marrow cultures again showing very low titer.
  • Samulski et al. (1987, J. Virol.. 61:3096-3101) constructed a plasmid called pSub201 which was an intact AAV genome in a bacterial plasmid but which had a deletion of 13 nucleotides at the extremity of each ITR and thus was rescued and replicated less efficiently than other AAV plasmids that contained the entire AAV genome.
  • Samulski et al. (1989, J ⁇ Virol.. 63:3822-3828) constructed AAV vectors based on pSub20l but deleted for rep and cap and containing either a hyg or neo gene expressed from an SV40 early gene promoter.
  • pAAV/Ad packaged these vectors by co-transfection with a packaging plasmid called pAAV/Ad which consisted of the entire AAV nucleotide sequence from nucleotide 190 to 4490 enclosed at either end with one copy of the adenovirus ITR.
  • pAAV/Ad a packaging plasmid which consisted of the entire AAV nucleotide sequence from nucleotide 190 to 4490 enclosed at either end with one copy of the adenovirus ITR.
  • the AAV rep and cap genes were expressed from the natural AAV promoters p5, pl9 and p40.
  • the function of the adenovirus ITR in pAAV/Ad was thought to be to enhance the expression level of AAV capsid proteins.
  • rep is expressed from its homologous promoter and is negatively regulated and thus its expression is limited.
  • Chatterjee et al., and Wong et al. used a packaging system known to give only low titer and which can lead to generation of wild-type AAV genomes because of the overlapping homology in the vector and packaging sequences.
  • Other reports have described the use of AAV vectors to express genes in human lymphocytes (Muro-Cacho et al., 1992, J. Immunotherapy. 11:231-237) or a human erythroid leukemia cell line (Walsh et al., 1992, Proc. Natl. Acad. Sci. USA. 89:7257-7261) with vectors based on the pSub201 vector plasmid and pAAV/Ad packaging plasmid. Again, titers of vector stocks were not reported and were apparently low because a selective marker gene was used to identify those cells that had been successfully transduced with the vector.
  • AAV vectors may have potential utility as vectors for treatment of human disease by gene therapy.
  • the ability to generate sufficient amounts of AAV vectors has been a severe limitation on the development of human gene therapy using AAV vectors.
  • One aspect of this limitation is that there have been very few studies using AAV vectors in in vivo animal models (see, e.g., Flotte et al., 1993b; and Kaplitt et al., 1994, Nature Genetics 8:148-154). This is generally a reflection of the difficulty associated with generating sufficient amounts of AAV vector stocks having a high enough titer to be useful in analyzing in vivo delivery and gene expression.
  • AAV gene therapy has been the relative inefficiency of the vector packaging systems that have been used. Because of the lack of cell lines expressing the AAV trans complementing functions, such as rep and cap, packaging of AAV vectors has been achieved in adenovirus-infected cells by co-transfection of a packaging plasmid and a vector plasmid. The efficiency of this process may be limited by the efficiency of transfection of each of the plasmid constructs, and by the level of expression of Rep proteins from the packaging plasmids described to date. Each of these problems appears to relate to the biological activities of the AAV Rep proteins. In addition, as noted above, all of the packaging systems described above have the ability to generate wild-type AAV by recombination.
  • Lebkowski et al. introduce rep and cap genes into the cell genome but the method again requires the use of episomal AAV transducing vectors comprising an Epstein-Barr virus nuclear antigen (EBNA) gene and an Epstein-Barr virus latent origin of replication; and, again, the only information relative to titer showed a fairly low titer.
  • EBNA Epstein-Barr virus nuclear antigen
  • AAV vectors can achieve in vivo gene transfer in the respiratory tract, for example, but high titers are critical so as to allow for the delivery of sufficiently high multiplicity of vector in as small a volume as possible.
  • Stable, helper-free AAV packaging cell lines have been elusive, mainly due to the activities of Rep protein, which down-regulates its own expression and can negatively affect the host cell.
  • Rep protein which down-regulates its own expression and can negatively affect the host cell.
