AAV Rep Research Articles

Transient expression of AAV rep protein facilitates targeted integration of a 200 kb BAC containing the entire beta-globin locus

Transient expression of AAV rep protein facilitates targeted integration of a 200 kb BAC containing the entire beta-globin locus

Combination of Baculovirus-Mediated Gene Delivery and Packed-Bed Reactor for Scalable Production of Adeno-Associated Virus

The production of recombinant adeno-associated virus (rAAV) commonly requires plasmid cotransfection, which hinders its mass production. Herein we describe the development of a novel process for rAAV production by combining the advantages of baculovirus-mediated gene delivery and BelloCell bioreactor (a novel packed-bed reactor for animal cell culture; CESCO Bioengineering, Hsinchu, Taiwan). We constructed three baculoviral vectors: Bac-LacZ carries the lacZ gene flanked by AAV inverted terminal repeats, Bac-RC harbors AAV rep and cap genes, and Bac-Helper carries helper genes derived from adenovirus. Cotransduction of HEK-293 cells with these three baculoviruses resulted in successful production of rAAV, and the protein and rAAV yield did not decrease with Bac-RC passage for up to four passages. By adjusting the dose ratio of Bac-LacZ to Bac-RC, adding sodium butyrate, and transferring the production process to the BelloCell-500-AP (500 ml), which allowed for high-density culture and effective baculovirus-mediated transduction of HEK-293 cells, the maximal specific rAAV yield reached approximately 3.8 x 10(4) vector genome (VG) or 247 infectious viral particles (IVP) per cell, which corresponded to approximately 1 x 10(14) VG or 8.5 x 10(11) IVP per reactor run. The yield was comparable or superior to those obtained with other production systems. Baculoviral transduction is simple and cost-effective and the BelloCell-500-AP offers high-density culture of HEK-293 cells. Altogether, the combination of baculoviral transduction and BelloCell reactor culture provides a novel and economically viable approach for rAAV production.

852. AAV Rep78 Interacts with the E1 Helper Protein of HPV and Enhances Its ATPase Activity

852. AAV Rep78 Interacts with the E1 Helper Protein of HPV and Enhances Its ATPase Activity

Efficient Expression of the Adeno-Associated Virus Type 5 P41 Capsid Gene Promoter in 293 Cells Does Not Require Rep

Efficient expression of the adeno-associated virus type 5 (AAV5) P41 capsid gene promoter required adenovirus E1A and/or E1B; however, in contrast to what was observed for expression of the AAV2 capsid gene promoter (P40), neither adenovirus infection nor the large Rep protein was required. Although both the AAV2 and the AAV5 large Rep proteins efficiently bound the (GAGY)(3) Rep-binding element, the AAV5 large Rep protein transactivated transcription of the inducible AAV2 P40 promoter much less well than AAV2 large Rep. Differences in their activation potentials were mapped to the amino-terminal region of the proteins, and the poorly transactivating AAV5 Rep protein could competitively inhibit AAV2 Rep transactivation.

Chimeric Herpes Simplex Virus/Adeno-Associated Virus Amplicon Vectors

Chimeric or hybrid herpes simplex virus type 1/adeno-associated virus amplicon vectors combine the large transgene capacity of HSV-1 with the potential for site-specific genomic integration and stable transgene expression of AAV. These chimeric vectors have been demonstrated to support transgene expression for significantly longer periods than standard HSV-1 amplicons. Moreover, HSV/AAV hybrid vectors can mediate integration at the AAVS1 pre-integration site on human chromosome 19 at a relatively high rate, although random integration has also been observed. One major remaining hurdle of HSV/AAV hybrid vectors is the low packaging efficiency and titers when AAV rep sequences are included in the amplicon vector. In the conditions prevalent during the replication/packaging of HSV/AAV hybrid amplicons into HSV-1 virions, in particular the presence of HSV-1 replication factors and AAV Rep protein, at least three different viral origins of DNA replication are active: the HSV-1 ori, the AAV inverted terminal repeats (ITRs), and the p5 promoter/ori driving expression of the AAV rep gene. A detailed understanding of the properties of these origins of DNA replication and the molecular mechanisms of interactions between them, may allow designing novel hybrid vectors that allow the efficient and precise integration of large transgenes in the human genome.

