Generation of high titers of recombinant aav vectors

ES 2 216 005 T3

DESCRIPTION

Production of high titers of recombinant AAV vectors.

Technical field of the invention

This invention relates to gene therapy, and more specifically to materials and methods used for the generation of high titer recombinant AAV vectors for use in gene therapy procedures.

Background of the invention

AAV vectors may have utility for gene therapy but thus far a significant obstacle has been the inability to generate sufficient quantities of such recombinant vectors in quantities that would be clinically useful for the application of gene therapy in humans. 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 that have been shown to have utility as gene transfer agents in vivo (reviewed by Carter, 1992, Current Opinion in Biotechnology, 3: 533-539, Muzcyzka, 1992, Curr. Top. Microbiol. Immunol. 158: 97-129) and are therefore potentially of great importance for gene therapy in humans. AAV vectors are susceptible to stable high-frequency DNA integration and expression in a variety of cells including cystic fibrosis (CF) bronchial and nasal epithelial cells (Flotte et al., 1992a, Am. J. Respir. Cell Mol. Biol. 7: 349-356; Egan et al., 1992, Nature, 358: 581-584; Flotte et al., 1993a, J. Biol. Chem. 268: 37813790; Flotte et al. , 1993b, Proc. Natl. Acad. Sci. USA, in press), human bone marrow derived erythroleukemia cells (Walsh et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 7257-7261), and many others. AAV does not require active cell division for stable expression, which could be a clear advantage over other viruses, especially in tissues such as the human airway epithelium where most cells are terminally differentiated rather than dividing.

AAV is a defective parvovirus that grows only in cells in which certain functions are provided by a coadjuvant virus that infects simultaneously (see Fig. 1). General reviews of AAV can be found in Carter, 1989, Handbook of Parvoviruses, Vol. I, pp. 169-228, Carter, 1989, Handbook of Parvoviruses, Vol. I, pp. 169-228, Bernes, 1990, Virology, pp. 1743-1764, Raven Press, (New York). Examples of co-infecting viruses that provide adjuvant functions for the growth and replication of AAV are adenoviruses, herpesviruses, and in some cases poxviruses such as vaccinia. The nature of the helper function is not known but there appears to be some indirect effect of the helper virus that makes the cell permissive for AAV replication. This concept is supported by the observation that in certain cases AAV replication can occur at a low level of efficacy in the absence of coadjuvant virus infection if cells are treated with agents that are either genotoxic or disrupt the virus. cellular cycle.

Although AAV can replicate to a limited extent in the absence of helper virus under 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 AAV integration. in the host cell genome. The integrated AAV genome can be rescued and replicated to yield a burst of AAV particles from infectious progeny if cells containing an integrated AAV provirus are superinfected with a helper virus such as an adenovirus. Because AAV integration appears to be an efficient event, this suggests that AAV could be a useful vector for introducing genes into cells for stable expression for uses such as gene therapy in humans. The most recent results (Kotin & Berns, 1989, Virology 170: 460-467; Kotin et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 2211-2215; Samulski et al., 1991, EMBO J 10: 3941-3950) have suggested that AAV may manifest a preference for integration at a site on human chromosome 19 but the generality and mechanism of this phenomenon have not been fully elucidated.

AAV has a very broad host range with no obvious species or tissue specificity and will replicate in virtually any cell line of human, simian or rodent origin as long as an appropriate adjuvant is present. AAV is ubiquitous and has been isolated from a wide variety of animal species including most mammals and various species of birds.

AAV has not been associated with the cause of any disease. AAV is not a transforming or oncogenic virus. The integration of AAV into the chromosomes of human cell lines does not cause any significant alteration in the growth properties or in the morphological characteristics of the cells. These properties of AAV also recommend it as a potentially useful vector for human gene therapy 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 made up of a protein capsid that has three capsid proteins, VP1, VP2, and VP3, which comprise a DNA genome. The AAV DNA genome is a linear single-stranded DNA molecule that has a molecular weight of approximately 1.5 x 10⁶ daltons or approximately 4680 nucleotides of

ES 2 216 005 T3 longitude. Strands of any complementary sense, "more" or "less" strands, are packed into individual particles but each particle has only one DNA molecule. The same number of AAV particles contain one more strand or one less strand. Any strand is equally infectious and replication occurs by conversion of the parental infectious single strand into a duplex form and subsequent amplification of a large pool of duplex molecules from which the single strands of progeny move and pack into capsids. . Duplex or single-stranded 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 AAV replication cycle is outlined in Figure 1.

The AAV2 genome has a 145 nucleotide long ITR (inverted terminal repeat) copy of each end and a single region of the sequence approximately 4470 nucleotides long (Srivastava et al., 1983, J. Virol., 45 : 555-564) containing two main open reading frames for the rep and cap genes (Hermonat et al., J. Virol, 51: 329-339; Tratschin et al., 1984a, J. Virol., 51: 611-619). The unique region contains three transcription promoters p5, p19 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 are also sufficient to provide the signals required for integration into the cellular genome as well as for efficient cleavage and rescue from cells. chromosomes from the host cell or from recombinant plasmids. Furthermore, it has been shown that ITR can function directly as a transcriptional promoter in an AAV vector (Flotte et al., 1993, vide supra).

The rep and cap genes are required in trans to provide functions for the replication and packaging of the viral genome respectively. The rep gene is expressed from two promoters p5 and p19. Transcription of p5 yields an unspliced ​​4.2 kb mRNA encoding a protein, Rep78, and a 3.9 kb spliced ​​mRNA encoding a Rep68 protein. Transcription of p19 yields a non-emplaced mRNA encoding Rep52 and a 3.3 kb spliced ​​mRNA encoding Rep40. Thus, the four Rep proteins comprise a common inner region sequence but differ with respect to their amino and carboxyl terminal regions. Only Rep78 and Rep68 are required for AAV duplex DNA replication, but Rep52 and Rep40 appear to be required for accumulation of single-stranded progeny DNA. Mutations in Rep78 and Rep68 are phenotypically Rep^- while the mutations that affect only Rep52 and Rep40 are Rep⁺ but Ssd. Rep68 and Rep78 specifically bind to the hairpin conformation of the AAV ITR and possess various enzymatic activities required to resolve replication at the AAV ends. Rep52 and Rep40 have neither of these properties.

Rep proteins, mainly Rp78 and Rep68, show numerous 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. Biol. 6: 28842894; Labow et al., 1987, Mol. Cell. Biol., 7: 1320-1325; Khleif et al., Virology, 181: 738-741). The AAV p5 promoter is downregulated by Rep78 and Rep68 (Tratschin et al., 1986, Mol. Cell. Biol. 6: 2884-2894). Due to the inhibitory effects of rep expression on cell growth, constitutive expression of rep has not been easily achieved in cell lines. For example, Mendelson et al. (1988, Virology, 166: 154-165) reported a very low level of expression of certain Rep proteins in certain cell lines after stable integration of AAV genomes.

The VP1, VP2, and VP3 proteins all share a common overlapping sequence but differ in that VP1 and VP2 contain additional amino terminal sequences. All three are encoded from the same reading frame of the cap gene expressed from a 2.3 kb spliced ​​mRNA transcribed from the p40 promoter. VP2 and VP3 are generated from the same mRNA by using alternate initiation codons. VP1 is encoded from a minor mRNA using the 3 'donor site that is 30 nucleotides upstream of the 3' donor used for the major mRNA encoding VP2 and VP3. VP1, VP2, and VP3 are required for capsid production. Mutations that remove all three proteins (Cap^- ) prevent AAV DNA accumulation in single-stranded progeny while mutations in the amino end of VP1 (Lip^- Inf^- ) allow the production of the single strand but avoid the assembly of stable infectious particles.