  • the approaches described in this invention effectively circumvent these problems and have allowed for substantial improvements in packaging efficiency.
  • a method of producing a mammalian cell capable of high efficiency packaging of a recombinant AAV (rAAV) vector comprising the steps of: (a) providing a mammalian cell which comprises a stably integrated AAV cap gene operably linked to a promoter, and a stably integrated AAV rep gene operably linked to a heterologous promoter; (b) replicating the cell of step (a) to produce a population of cells; (c) introducing a helper virus to the population of cells of step (b) ; and (d) selecting a cell exhibiting helper-virus-inducible rep protein activity.
  • rAAV recombinant AAV
  • step (a) comprises the combined rep and cap genes of AAV in which the p5 promoter has been replaced by a heterologous promoter.
  • heterologous promoter is a mouse metallothionein I (mMT-I) promoter.
  • mMT-I mouse metallothionein I
  • a mammalian cell capable of high efficiency packaging of a recombinant AAV (rAAV) vector, said cell comprising a stably integrated cap gene operably linked to a promoter, and a stably integrated rep gene operably linked to a heterologous promoter; wherein said cell exhibits helper- virus-inducible rep protein activity.
  • rAAV recombinant AAV
  • said heterologous promoter is a mouse metallothionein I (mMT-I) promoter.
  • ITR inverted terminal repeat
  • a method of packaging a recombinant AAV vector comprising the steps of: (a) providing an AAV packaging cell of embodiment 10; (b) introducing a recombinant AAV vector, said vector comprising a polynucleotide sequence of interest located between two AAV inverted terminal repeat (ITR) regions; (c) introducing a helper virus; and (d) incubating the cell under conditions suitable for replication and packaging of AAV.
  • ITR inverted terminal repeat
  • a method of packaging a recombinant AAV vector comprising the steps of: (a) providing an AAV packaging cell of embodiment 15 which comprises a stably integrated rAAV vector; (b) introducing a helper virus; and (c) incubating the cell under conditions suitable for replication and packaging of AAV.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Figure 1 is a diagram of plasmid pMt-rep/cap//pKO-neo, as described in Example l.
  • Figure 2 is a reproduction of Southern blots demonstrating that packaging cells produced according to the present invention have sufficient rep activity to replicate an incoming rAAV vector, as described in Example 4.
  • Figure 3 is a reproduction of Southern blots demonstrating that packaging cells produced according to the present invention are capable of replicating an AAV genome in the presence of adenovirus, and that this activity can be used to quantify the number of infectious viral particles present in a given sample, as described in Example 5.
  • Figure 4 is a reproduction of Southern blots demonstrating that packaging cells produced according to the present invention express rep protein and are able to replicate recombinant AAV plasmid DNA genomes introduced by transfection, as described in Example 6.
  • Figure 5 is a reproduction of Southern blots demonstrating that the infectious rAAV titering assay described in Example 5 had proceeded for a sufficient amount of time to reach a maximum, as described in Example ' 7.
  • Figures 6 and 7 are reproductions of Southern blots demonstrating that packaging cells produced according to the present invention can replicate and package rAAV vector genomes into infectious virions by either transfection or infection, as described in Examples 9 and 10.
  • Figure 8 is a reproduction of a Southern blot demonstrating that packaging cells produced according to the present invention possess sufficient rep activity to recognize, excise and amplify an integrated rAAV vector, as described in Example 11. DETAILED DESCRIPTION OF THE INVENTION
  • AAV vectors are recombinant constructs of the AAV virus comprising AAV components necessary for replication and encapsidation, along with a heterologous polynucleotide encoding a protein of interest. These recombinant AAV vectors are potentially powerful tools for human gene therapy, particularly for diseases such as cystic fibrosis and sickle cell anemia.
  • a major advantage of AAV vectors over other approaches to gene therapy is that they do not require ongoing replication of the target cell in order to integrate permanently into the cell's genome.
  • the invention described herein provides methods and materials for use in the production of high titers of recombinant AAV vectors for use in gene therapy. It also provides methods and materials for determining the relative infectious titer of rAAV preparations.