676. AAV-Rep 78 Mediated Rescue and Integration of a 27kb Transgene Cassette

Random integration of viral gene therapy vectors and subsequent activation or disruption of cellular genes poses safety risks. Major efforts in the field are aimed toward targeting vector integration to specific sites in the host genome. AAV Rep78 is able to target AAV integration vectors to a specific site on human chromosome 19 called AAVS1. We studied whether this ability can be harnessed to achieve site-specific integration of a 27 kb transgene cassette into a model cell line for hematopoietic cells (Mo7e). To deliver Rep78 and the transgene to MO7e cells we used helper-dependent adenovirus vectors containing Ad serotype 35 fibers (HD-Ad). An HD-Ad containing the rep78 gene conferred efficient Rep 78 expression in target cells. Upon co-infection of HD-Ad-rep78 with an HD-Ad vector containing a 27k |[beta]|-globin-LCR-GFP transgene cassette flanked by AAV ITRs (HD-Ad.AAV-LCR), efficient Rep78-mediated rescue/excision of the transgene cassette was observed in 293 cells but not in MO7e cells. We found that rescue in 293 cells was enhanced due to expression of viral proteins, specifically proteins encoded by Ad5 E2a and E4orf6. Although no rescue of the LCR-GFP cassette was detectable in MO7e cells, co-infection of HD-Ad.rep78 increased the overall stable transduction frequency of HD.Ad.AAV-LCR and mediated integration of the transgene cassette into AAV-S1. (40% of all analyzed vector junction were in AAVS1). Interestingly, analysis of integration junctions revealed that Ad integration occurred in 80% of sites via the Ad ITR and only in 20% of sites via the AAV ITR. No rearrangements within AAV-S1 or AAV-S1 specific transgene integration were observed when HD- Ad.AAV-LCR was infected onto MO7e cells without the Rep78-expressing HD Ad vector. Our data indicate that rescue of the AAV-ITR flanked transgene cassette is not necessary for integration into AAV-S1. We are currently evaluating whether rescue can enhance the frequency of site-specific integration. This study provides a basis for the construction of vectors that allow for preferential site-specific transgene integration in hematopoietic cells.

506. Production of Recombinant Adeno- Associated Virus Type 1 Vector Using a Herpes Simplex Virus Based System

Top of pageAbstract Recombinant adeno-associated virus (rAAV) is an attractive vector for use in gene therapy treatment of various diseases. However, attaining high-level of rAAV has so far been proven problematic and the usefulness of rAAV vectors has been limited by the low production yield with current production methods. Previously we reported a highly scalable method for rAAV serotype 2 (AAV-2) production using two recombinant herpes simplex viruses (rHSV). One rHSV (rHSV-rep2cap2) expresses rep and cap of AAV-2 and provides helper function for AAV replication and packaging, and the other rHSV (rHSV-TR-GFP) contains the reporter gene green fluorescent protein (GFP) flanked by the inverted terminal repeats (ITR) of AAV-2. Both rHSV vectors are replication defective in non complementing cell lines because of the deletion of an essential gene. This rHSV-based system markedly increased the yield of rAAV production when compared to other traditional AAV production methods. It is well known that different serotypes of AAV have distinct host ranges and cell type preferences, and AAV vectors pseudo- typed with serotype one capsid have been shown to be particularly useful in transducing muscle cells. Here we report the development and initial characterization of an rHSV based production system for rAAV-1. Using a replication defective rHSV as the backbone, the AAV-2 Rep and AAV-1 Cap genes were engineered into rHSV by homologous recombination to generate the helper virus rHSV- rep2cap1. The ability of this virus to produce rAAV-1 was tested by co-infecting HEK-293 cells with rHSV-rep2cap1 and rHSV-TR- GFP. AAV genome titers determined by real time PCR showed that rAAV-1-GFP was packaged with a similar efficiency as the positive control rAAV-2-GFP. The infectivity of the produced rAAV-1-GFP was also examined and the results showed that rAAV-1-GFP was able to transduce C12 cells at an expected efficiency when compared to that of AAV-2-GFP. Studies are currently underway to evaluate these rAAV-1 vectors for gene delivery in vivo. In conclusion, this study suggests that the rHSV-based rAAV production system has a great potential for large scale rAAV production of various serotypes and this system may be able to generate sufficient quantities of rAAV for preclinical and clinical applications.