The AAV genetic analysis described above was based on mutational analysis of AAV genomes that were molecularly cloned into bacterial plasmids. In early work, molecular clones of infectious AAV genomes were constructed by inserting AAV double-stranded molecules into plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79: 2077-2081), the addition of synthetic linkers containing restriction endonucleases (Laughlin et al., 1983, Gene, 23: 65-73) or by direct blunt end ligation (Senapathy & Carter, 1984, J Biol. Chem., 259: 46614666). It was later shown that transfection of such recombinant AAV plasmids from mammalian cells that were also infected with an appropriate helper virus, such as adenovirus, resulted in the rescue and excision of the AAV genome free of any plasmid sequences and replication. of the rescued genome and the generation of a harvest of infectious AAV particles from the progeny (see Fig. 1). This provided the basis for performing AAV genetic analysis as outlined above and allowed the construction of AAV transducer vectors.

Based on the genetic analysis, the general principles of AAV vector construction were defined as recently reviewed (Carter, 1992, Current Opinions in Biotechnology, 3: 533-539; Muzyczka, 1992, Current Topics in Microbiology and Immunology, 158: 97-129). AAV vectors were constructed in plasmids

ES 2 216 005 T3 recombinants of AAV by substituting the portions of the AAV coding sequence for the foreign DNA to generate a vector plasmid. In the vector plasmid, the terminal portions (ITR) of the AAV sequence must be preserved intact because these regions are required in cis for numerous functions including excision of the plasmid after transfection, vector genome replication and integration and the rescue of the host cell genome. The vector can then be packaged into an AAV particle to generate an AAV transducer virus by transfection of the vector plasmid into cells that are infected by an appropriate helper virus such as adenovirus or herpesvirus. In order to achieve replication and encapsidation of the vector genome into AAV particles, the vector plasmid must be complemented for any of the AAV functions required in trans, that is, rep and cap, which were suppressed in plasmid construction. vector.

There are at least two desirable traits of any AAV vector that is designed for use in human gene therapy. First, the transducer vector must be generated with sufficiently high titers so that it is practicable as a delivery system. This is especially important for gene therapy stratagems aimed at in vivo delivery of the vector. It is likely that for many desirable applications of AAV vectors, such as the treatment of cystic fibrosis by direct in vivo delivery to the airways, the required dose of transducer vector may exceed 10¹⁰ . Second, vector preparations must be free of wild-type AAV virus. Achieving high titer of AAV vectors has been difficult for a number of reasons including the preferential packaging of wild-type AAV genomes if present or generated by recombination, and the inability to generate sufficient complementary functions such as rep or cap. Useful cell lines expressing such complementary functions have not been generated, in part, due to the various inhibitory functions of the rep gene.

The first AAV vectors to be described contained foreign reporter genes such as neo or cat or dhfr that were expressed from AAV transcription promoters or from 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 and col., 1988, J. Virol., 62: 1963-1973; Lebkowski et al., 1988 Mol. Cell. Biol. 7: 349-356). These vectors were packaged into AAV transducer particles by simultaneous transfection into adenovirus infected cells together with a second packaging plasmid containing the rep and cap genes expressed from wild-type AAV transcription promoters. In an attempt to avoid packaging the packaging plasmid containing the AAV rep and cap genes expressed from wild type AAV transcription promoters. In an attempt to avoid packaging of the packaging plasmid into AAV particles, various approaches were taken. In some cases (Hermonat & Muzyczka, 1984; Mclaughlin et al., 1988) the packaging plasmid had a large region of bacteriophage lambda DNA inserted into the AAV sequence to generate an oversized genome that could not be packaged. In other cases, (Tratschin et al., 1984b; Tratschin et al., 1985, Lebkowski et al., 1988), the packaging plasmid had the ITR regions of AAV deleted so that it could not be cleaved and replicated. and therefore could not be packaged. All of these approaches failed to avoid the generation of particles containing wild-type AAV DNA and also failed to generate efficient high titers of AAV transducer particles. In fact, Hermonat & Muzyczka, 1984, cited titles of no more than 10⁴ ml. The production of wild-type AAV particles in these studies was likely due to the presence of overlapping homology between the AAV sequences present in the vector and the packaging plasmids. Senapathy and Carter (1984, J. Biol. Chem. 259: 4661-4666) showed that the degree of recombination in such a system is roughly equivalent to the degree of overlap of the sequence. It was suggested in a review of the first paper (Carter 1989, Handbook of Parvoviruses, Vol. II, pp. 247-284, CRC Press, Boca Raton, FL) that 10 titles could be obtained.⁶ per ml, but this was based on the studies cited above in which large amounts of wild-type AAV contaminated the vector preparation. Such vector preparations containing wild-type AAV were not useful in human gene therapy. Furthermore, these early vectors showed low transduction efficiencies and did not transduce more than 4 or 2% of cells from cultures of various human cell lines even though the vectors were delivered at multiplicities of up to 50,000 particles per cell. This may have partly reflected contamination with wild-type AAV particles and the presence of the AAV rep gene in the vector. Furthermore, Samulski et al. (1989, J. Virol.63: 3822-3828) showed that the presence of wild-type AAV significantly enhanced the yield of packaged vector. Thus, in packaging systems in which wild-type AAV production is eliminated, the yield of packed vector can actually be decreased. However, for use in any human clinical application it will be essential to eliminate wild-type AAV production.

Additional studies (McLaughlin et al., 1988: Lebkowski et al., 1988) to generate AAV vectors that did not contain the AAV rep or cap gene still encountered wild-type AAV generation and still produced transduction frequencies. very low in human cell lines. Thus, McLaughlin et al., 1988 reported that rep vectors^- chap^- AAV containing the neo gene packaged with the same packaging plasmid used earlier by Hermonat & Muzyczka (1984) still contained wild-type AAV. As a consequence it was only possible to use this virus at a multiplicity of 0.03 particles per cell (ie 300 infectious units per 10,000 cells) to avoid double hits with the vector and wild-type particles. When the experiment was carried out in this way, infecting 32,000 cells with 1,000 infectious units, an average of 800 geneticin resistant colonies were obtained. Although this was interpreted as a demonstration that the virus was capable of yielding a transduction frequency of 80%, in fact only 2.5% of the cells were transduced. Thus the effectively useful titer of this vector was limited. Furthermore, this study did not demonstrate that the actual titer of the vector preparation was slightly higher than those previously obtained by Hermonat & Muzyczka (1984). From

ES 2 216 005 T3 In a similar way, Lebkowski et al., 1988, packaged AAV vectors that did not contain the rep or cap gene and used an ori packaging plasmid^- pBa1A identical to that used earlier by Tratschin et al., (1984b, 1985) and reported transduction frequencies that were similarly low, since for many human cell lines no more than 1% of the cells could be transduced for resistance to geniticin even with its most concentrated vector stock solutions. Lebkowski et al., (1988) did not report the actual vector titers in a significant way but biological analyzes showing a transduction frequency of no more than 1% when 5 x 10⁶ cells were exposed to three ml of vector preparation indicate that the titer was less than 2 x 10⁴ . Also, the packaging plasmid pBa1 contains overlapping homology to the ITR sequence and leads to the generation 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 that again showed a titer very low.