  • polypeptide polypeptide
  • peptide protein
  • proteins that are post- translationally modified through reactions that include glycosylation, acetylation and phosphorylation.
  • Polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. This term refers only to the primary structure of the molecule. Thus, double- and single-stranded DNA, as well as double- and single- stranded RNA are included. It also includes modified polynucleotides such as methylated or capped polynucleotides.
  • Recombinant as applied to a polynucleotide, means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.
  • Sequence overlap occurs when the nucleotides share a homologous sequence of sufficient length and identity that recombination is facilitated.
  • the level of homology and corresponding frequency of recombination increase with increasing length of the homologous sequences and with their level of shared identity.
  • the level of homology that will pose a concern in a given system can be determined theoretically and confirmed experimentally, as is known in the art.
  • recombination can be substantially reduced or eliminated if the overlapping sequence is less than about a 25 nucleotide sequence if it is at least 80% identical over its entire length, or less than about a 50 nucleotide sequence if it is at least 70% identical over its entire length.
  • a “vector” refers to a recombinant plasmid or virus that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo.
  • the polynucleotide to be delivered sometimes referred to as a "target polynucleotide”, may comprise a coding sequence of interest in gene therapy.
  • a “recombinant AAV vector” refers to a vector comprising one or more polynucleotides of interest that are flanked by AAV inverted terminal repeat sequences (ITRs) .
  • ITRs AAV inverted terminal repeat sequences
  • Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus and is expressing the AAV rep and cap genes.
  • AAV "rep” and “cap” genes (encoding replication and encapsidation proteins, respectively) have been found in all AAV serotypes examined, and are described above and in the references cited therein. Typically, the rep and cap genes are found adjacent to each other in the AAV genome, and they are generally conserved among AAV serotypes.
  • helper virus for AAV refers to a second virus that allows wild-type AAV (which is a "defective" parvovirus) to be replicated and packaged by a host cell.
  • helper viruses include adenoviruses, herpesviruses and poxviruses such as vaccinia.
  • Packaging refers to a series of subcellular events that results in the assembly and ' encapsidation of an rAAV vector. Thus, when a suitable vector plasmid is introduced into a packaging cell line under appropriate conditions, it will be assembled into a vector viral particle.
  • Heterologous means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared.
  • a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide) .
  • a promoter that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous promoter.
  • Promoter refers to a genomic region that enhances the transcription of a gene or coding sequence to which it is operably linked.
  • operably linked refers to a juxtaposition, wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter promotes transcription of the coding sequence.
  • An operably linked promoter is usually in cis configuration with the coding sequence, but is not necessarily contiguous with it.
  • “Host cells”, “cell lines”, “cell cultures”, and other such terms denote higher eukaryotic cells, most preferably mammalian cells, which can be used as recipients for recombinant vectors or other transfer polynucleotides, and include the progeny of the original cell that was transduced. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or in genomic complement) to the original parent cell.
  • “Stable integration" of a polynucleotide into a cell means that the polynucleotide has been introduced into a chromosome or mini-chromosome of the cell and, therefore, becomes a relatively permanent part of the cellular genome.
  • “episomes” such as plasmids can sometimes be maintained for many generations (particularly if kept under selective pressure)
  • genetic material carried episomally is generally more susceptible to loss than chromosomally- integrated material.
  • the chromatin structure of eukaryotic chromosomes can influence the level of expression of an integrated polynucleotide; and we believe that such effects can sometimes prove beneficial in situations such as those described herein (in which the level of expression of the AAV rep gene can have negative effects upon cellular metabolism) .
  • the selection of stable cell lines having properties that are particularly desirable in the context of the present invention are described in the Detailed Description and Examples below. "Efficiency" when used in describing a cell line refers to the useful properties of the line; in particular, the growth rate, and (for packaging cell lines) the number of virus particles produced per cell. "High efficiency packaging" indicates production of at least 100 viral particles per cell.
  • the method for producing high titers of recombinant•AAV vectors comprises several steps.