511. An Enzyme Linked Immunosorbent Assay for the Detection of Herpes Simplex Virus Type 1 Proteins in a Novel rAAV Production and Purification Process

AGTC has developed a recombinant herpes simplex virus (rHSV-1)-based adeno-associated virus (AAV) production process. This method requires two rHSV-1 viruses; rHSV-1-rep/cap which expresses the AAV2 rep and cap proteins, and a second rHSV-1 that contains the therapeutic gene of interest flanked by AAV2 inverted terminal repeats (ITRs) necessary for rAAV production. Following co-infection of these two helper viruses onto HEK-293 cells, the rAAV DNA is replicated and packaged into rAAV capsids. rAAV is then liberated from the cells and purified. Our proprietary rAAV purification scheme removes all contaminants including HSV-1 proteins; however, it is necessary to develop a sensitive and quantitative method to confirm the absence of HSV-1 proteins within a level of detection, in the final rAAV preparation.

Transduction of Pancreatic Islets with Pseudotyped Adeno-Associated Virus: Effect of Viral Capsid and Genome Conversion

Recombinant adeno-associated viral (rAAV) vectors currently show promise for islet gene therapy. In the presence of complementing AAV2 Rep proteins, AAV2 genomes can be packaged with other serotype capsids to assemble infectious virions. During transduction, the ssDNA to dsDNA conversion is one of the major rate-limiting steps that contribute to the slow onset of transgene expression. Using pseudotyping strategy, we produced double-stranded (dsAAV) and single-stranded (ssAAV) rAAV2 genomes carrying the GFP reporter gene packaged into AAV1, AAV2, and AAV5 capsids. The ability of cross-packaged AAV1, AAV2, and AAV5 at the same genome containing particle (gcp) concentration to transduce murine and human pancreatic islets was evaluated by GFP positive cell percentage. Transgenic expression was also determined by transplant transduced human islet into SCID mice. Pseudotyped rAAV2/1 based vectors transduced murine islets at greater efficiency than either rAAV2/2 or rAAV2/5 vectors. For human islets transduction, the rAAV2/2 vector was more efficient than rAAV2/1 or rAAV2/5 vectors. rAAV2/2 transduced human islets more efficiently than murine islets, while rAAV2/1 transducted murine islets more efficiently than human islets. dsAAV, which do not require second strand synthesis and thus are potentially more efficient, evidenced 5 fold higher transduction ability than ssAAV vectors. Pseudotyped rAAV transduced islet grafts maintained normal function, expressed transgenic product persistently in vivo, and reversed diabetes. The transduction efficiency of rAAV vectors was dependent on the cross-packaged capsid. The vector capsids permit species-specific transduction. For human islets, dsAAV2/2 vectors may be the most efficient vector for clinical development.

Adeno-associated virus-mediated bone morphogenetic protein-7 gene transfer induces C2C12 cell differentiation into osteoblast lineage cells1

To investigate the effects of bone morphogenetic protein-7 (BMP7)-expressing recombinant adeno-associated virus (AAV) vector on the differentiation of C2C12 cells. AAV-BMP7 was packaged by infecting the stable cell clone BHK-21 (integrated with recombinant AAV vector plasmid pSNAV-BMP7) with recombinant herpes simplex virus type 1, which expresses AAV-2 Rep and Cap and possesses AAV packaging functions. Following infection with AAV-BMP7 at multiplicities of infection of 1 x 10(5) vector genomes per cell and subsequent culture, C2C12 cells were assessed qualitatively for BMP7 production, alkaline phosphatase activity, osteocalcin production and Cbfal and MyoD expression. C2C12 cells transduced with AAV-BMP7 could produce BMP7 protein until d 28. Alkaline phosphatase in the cultured C2C12 cell lysate was elevated. Secreted osteocalcin in the culture medium was detectable at d 12 and Cbfal mRNA expression level was upregulated, coinciding with downregulation of MyoD in a temporal manner. The present in vitro study demonstrated that AAV-BMP7 could infect and efficiently convert C2C12 cells from myoblasts into osteoblast lineage cells.