Samulski et al., (1987, J. Virol., 61: 3096-3101) constructed a plasmid named pSub201 that was an intact AAV genome in a bacterial plasmid but had a 13 nucleotide deletion at the end of each ITR and thus it was rescued and replicated less efficiently than other AAV plasmids containing the complete AAV genome. Samulski et al. (1989, J. Virol., 63: 3822-3828) constructed AAV vectors based on pSub201 but with rep and cap deleted and containing a hyg or neo gene expressed from an early SV40 gene promoter. They packaged these vectors by simultaneous transfection with a packaging plasmid called pAAV / Ad that consisted of the entire AAV nucleotide sequence from nucleotide 190 to 4490 included at either end with a copy of the adenovirus ITR. In this packaging plasmid the AAV rep and cap genes were expressed from the natural AAV promoters p5, p19 and p40. The role of the adenovirus ITR in pAAV / Ad was thought to be to enhance the expression level of AAV capsid proteins. However, rep is expressed from its homologous promoter and is down-regulated and thus its expression is limited. Using their packaging system Samulski et al., 1989, generated AAV vector stock solutions that were substantially free of wild-type AAV but had transduction titers of only 3 x 10⁴ hygromycin resistant units per ml of supernatant. When a wild-type AAV genome was used in the packaging plasmid the prep titer of the AAV vector increased to 5 x 10⁴ . The low titer produced in this system therefore appears to have been due in part to the defect in the ITR sequences of the basic plasmid pSub201 used for the construction of the vector and in part due to the limiting expression of the AAV genes from pAAV / Ad . In an attempt to increase the titer of the AAVneo vector preparation, Samulski et al., 1989, generated vector stock solutions by mass transfecting thirty 10 cm plates of 293 cells and concentrating the vector stock solution by mass formation. of bands in CsCl. This produced an AAVneo vector stock solution containing a total of 10⁸ particles measured by DNA point hybridization analysis. When this vector stock solution was used at multiplicities of up to 1,000 particles per cell, a transduction frequency of 70% was obtained. This suggests that the particle-to-transduction ratio is approximately 500 to 1,000 particles since at the rate of one unit of transduction per cell, the expected proportion of cells that should be transduced is 63% according to the Poisson distribution.

Although the system of Samulski et al., 1989, using the vector plasmid pSub201 and the packaging plasmid pAAV / Ad had no overlapping homology in the AAV sequence between the two plasmids, there is overlapping homology at the sites XbaI and recombination of these sites leads to the generation of full-length wild-type AAV. That is, although overlapping AAV sequence homology is not present, the entire AAV sequence is contained in the two plasmids, and therefore recombination can generate wild-type AAV, which is undesirable. That this kind of recombination occurs in AAV plasmids was shown by Senapathy & Carter (1984, J. Biol. Chem. 259: 4661-4666). Therefore, due to problems of low titer and the ability to generate wild-type recombinants, the system described by Samulski et al., 1989, has no utility for human gene therapy.

AAV vectors have been described in various other reports. Srivastava et al., (1989, Proc. Natl. Acad. Sci. USA, 86: 8078-8082) describe an AAV vector based on the plasmid pSub201 of Samulski et al., (1987), in which the coding sequences AAV were replaced by the coding sequences of another parvovirus, B19. This vector was packaged into AAV particles using the packaging plasmid pAAV / Ad and generated a functional vector, but titers were not reported. This system was based on pSub201 and therefore suffers from the defects described above for this plasmid. Second, the vector and the packaging plasmid both contained the overlapping AAV sequences (the ITR regions) and thus recombination to contaminating wild-type virus was highly likely.

WO92 / 10574 is concerned with the inhibition of HIV. This document refers to plasmids that have a rep gene operably linked to the HIV-LTR promoter and that contain an A17VITR sequence.

Chaterjee et al., (1991, Vaccines 91, Cold Spring Harbor Laboratory Press, pp. 85-89), Wong et al., (1991 Vaccines 91, Cold Spring Harbor Laboratory Press, pp. 183-189) and Chaterjee et al. . (1992, Science, 258: 1485-1488) describe AAV vectors designed to express antisense RNA directed against infectious viruses such as HIV or Herpes Simplex Virus. However, these authors did not report on any of the titles of their AAV stock solutions. Furthermore, they packaged their vectors using an Ori packaging plasmid analogous to that used by Tratschin et al. (1984b, 1985) containing fragment Ba1Adelgenome of AAV and forlotantosu

ES 2 216 005 T3 packaging plasmid contained AAV vector sequences that had homology to AAV sequences that were present in its vector constructs. This will also lead to the generation of wild-type AAVs. Thus, Chaterjee et al., And Wong et al., Used packaging systems known to provide only low titers and which may lead to the generation of wild-type AAV genomes due to the overlapping homology of the vector and the packaging sequences. .

Other reports have described the use of AAV vectors to express genes in human lymphocytes (MuroCacho et al., 1992, J. Immunotherapy, 11: 231-237) or in a human erythroid leukemia cell line (Walsh et al. , 1992, Proc. Natl. Acad. Sci. USA, 89: 7257-7261) with vectors based on the vector plasmid pSub201 and the packaging plasmid pAAV / Ad. Again, the titers of the starting solutions 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.

The transduction of human airway epithelial cells, developed in vitro from a patient with cystic fibrosis, with an AAV vector expressing the selective neo marker gene from the AAV p5 promoter was reported (Flotte et al. ., 1992, Am. J. Respir. Cell. Mol. Biol. 7: 349-356). In this study the AAVneo vector was packaged into AAV particles using the packaging plasmid pAAV / Ad. Up to 70% of the cells in the culture could be transduced for resistance to geneticin and the particle-transduction ratio was similar to that reported by Samulski et al., (1989). Thus to obtain a transduction of 70% of the cells, a multiplicity of up to several hundred vector particles per cell was required. The transduction of human airway epithelial cells in in vitro culture using an AAV transduction vector expressing the CFTR gene from the AAV ITR promoter showed that the cells could be functionally corrected for the electrophysiological defect in the function of the chloride channel that exists in the cells of cystic fibrosis patients (Egan et al., Nature, 1992, 358: 581-584; Flotte et al., J. Biol. Chem. 268: 37813790).

The studies cited above suggest that AAV vectors may have potential utility as vectors for the treatment of human diseases by gene therapy. However, the ability to generate sufficient amounts of AAV vectors has been a serious limitation of the development of gene therapy in humans using AAV vectors. One aspect of this limitation has also resulted in the prior absence of any study using AAV vectors in in vivo animal models. This is generally reflective of the difficulty associated with generating sufficient amounts of AAV vector stock solutions that have a sufficiently high titer to be useful in the analysis of in vivo release and gene expression. One of the limiting factors for AAV gene therapy has been the relative inefficiency of the vector packaging systems that have been used. Due to the lack of cell lines that express the trans-complementing functions of AAV, such as rep and cap, packaging of AAV vectors into adenovirus-infected cells has been achieved by simultaneous transfection of a packaging plasmid and a vector plasmid. . The efficiency of this procedure may be limited by the transfection efficiency of each of the plasmid constructs, and by the level of expression of the Rep proteins from the packaging plasmids described to date. Each of these problems appears to correspond to the biological activities of the AAV Rep proteins. Furthermore, as noted above, all of the packaging systems described above have the ability to generate wild-type AAV by recombination.

The lack of cell lines stably expressing functional Rep apparently reflects a cytotoxic or cytostatic function of Rep as demonstrated by inhibition by Rep of neo-resistant colony formation (Labow et al., 1987; Trempe et al., 1991) . This also appears to be related to the tendency of Rep to reverse the immortalized phenotype in cultured cells, which has made the production of cell lines that stably express functional Rep extremely difficult. Numerous attempts have been made to generate cell lines that express Rep. Mendelsonycol., (1988, Virology, 166: 154-165) reported obtaining in a cell line a somewhat low level of the expression of AAV Rep52 but not of Rep78 or Rep68 protein after stable transfection of HeLa or 293 cells with plasmids containing the AAV rep gene. Due to the absence of the Rep78 and Rep68 proteins, the vector could not be produced in the cell line. Another cell line made a barely detectable amount of Rep78 that was not functional.

Vincent et al. (1990, Vaccines 90, Cold Spring Harbor Laboratory Press, pp. 353-359) attempted to generate cell lines containing the AAV rep and cap genes from normal AAV promoters, but these attempts were unsuccessful because vectors were contaminated with 100 times more wild-type AAV particles or because the vectors were produced only at very low titers of less than 4 x 10³.