  • the general strategy involves preparation of mammalian packaging cell lines that comprise a stably integrated AAV cap gene operably linked to a promoter, and a stably integrated AAV rep gene operably linked to a heterologous promoter.
  • Packaging cells are then infected or transfected with a plasmid comprising the AAV ITR regions and the target polynucleotide.
  • suitable conditions including suitable growth conditions and infection with a competent helper virus
  • expression of the rep and cap genes of the packaging cell results in the synthesis of rep and cap proteins which mediate replication and encapsidation of the AAV vector, respectively.
  • Providing a polynucleotide of interest (also referred to as a "target polynucleotide”) in- between the AAV ITR sequences of the rAAV vector, thus results in packaging of the target polynucleotide into an infectious rAAV particle which can be used to deliver the polynucleotide to a desired host cell.
  • the proportion of wild-type AAV i.e., particles not containing the target polynucleotide
  • the presence of contaminating wild-type AAV limits the therapeutic potential of rAAV vector preparations.
  • the p5 promoter region is replaced with a different promoter.
  • the packaging cell lines of the present invention enable the efficient production of rAAV preparations that are of high titer and are substantially free of any contaminating wild- type AAV; attributes that are especially useful in the context of AAV-mediated gene therapy.
  • the degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to ITRs.
  • the similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.
  • the parental lines from which packaging cells are generated may be obtained from any cell line that is susceptible to AAV infection, and amenable to culture in vitro.
  • AAV has a very broad host range and has been isolated from a variety of mammalian cell types, including simian, human and rodent cells.
  • human cell lines in which appropriate helper functions can be expressed are typically preferred.
  • Such human cell lines from which the packaging cell lines may be derived include, for example, Hela, A549, 293, KB, Detroit, and WI38 cells. We initially selected both Hela cells and A549 cells for demonstrations of the present invention. As described in the Examples below, we were readily able to generate packaging cells from both parental lines tested.
  • the rep gene is under regulation of the p5 promoter, which is itself strongly down-regulated by rep expression.
  • the cells are provided with a stably integrated AAV cap gene operably linked to a promoter, and a stably integrated AAV rep gene operably linked to a heterologous promoter; as described and illustrated herein. Any heterologous promoter that is not strongly down-regulated by rep gene expression is suitable; but inducible promoters are preferred because constitutive expression of the rep gene can have a negative impact on the host cell.
  • inducible promoters are known in the art; including, by way of illustration, heavy metal ion inducible promoters (such as metallothionein promoters) ; steroid hormone inducible promoters (such as the MMTV promoter or growth hormone promoters) ; and promoters such as those from T7 phage which are active in the presence of T7 RNA polymerase.
  • inducible promoters are those that are induced by the helper virus that is used to complement the replication and packaging of the rAAV vector.
  • helper-virus-inducible promoters include for example, the adenovirus early gene promoter which is inducible by adenovirus E1A protein; the adenovirus major late promoter; the herpesvirus promoter which is inducible by herpesvirus proteins such as VP16 or 1CP4 ; as well as vaccinia or poxvirus inducible promoters.
  • the Examples below illustrate a generally applicable method that can be used to test putative promoters to readily determine whether or not they are helper-virus-inducible and whether or not they will be useful in the generation of high efficiency packaging cells.
  • the method involves replacing the p5 promoter of the AAV rep gene with the putative helper-virus-inducible promoter (either known in the art or identified using well-known techniques such as linkage to promoter-less "reporter" genes) .
  • the AAV rep-cap genes (with p5 replaced) , preferably linked to a positive selectable marker such as an antibiotic resistance gene, are then stably integrated into a suitable host cell (such as the Hela or A549 cells exemplified below) .
  • Cells that are able to grow relatively well under selection conditions are then tested for their ability to express the rep and cap genes upon addition of a helper virus.
  • As an initial test for rep and/or cap expression cells can be readily screened using immunofluorescence to detect rep and/or cap proteins (as illustrated in the Examples below) . Confirmation of packaging capabilities and efficiencies can then be determined by functional tests for replication and packaging of incoming rAAV vectors (also illustrated below) .