404. Roles of Inverted Terminal Repeats (ITRs) and Capsid Proteins from Novel NHP AAVs in rAAV Mediated Gene Transfer

Recombinant AAV2 genomes transcapsidated with the capsid from a number of novel primate AAVs including AAV8 and rh.39 have been proven to be more robust gene transfer vehicles than the original AAV2 vector. We have recently isolated and characterized infectious molecular clones of AAV8 and AAVrh.39 and created recombinant AAV vector genomes carrying ITRs from AAV8 and AAVrh.39. Our study on the cleavability of those ITRs by AAV2 Rep in an in vitro nicking assay suggested the possibility of packaging those novel vector genomes with chimeric packaging plasmids consisting of the rep gene from AAV2 and cap genes from other AAVs.

716. Transducing Efficiency Screening for rAAV2, rAAV1, and rAAV5 in 56 Human Tumor Cell Lines

Recombinant adeno-associated viral vectors (rAAV) can efficiently transfer genes to a broad range of mammalian cell types leading to high levels of stable and long-term expression. Using rAAV to deliver therapeutic genes has been highly effective in both research and pre-clinical studies. AAV2 capsids utilize heparan sulfate proteoglycans (HSPG) as a cell surface ligand and possibly fibroblast growth factor1 and alpha V integrins as receptors that mediate cellular entry, whereas other AAV serotypes appear to use different attachment receptors, such as alpha 2-3 O-linked sialic acid and N-linked sialic acid for AAV4 and AAV5 respectively. The receptors and cell surface ligands for other serotypes have not been determined. Therefore, matching a particular rAAV serotype to an appropriate target cell is necessary to achieve efficient transduction. To explore the broad range applications of rAAV delivery in tumor therapy, we analyzed the ability of three diverse AAV serotype vectors to transduce 56 human tumor cell lines. Three different isogenic rAAV serotype vectors with the EGFP reporter gene were produced using AAV2 ITRs and Rep protein, and structural proteins from AAV1, AAV2, and AAV5 respectively, in Sf9 cells by using baculovirus expression vectors. The 56 cell lines are derived from nine distinct tumor types. Tumor cell lines were transduced 24 hr post-plating using high MOI (Multiplicity Of Infection, based on particles per cell) of |[ge]|10,000 virus particles per cell. The EGFP transduced cells were observed by fluorescence microscopy and analyzed quantitatively by flow cytometry. Our results showed the majority of melanoma cell lines and glioma cell lines are readily transduced with rAAV. Some lung and breast tumor cell lines have a relatively high rAAV transducing efficiency: the EGFP intensity was about the same as 293 cells, but lower than most of the melanoma cell lines. We found low rAAV transduction for tumor cell lines derived from prostate, renal, and ovarian cancers while most of the cells from cancer of breast, colon, and blood cells (leukemia) were poorly trnansduced with rAAV. Of the three AAV serotypes tested, we found that in general, rAAV2 had the earliest EGFP expression onset. Of the cell types that were transduced with rAAV, the three serotypes the highest transduction rate ranking was observed: AAV2>AAV5>AAV1, or AAV5>AAV2>AAV1, with some exceptions. These results suggest that AAV vectors have potential use as gene delivery vectors, particularly for melanoma and CNS tumors.