In an alternative approach, Lebkoswski et al. (US Patent 5,173,414, issued December 22, 1992) constructed cell lines containing AAV vectors on an episomal plasmid. These cell lines could then be infected with adenovirus and transfected with the complementary AAV functions in trans rep and cap to generate AAV vector preparations. This is claimed to allow higher titers of AAV starting solutions to be produced. However, in the examples shown, the only information regarding the titer shown is that a human cell line, K562, could be transduced only at efficiencies of 1% or less, which does not indicate the production of high titer of any vector. by AAV. In this system the vector is carried as an episome (non-integrated construct), and it is stated that integrated copies of the vector are not preferred.

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The approach to packaging AAV vectors described by Lebkowski et al., 1992, has several undesirable aspects. First, maintaining the vector in the form of a high copy number episomal plasmid not integrated into a cell line is undesirable because the copy number per cell cannot be tightly controlled and episomal DNA is much more likely to undergo a transposition leading to the production of defective vectors. Second, in this system, the vector must be packaged by infecting the cell line with adenovirus and introducing a plasmid containing the AAV rep and cap genes. The plasmid used by Lebkowski et al., 1992 was again pBa1 which, as noted above, has overlapping homology to vector ITR sequences and will result in the generation of wild-type AAV. Third, in the packaging plasmid pBa1 used by Lebkowski et al., 1988, 1992, the rep gene is expressed outside its homologous p5 promoter and is thus negatively self-regulated and therefore expression of rep is likely to be limited. .

The problem of suboptimal levels of expression of rep after transfection with the plasmid may be related to another biological activity of these proteins. There is evidence (Tratschin et al., 1986, Mol. Cell. Biol. 6: 28842894) that AAV Rep proteins down-regulate their own expression from the AAV p5 promoter that has been used in all previously described packaging constructs such as pAAV / Ad (Samulski et al., 1989) or pBa1 ( Lebkowski et al., 1988, 1992)

Compendium of the invention

One of the basic challenges for gene therapy has been the development of strategies for the transduction of cells and tissues that cannot be easily manipulated ex vivo or that are not actively dividing. AAV vectors can achieve gene transfer in vivo in the respiratory tract, for example, but high titers are critical so as to allow a sufficiently high multiplicity of vector for release in as small a volume as possible. This makes optimal packaging methodology of central importance in determining the feasibility of an AAV-based gene therapy. Stable adjuvant-free AAV packaging cell lines have been difficult to find, mainly due to the activity of the Rep protein, which downregulates its own expression and reverses cell immortalization. The approaches described in this invention effectively avoid these problems and have enabled substantial improvements in packaging efficiency.

The use of an HIV-LTR promoter to express high levels of AAV Rep proteins has been reported elsewhere (Antoni et al., 1991), but the application of this expression system to the packaging of recombinant AAV vectors is a new progress. In fact, Rep expression levels have not previously been shown to be limiting in the process of packaging the AAV vector by simultaneous transfection. The fact that a 100-fold increase in packing titer is achieved by increasing Rep expression provides direct evidence that Rep levels are limiting in this circumstance. The fact that simultaneous transfection with pARtat did not further increase the efficiency of packaging may indicate that (1) the expression level of the HIV promoter in 293 cells was maximized even in the absence of tat or (2) that the levels of Rep achieved with pRS5 alone were sufficient to ensure that Rep expression was no longer limiting for packaging efficiency. It would now be obvious that promoters other than AAV can be used to generate Rep in packaging plasmids analogous to pRS5.

Similarly, the phenomenon of rescue of integrated recombinant AAV genomes is known (Tratschin et al., 1985; Flotte et al., 1993a), but has never before been applied to produce a vector-producing cell line as described above. described here.

The overall packaging efficiency of the pRS5 vector cell line system was at least 10⁴ particles per packaging cell, which will be more than sufficient to allow the production of reagents for recombinant clinical-grade AAV vectors. Generation of wild-type AAV has not been observed with this method, which is an additional advantage over most simultaneous transfection methods. These improvements make the production of clinical grade recombinant AAV vectors for use in gene therapy feasible.

Described herein are procedures and constructs that allow the production of high titer AAV vectors in the absence of wild-type AAV generation.

Accordingly, one embodiment of the invention is a method for the generation of high titer recombinant AAV vectors comprising:

(a) providing cells containing at least one intact copy of a stably integrated recombinant AAV vector, wherein the AAV vector consists of AAV inverted terminal repeat (ITR) regions and a transcriptional promoter operably linked to a polynucleotide target, and where the expression of the rep gene is limiting in said cells;

(b) provide an AAV packaging plasmid that allows expression of the rep gene product, where in the plasmid the rep gene is operably linked to a heterologous promoter, and where the packaging plasmid lacks overlapping homology to the sequences of AAV from the cell vector provided in (a);

ES 2 216 005 T3 (c) inserting the AAV packaging plasmid into the cell and incubating the cell under conditions that allow AAV replication; and (d) isolating the recombinant AAV vectors produced in step (c).

Included in this embodiment are methods in which the promoter of the packaging plasmid to which Rep is operably linked is HIV-LTR, and those methods in which the packaging plasmid is pRS5.

Also included in this embodiment are methods in which the target polynucleotide encodes a polypeptide that can function as a cystic fibrosis transmembrane conductance regulator (CFTR).

Another embodiment of the invention is a packaging system for the generation of high titer recombinant AAV vectors comprising:

(a) cells containing at least one intact copy of a stably integrated recombinant AAV vector, where the AAV vector consists of AAV inverted terminal repeat (ITR) regions and a transcriptional promoter operably linked to a target polynucleotide , and where the expression of the rep gene is limiting in said cells; and (b) an AAV packaging plasmid that allows expression of the rep gene product, where in the plasmid the rep gene is operably linked to a heterologous promoter, and where the packaging plasmid lacks overlapping homology with the sequences of AAV from the cell vector provided in (a).

Yet another embodiment of the invention is a packaging plasmid for use in the production of high titer generation recombinant AAV vectors that allow expression of the rep gene product, where the plasmid consists of the rep gene operably linked to a heterologous promoter.

Brief description of the figures

Figure 1 is a diagram of the AAV life cycle.

Figure 2 is a schematic showing the production of the packaging plasmid pRS5, and the relationship of the HIV-LTR promoter and the coding sequence of the AAV2 rep and cap genes.

Figure 3 is a halftone reproduction of Southern hybridizations of Hirt extraction DNA samples showing salvageable intact AAV-neo genomes in stable cell lines.

Figure 4 is a halftone reproduction of point hybridizations comparing neo detection in control and packaged AAV-neo genomes.

Figure 5 is a halftone reproduction of Southern hybridizations of Hirt extraction DNA samples showing rescue of the AAV CFTR vector (TRF42).^- of vector producing clones in 293 cells.

Figure 6 is a graph of the percentage of lung carcinoma cells stained for the CD44 marker, as determined by antibody staining and FACS analysis.

Detailed description of the invention

AAV vectors have relevance for gene therapy in humans, particularly for diseases such as cystic fibrosis and sickle cell anemia. The invention described herein provides methods and materials for use in the production of high titer recombinant AAV vectors for use in gene therapy.

In the practice of the present invention, conventional mechanisms of molecular biology, microbiology, DNA recombination, and immunology will be employed, unless otherwise indicated, which are within the logical judgment of the art. Such mechanisms are explained in detail in the literature. See, eg, Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989), Oligonucleotide Synthesis (MJ Gail Ed., 1984), Animal Cell Culture (RI Freshney, Ed., 1987), Methods in Enzymology series (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos eds. 1987), Handbook of Experimental Immunology, (DM Weir and CC Blackwell, Eds.), Current Protocols in Molecular Biology (FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Siedman, JA Smith, and K. Struhl, eds., 1987), and Current Protocols in Immunology (JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach, and W. Strober, eds., 1991).

The generation of high-titer recombinant AAV vectors of heterologous polynucleotides requiring transcription is completed by the following method.