  • the AAV cap gene is also stably integrated into the packaging cell line.
  • the rep and cap genes are introduced into the parental line together, by using a plasmid that contains them both (essentially as they are arranged in the AAV genome, except for replacement of the sequences upstream of rep, i.e. the p5 promoter region) .
  • a plasmid designated pMt-rep/cap//pKO-neo shown in Figure 1 .
  • the plasmid contains a heterologous promoter linked to a region containing the rep-cap genes. The rest of the rep-cap region, including the pl9 promoter and the p40 promoter are retained.
  • the plasmid also contains an AAV polyadenylation signal.
  • the components of native AAV that are not present in the plasmid include the p5 promoter region (which has been substituted by the heterologous promoter) and the ITRs (which are present in the vector plasmid to be introduced separately) .
  • Cells transfected with rep and cap genes as described above are then selected from untransfected cells according to methods that are routine in the art. Most conveniently, selection is accomplished by linking the rep and cap genes to one or more selectable markers (such as antibiotic resistance genes) .
  • selectable markers such as antibiotic resistance genes
  • the neo-resistance gene was included next to the rep-cap sequences.
  • selectable markers are driven by constitutive promoters; and preferably, such markers are introduced in an opposite orientation relative to the AAV rep-cap genes since that tends to reduce the potential for the promoter driving the selectable marker to effect expression of the rep gene (which can be detrimental to the host cell) .
  • the cell lines are exposed to the antibiotic for which resistance has been provided (geneticin was used in the case of the constructs referred to above) .
  • the selectable marker is included on the same plasmid as the rep-cap sequences; and both are stably integrated into the host genome.
  • the plasmid Mt-rep/cap//pKOneo for example, geneticin- resistant cells would be expected to possess an integrated copy of the neo gene as well as pMt-rep/cap. Since the rep sequences cannot readily be lost in our system, the prior art would predict that the recipient cells would exhibit reduced growth rates.
  • our constructs were introduced into exemplary mammalian host cells (Hela and A549) , the rate of proliferation of the geneticin-resistant clones was not significantly affected in either of the cell lines.
  • packaging cells that are capable of replicating at least one half as rapidly as the parental cells, and capable of producing more than 100 rAAV particles/cell.
  • the cells grow at least two-thirds as rapidly as the parental line, and produce more than 250 rAAV particles/cell.
  • packaging cells that replicate substantially as rapidly as the parent cells (at least about 80% of the rate), and that produce more than about 500 rAAV particles per cell.
  • the packaging cell line is supplied with a recombinant AAV vector comprising AAV inverted terminal repeat (ITR) regions surrounding one or more polynucleotides of interest (or "target" polynucleotides) .
  • ITR inverted terminal repeat
  • the target polynucleotide is operably linked to a promoter, either its own or a heterologous promoter.
  • a promoter either its own or a heterologous promoter.
  • suitable promoters are known in the art, the choice of which depends on the desired level of expression of the target polynucleotide; whether one wants constitutive expression, inducible expression, cell-specific or tissue-specific expression, etc.
  • the rAAV vector will also contain a positive selectable marker in order to allow for selection of cells that have been infected by the rAAV vector.
  • Negative selectable markers can also be included; as a means of selecting against those same cells should that become necessary or desirable.
  • those constructs involve direct translational fusions between a dominant positive selectable marker a negative selectable marker.
  • Preferred positive selectable markers are derived from genes selected from the group consisting of hph , neo, and gpt
  • preferred negative selectable markers are derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt
  • Especially preferred markers are bifunctional selectable fusion genes wherein the positive selectable marker is derived from hph or neo , and the negative selectable marker is derived from cytosine deaminase or a TK gene.
  • CFTR operably linked to a promoter.
  • CFTR polypeptides that are capable of reconstructing CFTR functional deficiencies in cells derived from cystic fibrosis patients.
  • Rich et al. (1991, Science, 253: 205-207) described a CFTR derivative missing amino acid residues 708-835, that was capable of transporting chloride and capable of correcting a naturally occurring CFTR defect.