880. AAV-Rep 78 Mediated Rescue of a 27kb Transgene Cassette from a Helper-Dependent Ad Vector Genome

AAV Rep78 is thought to increase site-specific integration of AAV vectors into the AAVS1 site within human chromosome 19. We have constructed helper-dependent (HD) adenovirus vectors containing a 27kb globin LCR-GFP expression cassette flanked by AAV ITRs (HD-Ad.AAV-GFP). Our goal was to test whether transient Rep78 expression would rescue this large expression cassette from the HD-Ad genome and increase its site-specific integration. We planned to use HD-Ad5/35 vectors to deliver the rep78 gene to our target cells, MO7e cells, which are a human erythroleukemic cell line. Construction of Rep78-expressing Ad vectors is problematic, however, because trace amounts of Rep78 inhibit Ad replication. We therefore placed the rep78 gene under the control of the â-globin promoter, which was found (in preliminary studies) to have low activity in 293 (producer) cells but high activity in target MO7e cells. We produced this vector (HD-Ad.LCR-rep78) at titers of 4.1x1012 genomes per ml. Western blot analysis with Rep-specific antibodies confirmed rep78 expression from HD-Ad.LCR-rep78. We then performed rescue assays in MO7e cells by infection with HD-Ad.LCR-rep78 and HD-Ad.AAV-GFP. While no rescue of the AAV-LCR-GFP cassette was detectable by Southern blot analysis upon infection with the two HD-vectors, addition of a first-generation Ad vector caused rescue. This effect was nullified by addition of hydroxyurea, an inhibitor of adenoviral DNA replication. We drew the conclusions that a large (27kb) transgene cassette can be excised from HD-Ad genomes by Rep78, and E1-deleted first-generation vectors are able to replicate their DNA and the DNA of co-infected HD-Ad vectors in MO7e cells, and Rep78 mediated rescue in MO7e cells requires Ad proteins and/or viral replication of HD-Ad.AAV genomes. We are currently attempting to dissect these two possibilities. We also performed transduction studies with HD-Ad.LCR-rep78 and HD-Ad.AAV-GFP vector. Co-infection with HD-Ad.LCR-rep78 increased the overall stable transduction frequency of HD.Ad.AAV-LCR in MO7e cells about two-fold. We are currently analyzing the integration frequency into AAV-S1 in the presence of HD-Ad.LCR-rep78 and the results will be presented.

512. The Cellular TATA-Binding Protein Is Required for Replication of a Minimal Adeno-Associated Virus Type 2 (AAV-2) p5 Promoter Element

It was previously reported that the p5 promoter region (nt 191 to 350 of wt AAV-2) of the AAV-2 rep gene contained a cis-acting element that behaved as a Rep-dependent origin of replication in the absence of ITR (Nony et al, J. Virol. 2001; Musatov et al., J. Virol. 2002). This region was also shown to promote AAV-2 site-specific integration into human chromosome 19 (Philpott et al., PNAS 2002). The present study was conducted to identify the minimal elements in the p5 region necessary for the Rep-dependent replication.

852. Human ANP32B Protein Binds AAV-2 Rep and Increases Viral Replication and rAAV Production

AAV Rep proteins play a fundamental role at every phase of the viral life cycle and have pleiotropic effects on diverse cellular pathways such as transcription, cell-cycle progression and apoptosis. For these reasons we focused on identifying novel cellular factors that bind Rep and modulate its biological activities. To this aim, we designed and implemented a tagged proteomic approach by expressing a Flag-tagged version of Rep in human cells. Flag-Rep was subsequently purified by affinity chromatography and the identity of several proteins co-purifying with Rep was determined by ESI-MS/MS.

Adeno-Associated Virus Vectors in Clinical Trials

Adeno-Associated Virus Vectors in Clinical Trials

870. Transfection of Mammalian Cells Using Linear Polyethylenimine Is a Simple and Effective Means of Producing Recombinant AAV

We have developed a very simple protocol to transfect mammalian cells with linear polyethylenimine (PEI). Linear PEI has been used to transfect nucleic acids into a wide variety of mammalian cells both in cell culture and in vivo. Almost all of these reports have used commercially available preparations of PEI. Our linear PEI protocol is as efficient as commercial reagents in the transfection of HEK293 and HeLa cells, but at only a fraction of the cost. Our protocol should be useful for many different applications. It should be particularly useful for large-scale production of recombinant proteins and viruses requiring transfection of mammalian cells in large batches. We have used this protocol to produce recombinant AAV, which requires transfection of cells with three plasmids simultaneously. One plasmid contains the recombinant AAV genome. The second plasmid expresses the AAV rep and cap genes. The third plasmid expresses several adenovirus genes that are required for AAV replication. We have assessed production of a recombinant AAV that expresses GFP both by quantitative PCR and by transduction of HeLa cells.