ES 2 216 005 T3

Cloned cells containing a suitable AAV vector plasmid are provided in the method. The suitable AAV vector plasmid consists of the ITR regions of AAV and a transcription promoter operably linked to a target polynucleotide. The transcriptional promoter that is connected to the target polynucleotide allows the formation of transcripts, and includes, for example, non-AAV promoters as well as AAV promoters such as p5, p19, p40, and AAV ITR promoters. The transcription and / or translation products of the target polynucleotide preferably find use in gene therapy. Thus, target polynucleotides include genes to be released for gene therapy, for example, those that encode chains of the coding subunit of hemoglobin, of enzymes, of proteins such as the transmembrane conductance regulator of cystic fibrosis ( CFTR), and the like. The target polynucleotides can also be polynucleotides that when transcribed have activity as antisense molecules, such as lures that bind to transcriptional or translation factors, such as ribozymes, and the like.

A requisite feature of the cloned cells provided for the method is that they contain at least one intact copy of the AAV vector plasmid that is stably integrated into the cell and can be rescued by infection of the transfected cell with a helper virus such as adenovirus when also Complementary AAV rep or rep and cap functions are provided.

In the examples shown below the authors of the present invention have used the AAVneo vector in which the initial selection of the cell line containing the vector was carried out by geneticin selection. However, it should be obvious to one of ordinary skill in the art that to generate cell lines containing a vector, such as an AAV vector containing a CFTR gene, in which a selective marker is not included, it is straightforward to transfect the cells together. with the desired vector plasmid and a second plasmid containing the selective marker. Upon selection of cell clones based on the selective marker, it would be obvious to use direct screening to easily identify those from which a vector can be rescued at a high titer. An example of this is reported by Flotte et al. (1992a), although in that example the cells were rescued by infection with AAV viruses and adenoviruses. As reported by Flotte et al. (1993a), producer cell lines containing a salvageable AAV vector that did not contain a selectable marker in the vector could also be obtained. In that example, the AAV vector plasmids comprised constructs containing the human CFTR cDNA operably linked to an AAV promoter made up of the ITRs. Cell lines containing stably integrated copies of these vectors were derived by simultaneous transfection of the human epithelial cell line IB-3 with the AAV-CFTR vector plasmid and a second plasmid containing the selectable marker neo. Upon selection of the colonies on geneticin, individual clones were obtained containing the stably integrated vector from which the vector could be rescued by subsequent infection with helper adenovirus and wild-type AAV particles. This clearly allows the generation of clones that have stably integrated copies of a vector in which the vector itself does not have a selectable marker.

The method also includes providing a complementing packaging plasmid from which the Rep or Rep and Cap proteins can be expressed from the rep or rep and cap genes. The packaging plasmid lacks overlapping homology to vector sequences between and including the AAV ITR sequences. On the other hand, the combination of the packaging plasmid and the vector cannot yield a complete AAV genome. In the example shown below, the 120 nucleotide long sequences around the AAV p5 promoter are absent from the packaging plasmid and vector. Furthermore, in the packaging plasmid the rep gene is not transcribed from the AAV p5 promoter, instead it is operably linked to a heterologous transcriptional promoter that is not strongly self-regulated in a negative way by expression from rep. .

In the preferred example of a packaging plasmid, such as pRS5, we have used HIV-LTR as a heterologous promoter but any heterologous promoter can be used, and preferably a constitutive or inducible promoter. The HIV-LTR is an example of both types of promoter. Generally, this promoter is inducible at a very high level of expression through the action of the tat protein. However, in the preferred example shown here, the HIV-LTR promoter shows high levels of constitutive expression of rep when used in 293 cells. This is because 293 cells express the adenovirus EIA gene product that is known to be transactive. the HIV-LTR promoter. Thus, in 293 cells (which are the preferred cell line for establishing vector-containing cells) further transactivation of the HIV-LTR promoter in pRS5 may not be necessary to obtain a maximum level of functional rep expression. If vector-producing cell lines are made in other cells, transactivation of pRS5 may be desirable, by adding a tat expression plasmid such as pARtat (Antoni et al. 1991). Such cell lines could include any of the human cell lines such as HeLa, A549, KB, Detroit, WI38 or any of the cell lines in which appropriate helper functions can be expressed. When a human adenovirus is used as an adjuvant, this could also include the desired monkey cell line, VERO. Alternatively, if herpesviruses or poxviruses such as vaccinia or avipox are used to provide adjuvant function, any appropriate human, rodent, or simian cell line may suffice as the vector-producing cell.

The packaging plasmid pRS5 serves as a model for either an inducible or a constitutive promoter. It would be obvious to one of ordinary skill in the art that many other inducible or constitutive promoters can be used in the packaging plasmid construct. The first characteristic of the packaging plasmid is that it does not contain the wild-type AAV p5 promoter and therefore is not strongly negatively self-regulated by rep. Examples of such promoters would be mutations of the wild-type p5 promoter that eliminate homology with the parent wild-type promoter or that inactivate negative regulatory elements of this promoter.

ES 2 216 005 T3 such as the YYI region of the p5 promoter. Examples of inducible promoters include: metal ion inducible promoters such as the metallothionein promoter; promoters inducible by steroid hormones such as the MMTV promoter; or the growth hormone promoter; promoters that would be inducible by the helper virus such as the adenovirus early gene promoter inducible by the adenovirus EIA protein, or the adenovirus major late promoter; the herpesvirus promoter inducible by herpesvirus proteins such as VP16 or 1CP4 or the promoters inducible by vaccinia or poxvirus or the promoters inducible by a poxvirus RNA polymerase or a bacterial promoter such as that of phage T7 that would be inducible by an RNA polymerase poxvirus or a bacterial promoter such as T7 RNA polymerase.

There are many strong constitutive promoters that will be suitable for use as a heterologous promoter for expression of rep in the packaging plasmid, including the adenovirus major late promoter, cytomegalovirus immediate early promoter, β-action promoter, or the / 1-globin. Promoters activated by RNA polymerase III could also be used.

The efficiency of the packaging plasmid for the complementation of the Rep and Cap functions for the AAV vector plasmid can be tested using AAV expression vectors that lack the Rep and / or Cap function, and further contain a marker. Such expression vectors are known in the art, and include, for example, the pAAVp5neo construct (Flotte et al., 1992). In the Examples, pAAVp5neo was used as a vector construct to test each of the described packaging mechanisms since it could be titrated for the number of particles both by point hybridization of the DNA (Samulski et al., 1989) and by titers. neo transduction.

The packaging plasmid is introduced into cells containing the integrated AAV vector plasmid by any suitable mechanism known in the art, including, for example, transfection, electroporation, and the like. Following introduction of the packaging plasmid, cells are grown for 3 to 5 days under conditions that allow AAV replication, lysates are prepared, and recombinant AAV vector particles are purified by techniques known in the art.

The examples presented below are provided as an additional guide to the practitioner of skill in the art, and are not intended to be limiting of the invention in any way.

The plasmid pRS5 from the E. coli DH5 cell line (E. coli :: strain pRS5) was deposited on November 9, 1993 in the American Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville, Maryland 20852, and It has been consigned with Access Number 69483. The consignment was made under the terms of the Budapest Treaty. Upon the grant and promulgation of this application as a United States Patent, any restriction on the availability of the consignment will be irrevocably removed; and access to the designated lockers will be available during the aforementioned request delay for the one determined by the Commissioner authorized to do so under 37 CFR § and 35 USC § 1.22. On the other hand, the designated consignments will be maintained for a period of thirty (30) years from the date of the consignment, or for five (5) years after the last request for the consignment; or for the life of the enforceable United States patent, whichever is longer. The listed materials mentioned herein are for the sole purpose of convenience, and it is not required to practice the present invention in light of the present disclosures.

Examples

Example 1

Packaging Plasmid pRS5

In the packaging plasmid pRS5 the AAV p5 promoter is replaced by a heterologous promoter so that the expression of the rep gene polypeptide does not negatively self-regulate its own synthesis.