  • Egan et al. (1993) described another CFTR derivative (comprising about 25 amino acids from an unrelated protein followed by the sequence of native CFTR beginning at residue 119) that was also capable of restoring electrophysiological characteristics of normal CFTR.
  • polynucleotides include, but are not limited to: (i) polynucleotides encoding proteins useful in other forms of gene therapy to relieve deficiencies caused by missing, defective or sub-optimal levels of a structural protein or enzyme; (ii) polynucleotides that are transcribed into anti-sense molecules; (iii) polynucleotides that are transcribed into decoys that bind transcription or translation factors; (iv) polynucleotides that encode cellular modulators such as cytokines; (v) polynucleotides that can make recipient cells susceptible to specific drugs, such as the herpes virus thymidine kinase gene; and (vi) polynucleotides for cancer therapy, such as the wild-type p53 tumor suppressor cDNA for replacement of the missing or damaged p53 gene associated with some lung and breast cancer
  • the same packaging cell line can be used for any of these applications.
  • the plasmid comprising the specific target polynucleotide is introduced into the packaging cell for production of the AAV vector by one of several possible methods; including, for example, electroporation.
  • Helper virus can be introduced before, during or after introduction of the rAAV vector. As illustrated in Example 10, the plasmid can be co-infected into the culture along with the helper virus.
  • the cells are then cultured for a suitable period, typically 2-5 days, in conditions suitable for replication and packaging as known in the art (see references above and examples below) . Lysates are prepared, and the recombinant AAV vector particles are purified by techniques known in the art.
  • the recombinant AAV vector is itself stably integrated into a clone of the packaging cell line.
  • a stable, vector-containing packaging line can be grown and stored until ready for use.
  • the user simply infects the cells with helper virus and cultures the cells under conditions suitable for replication and packaging of AAV (as described below) .
  • the amount of helper virus and the incubation time influence the amount of rep activity, they can be readily optimized and kept constant, as illustrated below.
  • To conduct the assay aliquots of the packaging cell line are introduced with a standard amount of helper virus and serial dilutions of the rAAV preparation to be tested.
  • the relative infectious titer of the AAV is indicated by the amount of replicated AAV present in each aliquot after suitable incubation; and can be compared to a preparation of known titer.
  • the examples presented below are provided as a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the invention in any way.
  • Example 1 Construction of a plasmid encoding the rep-cap se ⁇ uences operably linked to a heterologous promoter
  • a plasmid containing the wild type rep and cap genes from deoxyribonucleotide 311 to 4493 of the AAV genome
  • mMt-I mouse metallothionein I
  • This construction effectively removes both ITR's and substitutes the mMt-I promoter for the p5 promoter while maintaining all of the AAV reading frames, the pl9 and p40 promoters and the polyadenylation signal.
  • pKOneo contains the neo gene (providing resistance to neomycin and gentamicin) under control of the SV40 early promoter; as well as SV40 small t intron and SV40 polyadenylation signal oriented in the opposite transcriptional direction relative to pMt-rep/cap (Ito et al. 1994 Cancer Lett. 76:33-39).
  • pMt-rep/cap//pKO-neo The resulting plasmid, designated pMt-rep/cap//pKO-neo, is shown in Figure 1.
  • Example 2 Integration of the rep-cap genes into mammalian cell lines
  • DMEM Dulbecco's modified Eagle's medium
  • the cells were plated at low density in the presence of 1 mg/ml active component geneticin (Gibco-BRL) . Individual colonies were ring cloned, expanded and maintained in 1 mg/ml geneticin.
  • the selectable marker is included on the same plasmid as the rep-cap sequences; and both are stably integrated into the host genome.
  • the plasmid Mt-rep/cap//pKOneo for example, geneticin- resistant cells would be expected to possess an integrated copy of the neo gene as well as pMt-rep/cap. Since the rep sequences cannot readily be lost in our system, the prior art would predict that the recipient cells would exhibit reduced growth rates.