The plasmid pSR5 was constructed by ligating the HindIII fragment to the large SphI (5 kb) of the plasmid pHIVrep described above (Antoni et al., J. Virol., 65: 396-404) (containing the HIV-LTR promoter and the rep gene sequences including AAV nucleotides 263 to 1886 flanked by the plasmid sequence of pBR322) with the HindlII to Sphl fragment of a plasmid named pcapl ( containing the AAV cap gene from nucleotides 1886 to 4491 without the AAV ITR again flanked by the sequences of pBR322) (see Fig. 2). After ligation of these two fragments, a packaging plasmid was produced, pRS5 in which the AAV rep gene (Rep proteins 68 and 78) is transcribed from the HIV-LTR promoter, transcribing the p promoters.₁₉ And p₄₀ from internal AAVs the smaller Rep products (40 kD and 52 kD Rep proteins) and capsid proteins, respectively. Thus pRS5 contains the complete AAV coding sequence in the AAV nucleotide sequence from nucleotide 263 to 4491 which includes the rep coding sequence for Rep 78 and Rep 68 operably linked to the heterologous HIV-LTR promoter and expresses Rep 52 and Rep 40 from from the AAV p19 promoter and AAV capsid proteins from the p40 promoter. The map of this construct is shown in Figure 1.

The pAAVp5neo construct (Flotte et al., 1992) was used as a vector construct to test each of the described packaging mechanisms since it could be titrated both in terms of the number of particles by point hybridization of the DNA (Samulski, et al., 1989) and by neo transduction titers.

ES 2 216 005 T3

Example 2

Cell Lines

Human 293-31 cells (Graham et al., 1967, J. Gen. Virol., 36: 59-72) were grown in Essential Medium

Modified Eagle's with 10% fetal calf serum at 37 ° C in CO₂ at 5%. The 293 cell line was used for both packaging of vector preparations and neo transduction experiments to verify neo transduction titers.

Example 3

Packaging of AAV virions with an HIV-LTR promoter-rep gene plasmid

The pRS5 construct was used to package the vector plasmid pAAVp5neo by simultaneous transfection into 293 cells infected with adenovirus type 5 (Ad5) (Flotte et al., 1992). A total of ten 10 cm plates were infected each containing 2 x 10⁶ 293 cells (semi-confluent) with 2 x 10⁶ pfu of Ad5 (moi = 1) 1 hour before transfection with 12.5 μg each of pRS5 and pAAVp₅neo. The cells were then incubated for 3 days at 37 ° C before harvesting. Cells were scraped and pooled by low speed centrifugation (4,000 rpm x 10 minutes).

The cell pellet was then resuspended in 4 ml of 10 mM Tris-HCl, pH 8.0, lysed by freeze-thaw three times, pushed through a 25 g needle several times to decrease the viscosity, and treated with micrococcal nuclease (40 µl of 300 µ / µζ stock solution incubated at 37 ° C x 20 minutes, then at 4 ° C for 10 minutes, then CsCl was added to a final density of 1.41 g / cc. Each tube was covered with 0.5 to 1.0 ml of mineral oil and centrifuged in an oscillating bucket rotor (SW50) at 35,000 rpm at 4 ° C for 12 hours.

Serial 0.5 ml fractions were then collected and each one was titrated by DNA point hybridization (Samulski et al., 1989), with dilutions to the tenth of each fraction transferred on nitrocellulose filters and hybridized with a DNA probe. marked with P³² prepared from a 2.3 kb neo gene fragment using the Boehringer-Mannheim Random Priming Kit. The selected fractions were then dialyzed against Ringer's balanced salt solution, pH 7.4, and stored at -20 ° C for use in transduction titration experiments.

Example 4

Determination of transduction titers of AAV-neo vector starting solutions

The AAV-neo vector stock solutions produced as outlined above were titrated in 293-31 cells by infecting 293-31 cells at ten-fold increasing particle multiplicities ranging from 10 to 1,000 particles per cell. In each case the cells were seeded in microtiter wells at 10⁴ cells per well, and then infected for 2 hours by direct inoculation of the vector into the medium. Cells in each well were then trypsinized 24 hours later and plated on 4-100 mm plates. Three plaques from each group were then selected in G418 at a dose of 200 µg / ml (active, starting 48 hours after original infection). The fourth plate in each group was grown without G418 as a control for plating efficiency. Cells were selected for 10-14 days, and then stained with Safranin Red. Geneticin resistant colonies were counted and the transduction frequency was determined by dividing the mean number of gene colonies in each group by the number of colonies observed on the plating efficiency control plate. A transduction unit (tu) was then defined in this analysis as the volume of inoculum per cell required to transduce 63% of the cells for geneticin resistance.

Example 5

Improvement of AAV vector packaging by simultaneous transfection using an expression vector with the HIV-LTR promoter

In Table 1 (experiments 1 to 3) the results of the titrations by point hybridization of the DNA of the AAV-neo particle preparations produced by the mechanism of simultaneous transfection pAAV / Ad established above and a simultaneous transfection with parallel pRS5 are shown.

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TABLE 1

Particle Title of AAV Vector Preparations

<td>Exp. #</td><td></td><td colspan="2">Vector Production Method³</td><td>Vector Particles Title¹³</td>

<td></td><td>Cells</td><td colspan="2">Transfected with DNA</td><td>Qualification</td>

<td></td><td></td><td>Plasmid Vector</td><td>Plasmid Packaged.</td><td>(particles / ml)</td>

<td> 1</td><td></td><td>pSA206</td><td>pAAV / Ad</td><td>4.0 x 10⁹</td>

<td> 2</td><td> 293</td><td>pSA206</td><td>pRS5</td><td>3.0 x IO¹⁰</td>

<td> 3</td><td> 293</td><td>pSA206</td><td>pRS5 + pARtat</td><td>4.0 x IO¹⁰</td>

<td> 4</td><td>neo4-6</td><td> -</td><td>RS5</td><td>2, 6 x 10¹¹</td>

^to Vector preparations were derived as follows. All vector preparations represent vectorAAVneo derived from plasmid vectorpAAVp5neo (pSA206). In experiments 1, 2, and 3 the vector preparation was derived by infection of 293 cells with aadenovirus as adjuvant and simultaneous infection of the infected cells with the plasmid vectorpAAVp5neo (pSA206) ​​and the packaging plasmid was pAAV / Ad, pRS5 or pRS5 plus pARtat as indicated. In experiment 4 the vector was obtained by rescue of an integrated vector from the neo4-6 cell line by transfection of the cell line with the packaging plasmid pRS5 in the presence of an infecting adenovirus 5 as adjuvant. The neo4-6 cell line was derived by transfection of 293 cells with the vector plasmid pAAVp5neo (pSA206) ​​and selection with the antibiotic G418 (geneticin) for resistance to geneticin. All vector preparations were purified by CsCl banding.

^b The purified vector preparations were analyzed for particle titer by spot hybridization analysis.

The results in Table 1 show that the titers achieved with the pRS5 construct introduced by simultaneous transfection (experiment 4) were approximately five to ten times higher than those obtained using the packaging plasmid pAAV / Ad. The addition of the HIV trans-activator gene (tat) by tritransfection resulted in very little additional packaging efficiency. In the experiment shown in Figure 3, there was only a 30% increase in particle titer from the maximum titer fraction, from 3.0 x 10¹⁰with pRS5 only at 4.0 x 10¹⁰ with pRS5 + pARtat. Based on these results, it seemed highly probable that the expression levels of rep and cap were no longer limiting of this mechanism.

The experiment of Figure 3 was performed as follows.

Example 6

Rescue of intact AAV-neo genomes present in stable cell lines

In this example, a 2 x 10 crop⁶ Human 293 cells were transfected with 5 micrograms of the AAV vector plasmid pAAVp5 neo (pSA206). Individual colonies were selected for geneticin resistance using 200 micrograms of active G418 (Ginco-BRL) per ml of medium, beginning 48 hours after transfection. Individual G418 resistant colonies were isolated with sterile cloning cylinders and expanded into stable cell lines. From several of these cell lines, 2 x 10 were infected⁶ cells with both adenovirus type 5 (moi = 2) and wild-type AAV2 (moi = 2). Forty-eight hours later, low molecular weight DNA was selected using Hirt's high salt detergent procedure and analyzed by 0.7% agarose gel electrophoresis and Southern blotting with a labeled neo DNA probe. with P³² random barley. The results of Southern blot hybridization of Hirt extracted DNA are shown in Figure 3.

The Figure shows that from 7 to 8 individual cell lines examined (lanes A to G), the vector sequence could be rescued and replicated. Lane H shows an example of a geneticin resistant cell line from which the vector could not be rescued.

The expected intracellular replicating species of the vector genomes are indicated (mRF for the replicating form

ES 2 216 005 T3 monomeric duplexes and RFd for the replicating dimeric duplex form) and single-stranded (SS) genomes. In at least 6 of the 8 examples (Lanes A to D and Lanes F and G) untransposed copies of the vector were rescued.

Example 7

The use of cell lines with the integrated salvageable AAV vector improves packaging efficiency

After enhancing the expression of the AAV rep and cap genes with the pRS5 construct, the present inventors sought to further improve packaging efficiency by producing uniform cell populations containing integrated but salvageable copies of the AAV-neo vector genome. Combination of rep and cap pRS5 transfection with stable addition of vector pAAVp₅neo to cell lines resulted in a significant improvement in packaging efficiency.

Cell lines were produced by transfecting AAV-p5neo by infection with both wild-type AAV2 and Ad5 at a moi of 5 for each virus. Hirt extraction was used to isolate replicating viral DNA, and these DNA samples were analyzed by electrophoresis and Southern blot hybridization using a P-labeled neo probe.³². As shown in Figure 3, salvageable AAV-neo recombinants were present in almost all of these cell lines. The pattern of these bands, as expected, included single strands (blurred band near the top of the gel), 2.7 kb in size duplex monomers, 5.4 kb dimers, larger multimeric forms.

Two cell lines made in a similar way and named neo4-6 and neo4-9 were constructed and used for subsequent packaging experiments. A direct comparison was made between the simultaneous transfection of pRS5 and pAAVp5neo in 293 cells and the individual transfection of pRS5 in both neo46 and neo4-9 cell lines. As shown in Table 1, the neo4-6 cell line produced packaged AAV-neo titers that were 2.6 x 10¹¹. This particle titer represented a multiple-fold improvement over the 293 cell simultaneous transfection method, and yielded the highest titer of any method or combination of methods used. Total particle titer nearly 2.6 x 10¹¹ Particles are achieved starting with 2x10⁷cells and thus represents a yield of 10⁴ particles per cell.

The above analyzes are all based on a DNA / point hybridization mechanism that could also theoretically detect copies of AAV-neo genomes that had not been packaged into AAV particles. The results of two types of control experiments excluded that possibility. First, plasmids AAVp5neo and pRS5 were simultaneously transfected into 293 cells as before, but in the absence of Ad5 infection. The lysates of these cells were treated with any of the other preparations mentioned above. As shown in Figure 4, control dot blot hybridizations indicated that the neo signals reflected the packaged AAV-neo genomes.

The DNA dot blot hybridization was carried out as follows: Starting with 50 microliters of each fraction, five serial dilutions to the tenth were made in PBS. To lyse the virions, 1/10 of the volume (5 μυ of 3 N NaOH was added, and the mixture was incubated at 65 ° C for 1 hour. An equal volume (50 μ ^ of NH₄2M OAc, pH 7.0, and the total volume of 100 μl was transferred to 0.45 μm nylon filters with a Schleicher and Schuell Minifold I micro-sample filtration manifold. These filters were then hybridized with neo DNA probes. marked with P³² randomly primed under standard conditions.

Serial one-tenth dilutions of the control stock solutions prepared as described above were compared with dilutions of an AAV-neo stock solution prepared by transfection with pRS5 of the neo4-9 cell line after infection with adenovirus ( shown in column A). Column B shows pRS5 transfection of the neo4-9 cell line without adenovirus infection. Column C shows adenovirus infection of the neo4-6 cell line without transfection with pRS5. Column D shows adenovirus infection of 293 cells transfected by pSA206 without co-transfection with pRS5. In no case did the excess cell or plasmid DNA give a significant neo signal. The results in Figure 4 show that no detectable AAV-neo signal was present in DNA / dot blot hybridization from that experiment, clearly indicating that plasmid DNA was not present in these purified preparations.

A direct comparison was then made of the transduction titer of the AAVneo vector stock solutions produced in the dual plasmid transfection system or by vector rescue from stable lines. As indicated in Table 2, the transduction frequency obtained with an equivalent number of vector particles produced in any system was similar. This indicates that vector particles generated by vector rescue from stable cell lines had the same biological efficiency and transduction potential as those produced in dual transfection. Thus, the vectors rescued from the cell lines did not have any significant biological alteration.

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TABLE 2

Biological Equivalence of AAV-neo Vector Preparations^to

<td>Preparation of</td><td>Multiplicity of</td><td>Percentage</td>

<td>Vector^b</td><td>Particles⁰</td><td>Transduced^d</td>

<td>neo4-6</td><td>10 particles / cell</td><td> 23, 1%</td>

<td>neo4-9</td><td>10 particles / cell</td><td> 44,4%</td>

<td>pRS5 trf</td><td>10 particles / cell</td><td> 32, 9%</td>

<td>pRS5 + tat</td><td>10 particles / cell</td><td> 23, 7%</td>

^to The transduction efficiency of numerous AAV-neo vector preparations was compared in the 293 cell line.

^b Vector preparations were derived as follows: All vector preparations represent AAVp5neo vector derived from vector plasmid pAAVp5neo. The preparations called neo46 and neo4-9 were obtained by rescue of an integration vector of cell lines 46 or 4-9 by transfection of the cell line with the packaging plasmid pRS5 in the presence of an infecting adenovirus 5 as adjuvant. Cell lines 4-6 and 4-9 were derived by transfection of 293 cells with the vector plasmid pAAVp5neo and selection with the antibiotic G418 (geneticin) for geneticin resistance.

The vector preparation named pRS5 trf was derived by direct simultaneous transfection of 293 cells with the plasmid vectorpAAVp5neo and the packaging plasmid pRS5 in the presence of an infecting adenovirus 5 as adjuvant. The pRS + tat vector preparation was also obtained by direct transfection exactly as for pRS5 trf except that the simultaneous transfection also included the plasmid pARtat, which expresses the HIV tat transcriptional activator.

^c Cultures of 10⁴ 293 cells with 10⁵ particles of AAV-neo vector preparations. Thus, each vector preparation was used to infect 293 cells at a multiplicity of 10 vector particles per cell.

^d The infected cultures were then grown under conditions to select for geneticin resistant colonies. The percentage of 293 cells stably transduced for geneticin resistance (i.e., transduction frequency) was calculated as the number of geneticin resistant colonies from the individual culture divided by the number of colonies obtained in an untreated control culture. with geneticin.

The above results indicate that the combined modifications described herein can yield increases in vector titers of approximately 50 to 100 times. Also, compared to previously published reports (eg, Samulski et al., 1989), this procedure can provide vector particle titers of at least 2 x 10¹¹, which is three orders of magnitude higher than previously achieved from a similar number of cells (ie, human 293 cells grown in a total of ten 10 cm cell culture dishes). Example 8

Packaging of AAV vectors encoding CFTR

An AAV-CFTR vector, pTRF42, containing CFTR cDNA expressed from an AAV ITR as a promoter in the absence of a selectable marker was used to generate stable vector producer lines in the 293 cell line by simultaneous transfection with a plasmid. pSVneo. This vector was salvageable from stable cell lines, and the rescued vector was intact and unreorganized.