  • PBS phosphate buffered saline
  • the cells were then washed three additional times with PBS and incubated overnight with "WT" medium (1% nonfat dry milk, 0.5 mg/ml bovine serum albumin, 150 mM NaCl, 50 mM HEPES (pH 7.5), 0.1% Tween 20 and 1 mM NaN 3 ) .
  • WT "WT” medium
  • Anti-rep antibody (rabbit anti-Rep78.93; Trempe et al. 1987 Virology 161:18-28) was diluted 1:250 in WT and 100 ⁇ l added to each well for 1 hour at room temperature (RT) .
  • the cells were washed five times with WT and then incubated with 100 ⁇ l of a 1:100 dilution of anti-rabbit IgG FITC conjugate secondary antibody (Sigma Chemical Corp.) in the dark for l hour at RT.
  • the cells were then washed three times with WT and two times with PBS in the dark and examined with an Axioskop H fluorescence microscope (Zeiss, Germany) .
  • rep protein was detectable in a number of the cells examined (8 out of 23 A549 clones and 3 out of 28 Hela clones) .
  • the addition of heavy metals did not significantly affect the observed rep expression under any conditions.
  • helper-virus-inducible promoter is a general one that can be readily applied to any promoter of potential interest by simply swapping it into rep constructs and screening for colonies as we describe herein.
  • exemplary clones (of Hela and A549 origin) were tested for their ability to replicate recombinant AAV genomes after infection, as described below.
  • Replication activity of IF+ cells We examined whether the pMt-rep/cap//pKO-neo transfected cell lines exhibited functional replication activity.
  • MOI 25 pfu/cell
  • the culture medium from each well was removed to a labeled tube and any cells still attached to the culture dish were trypsinized and pooled with cells present in the media.
  • the cell suspension was centrifuged at 3000 rpm for 5 min. , after which the supernatant was removed and total nucleic acid was prepared from the cell pellet (according to Ausubel et al. (ed.) 1987 Current Protocols in Molecular Biology Greene Publishing Associates, Brooklyn, N.Y.) .
  • Negative controls for the experiment included the incubation of 1.2 x 10 8 AAVCFTR particles on either cell line without adenovirus. Fifteen micrograms of nucleic acid for each sample, as well as untreated Hela clone 37 DNA +/- 20 pg of AAVCFTR plasmid (positive control for Southern) , was digested with EcoRI, subjected to gel electrophoresis, transferred to nitrocellulose and probed with a 1.488 kb EcoRI fragment from within the CFTR cDNA. Lanes 15-18 (Fig.
  • a hybridization signal migrating at 1.488 kb is present in DNA isolated from both the Hela clone 37 and A549 clone 20 cell lines after infection by AAVCFTR virus and adenovirus (Fig. 2, lanes l, 2 and 8).
  • Example 5 rAAV infectious titer assays Additional rep activity assays were performed in order to determine whether there was a linear relationship between incoming AAVCFTR virus and replicated AAVCFTR sequences (which could be exploited as the basis of an rAAV infectious titer assay) .
  • Three log dilutions from 1.2 x 10 9 to 1.2 x 10 7 AAVCFTR particles, as determined by slot blot hybridization, were cultured in 2.5 ml media on 2.5 x 10 5 Hela clone 37 cells plus adenovirus (MOI 25 pfu/cell) for 48 hours in a 6 well culture dish.
  • MOI 25 pfu/cell
  • packaging cells produced according to the present invention are capable of replicating an AAV genome in the presence of adenovirus, and that this activity can be used to quantify the number of infectious viral particles present in a given sample.
  • the particle number was determined by slot blot hybridization of the AAVCFTR virus preparation and may reflect the contribution of infectious and defective AAVCFTR particles; whereas the infectious assay described above should only detect infectious particles.
  • AAV recombinant AAV
  • Current methods for the production of recombinant AAV (rAAV) virus include the transient transfection of plasmid vectors containing the rAAV sequences. One or more steps are undertaken to remove the plasmid DNA from a rAAV preparation.
  • AAVCFTR plasmid DNA was incubated directly onto packaging cells (Hela clone 37) +/- adenovirus to determine whether the above-described infection assay would detect non-viral DNA.