The construction of pTRF42 has been described (Flotte et al. 1993a, J. Biol. Chem. 268: 3781-3790). This construct contains an AAV-CFTR vector consisting of 145 nucleotides from the 5 'end of AAV (the ITR) followed by an in-frame ATG (Met) initiation codon, which reads directly into the CFTR coding sequence from amino acid 1119 The remainder of the CFTR cDNA is intact up to the native stop codon and up to nucleotide 4629 of the original sequence. This is followed by a synthetic polyadenylation signal, and then by AAV nucleotides 4490-4681 (3 'ITR). Four micrograms of this vector were simultaneously transfected with one microgram of plasmid pSV2neo, at 2 x 10⁶ human 293 cells, which were then selected with 200 µg of active G418 beginning 48 hours after transfection. G418 resistant clones were then isolated with cloning cylinders and expanded. Clones were analyzed by wild-type AAV2 rescue and Ad5 infection combined (moi = 2 for each) as was done for lines containing the neo vector. The low molecular weight DNA extracts (Hirt) were analyzed again by electrophoresis

ES 2 216 005 T3 on 0.7% agarose gel and Southern blot with a P-labeled CFTR cDNA probe³² random barley. Again, the cell lines were found to have intact, salvageable vector sequences of the predicted sizes for the monomeric and dimeric replicating (RF) forms, that is, 4.6 and 9.2 kb, respectively. This confirmed the usefulness of the approach using cell lines containing salvageable vectors with the clinically significant example of an AAV-CFTR vector.

The principles and teachings described above have been applied to produce additional illustrations of the present invention, some of which are described below, that further demonstrate the utility of the present invention.

Example 9

Integration of the lacZ gene in Pulmonary Airway Epithelium

The in vivo activity of packaged vectors was tested by the foregoing methods using the lacZ gene encoding an enzyme with e'-galactosidase activity. The AAVp vector₅lacZ was made by digesting the pAAVp5neo construct with HindIII and BamHI and ligating the large fragment (containing the AAV ITRs flanking the p5 promoter and a synthetic polyadenylation sequence) with a HindIII to BamHI fragment of pSVBgal containing the lacZ gene of E. coli. This initial construct was then modified by digestion with HindIII and KpnI, blunt-ended with T4 polymerase (Boehringer-Mannheim), and re-ligation of the large fragment. This final manipulation allowed the removal of an intermediate sequence segment containing four ATG codons out of frame with the lacZ coding sequence. The vector was then packaged using plasmid pRS5 as described above.

Aliquots of packaged AAVp5lacZ containing 10¹⁰ particles in 0.2 to 0.5 ml were administered by intraperitoneal injection into three weaned C57BL mice. Aliquots of the same vehicle solution against which the vector had been dialyzed were injected into three additional mice serving as controls. The weight of these mice was approximately 30 g each, with a total number of body cells estimated to be between 10⁸ and 10⁹ cells. The mean vector dose per cell was therefore between 10 and 100 particles per cell, depending on blood flow. The animals were sacrificed four days later by pentobarbital overdose, and samples from the abdominal wall, lungs, liver, spleen, kidneys, and pancreas were collected and fixed in 2.5% glutaraldehyde.

In situ PCR analysis was performed on 5-micron paraffin-embedded tissue sections, using vector-specific primers and a non-radioactive digoxigenin-dUTP, anti-digoxigenin, alkaline phosphatase immunodetection system as previously described (Flotte and col., 1993). The primers were selected from the lacZ sequence (5 'primer: 5'-ACAACTTTAACGCCGTGCGCT-3'; 3 'primer: 5'-TGCAGGAGCTCGTTATCGCTA-3'). After a heat start at 82 ° C, a 40-cycle PCR was performed with direct incorporation of digoxigenin-labeled dUTP (Boehringer-Mannheim) to the reaction products. Digoxygenin-labeled nucleotides were then detected with an anti-digoxigenin antibody with an alkaline phosphatase tag (Genius III kit, Boehringer-Mannheim), and immunohistochemical staining (NBT, X-fos).

Various tissue sections for the vector and control injected mice were examined by light microscopy. Cells containing vector DNA were indicated by a dark purple-brown reaction product covering the nucleus. Staining was clearly seen in sections of vector injected mice but not in controls. Vector DNA was detected in all cell types of the lungs, as well as in most hepatocytes and acinar pancreatic cells. After absorption by the lymphatic route, the vector particles would have entered the venous circulation and encountered the pulmonary circulation, which probably accounts for the very efficient gene transfer in the lung. After passing through the pulmonary circulation to the general circulation, the vector particles would probably have distributed more efficiently to other organs with high blood flow.

Example 10

LacZ Gene Expression In Vivo

Serial sections of the same tissue samples used for in situ PCR detection were stained for B-galactosidase activity with X-gal reagent (0.2%, 37 ° C, 16 hours). Sections from AAVp5lacZ injected mice were compared to vehicle injected controls. Endogenous' -galactosidase activity was not detectable in control animals in sections taken from lung, spleen, liver, pancreas, kidney, or peritoneum.

The lung sections showed expression of lacZ in entire segments of airway epithelium from vector injected animals but not from controls. In some of the airways,&gt; 75% of the 200 cells examined were positively stained. Approximately 25% of the airways demonstrated this degree of positive staining (4 to 6 airways examined in sections, one to two sections per animal), while other regions of the lung had lower levels of expression.

ES 2 216 005 T3

Spleen sections demonstrated ^ -galactosidase activity in non-lymphoid areas of vector injected animals, but not of controls. The lymphoid follicles of the spleen were essentially negative. Uncommon staining was observed in non-epithelial cells of the lungs or in cells of the liver, abdominal wall, and kidneys of vector-treated animals, while expression was not detectable in the pancreas.

The distribution of vector DNA, as detected by in situ PCR analysis, is compared to the distribution of ^ -galactosidase activity in Table 3, as follows (n = 3): (- -) no staining, (+ ) cells stained with X-gal &lt;1%, (++) 1-25% staining, (+++)&gt; 25% staining, (nr) not performed.

TABLE 3

Distribution in Transfer Tissues and Expression of the lacZ Gene

<td>Organ (Fabric)</td><td>In situ PCR</td><td>X-gal staining</td>

<td>Lung (Respiratory tract)</td><td> ++ +</td><td> + + +</td>

<td>Lung (Alveoli, vessels)</td><td> +++</td><td> +</td>

<td>Spleen (Non-lymphoid)</td><td> ++ +</td><td> + + +</td>

<td>Spleen (Lymphoid follicles)</td><td> +</td><td> --</td>

<td>Liver</td><td> + + +</td><td> +</td>

<td>Pancreas</td><td> + + +</td><td> --</td>

<td>Kidney</td><td>nr</td><td> + +</td>

<td>Peritoneum</td><td>nr</td><td> --</td>

The expression of ^ -galactosidase activity therefore depends on both the distribution of the DNA vector, and the tissue specificity of the promoter. For example, the p5 promoter is very active in airway epithelial cells, but is much less active in other cells that have been tested, such as those derived from the pancreas.

Example 11

Integration and expression of the CD44 gene in lung carcinoma cells

The pSacd44 vector, which expresses the CD44 cell surface marker from the p5 promoter, was subcloned directly from the pSA206 construct (AAVp5neo). Particle numbers were determined by spot hybridization analysis. With this vector and the packaging plasmid pRS5, the lung carcinoma cell lines H209 and H82 were simultaneously transfected. Various doses of the vector were tested at 4 x 10⁴ cells per aliquot. Forty-eight to 72 hours after transfection, cells were fluorescently stained for cell surface gene expression using an anti-CD44 antibody. The cells were then passed through a fluorescence activated cell counter to determine the proportion that was positively stained.

The results of this analysis are shown in Figure 6. Virus titers are expressed in units of 10⁴; thus, "1000" on the horizontal axis represents 250 vector particles per carcinoma cell. The high frequency of CD44 expression was achieved at doses as low as 100 particles per cell.