  • AAVCFTR plasmid (10 ⁇ g) was electroporated as previously described into 4 x 10° Hela clone 37 cells and then transferred to a 100 mm culture dish.
  • FIG. 4 shows the hybridization pattern of the endogenous CFTR gene and the migration of CFTR cDNA (20 pg) spiked into human genomic DNA when digested with EcoRI and probed with the 1.488 kb CFTR cDNA fragment. Electroporation of the AAVCFTR plasmid into Hela clone 37 cells resulted in a signal migrating at 1.488 kb (Fig.4, lane 1) and represents the amount of AAVCFTR plasmid present in these cells 24 hours after transfection.
  • Lanes 5-8 show the results of incubating 1 ⁇ g, 100 ng, 10 ng and 1 ng AAVCFTR plasmid, respectively, on Hela clone 37 cells in the presence of adenovirus.
  • Example 8 Adenovirus titration of rAAV infectious titer assay
  • MOI 2.5 ml culture media +/- adenovirus
  • a slight signal migrating at 1.488 kb can be detected in DNA isolated from Hela clone 37 cells incubated with supernatant derived from the minus adenovirus control and reflects a small amount of contaminating input AAVCFTR plasmid from the electroporation (Fig. 7, lane 1).
  • Supernatant derived from a duplicate well cultured with adenovirus and titered on Hela clone 37 cells revealed significantly more hybridization migrating at 1.488 kb relative to control conditions (Fig. 7, compare lanes 3 and 1) .
  • Example 11 Rescue and amplification of an integrated rAAV vector from packaging cells
  • Packaging cells derived from Hela clone 37
  • a recombinant AAV vector designated rAAV-CMV-Hygro
  • hygromycin resistance gene operably linked to the CMV enhance /promoter
  • a stable, polyclonal line was derived by selection in 300 ⁇ g/ml hygromycin.
  • the polyclonal, hygro-resistant Hela clone 37 line (2.5 x 10 5 cells/well) was seeded onto a 6 well dish for infection with adenovirus
  • Lane 1 represents DNA isolated from the parental Hela clone 37 cell line, and hence does not contain the hygro-resistance gene.
  • Lane 2 contains DNA from the polyclonal, hygro-resistant Hela clone 37 line which at this exposure time does not show the presence of the resistance gene which is present at an average of about 1 copy/well (data not shown) .
  • DNA isolated from a duplicate well containing the hygro-resistant Hela clone 37 cells treated with adenovirus was run in lane 3.
  • the hybridization present at 1048 bp represents material derived from the excision and amplification of the integrated AAVCMVHygro vector integrated in the Hela clone cells.
  • the addition of wild-type AAV to the adenovirus infection gave similar results (lane 4) .

Abstract

Cette invention concerne les vecteurs AAV (virus associé à un adénovirus), lesquels peuvent être utiles en thérapie génétique. Jusqu'à présent, toutefois, l'obstacle principal a été l'incapacité de produire de tels vecteurs recombinants en quantité suffisante pour qu'ils soient utiles, d'un point de vue clinique, lors d'applications de la thérapie génétique chez l'homme. Les lignées cellulaires stables d'encapsidation d'AAV étaient difficiles à isoler, notamment à cause de l'activité de la protéine rep, qui, par la rétro-régulation de sa propre expression, peut avoir des effets négatifs sur la cellule hôte. Cette invention propose des systèmes d'encapsidation, ainsi que des procédés d'encapsidation de vecteurs AAV, permettant d'éviter ces problèmes et d'accroître sensiblement l'efficacité d'encapsidation.
PCT/US1995/015892 1994-12-06 1995-12-06 Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants WO1996017947A1 (fr)

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JP8517764A JPH10511264A (ja) 1994-12-06 1995-12-06 高力価組換えａａｖベクターの生成のためのパッケージング細胞株
US09/731,941 US6924128B2 (en) 1994-12-06 2000-12-06 Packaging cell lines for generation of high titers of recombinant AAV vectors
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