Herpesvirus-based infectious titering of recombinant adeno-associated viral vectors

Studies in animals and human clinical trials demonstrate the safety and persistence of recombinant adeno-associated viral (rAAV) serotype 2 vectors in a variety of tissues. rAAV vectors of other serotypes are also being developed for efficient gene transfer. To date, the literature describing these vectors has relied on physical or transducing titers to determine dose, but few, if any, infectious titers have been presented. This is due in large part to the lack of reagents and methods that would facilitate the infectious titering of vectors other than serotype 2. Here, we describe reagents and methods for infectious titering of AAV2 ITR-containing vectors pseudotyped with other AAV capsid serotypes and demonstrate their utility by titering pseudotyped rAAV1 or rAAV5 vectors. Cell lines are screened for optimal transduction using a vector of a particular serotype that expresses a marker transgene. Once a cell line and vector serotype are matched, a recombinant herpes simplex virus vector expressing AAV2 rep and cap genes provides helper functions that amplify the rAAV vector genome. The vector genomes are then detected and a titer is calculated. These methods generate reliable infectious titers for AAV vectors of different serotypes, thus enhancing product characterization and reducing risk in future clinical applications.

The English strain of rat cytomegalovirus (CMV) contains a novel captured CD200 (vOX2) gene and a spliced CC chemokine upstream from the major immediate-early region: further evidence for a separate evolutionary lineage from that of rat CMV Maastricht

Sequence data for eight genes, together with time-course Northern blotting and 3'- and 5'-RACE (rapid amplification of cDNA ends) analysis for some mRNAs from a 12 kb region upstream from the major immediate-early (MIE) genes of the English isolate of rat cytomegalovirus (RCMV), are presented. The results identified important differences compared to both murine cytomegalovirus (MCMV) and the Maastricht isolate of RCMV. A striking finding is the presence of a highly conserved, rightwards-oriented homologue of the rat cellular CD200 (OX2) gene immediately to the right of the MIE region, which replaces either the leftwards-oriented AAV REP gene of RCMV (Maastricht) or the upstream spliced portions of the immediate-early 2 gene (ie2) in MCMV. From the presence of other homologues of MCMV- and RCMV-specific genes, such as the beta-chemokine MCK-2, SGG1 and an Fcgamma receptor gene, as reported here, the basic architecture of the MIE region (reported previously) and the level of IE2 and DNA polymerase (POL) protein conservation in phylogenetic analyses, it is clear that the English strain of RCMV is also a member of the genus Muromegalovirus, but is a beta-herpesvirus species that is very distinct from both MCMV and RCMV (Maastricht). Both the lack of a CD200 homologue in the other two rodent viruses and the depth of sequence divergence of the rodent CMV IE2 and POL proteins suggest that these three viruses have evolved as separate species in the genus Muromegalovirus since very early in the host rodent lineage.

710. Stem Cell Gene Therapy: Synergizing Improved Gene Repair/Modification & Targeted Integration of Transgenes to Transient In Vivo Topical Minigene Expression

With their self-renewing and homing/differentiative abilities, stem cells are the ideal targets for most gene therapy protocols: they are either long-term carriers of therapeutic genes or mobilized/recruted targets of transient regenerative processes (eg revascularization). Long term gene therapy is thus amenable to synergistic combinations where ex vivo/in vivo genetic engineering of stem cells associates to transient in vivo topical expression of minigenes encoding various homing/regenerative factors. Such a combination of long-term and transient gene therapy protocols stands as a powerful mean to tackle inherited and degenerative/acquired disorders. Indeed, transient magnification of stem cell regenerative potential, in which therapeutic homing is a key feature, should culminate in efficient targeted repopulation dynamics of engineered stem cells and differentiated derivatives in relevant tissues/organs, thereby providing appropriate gene therapeutics and life-long maintenance/replacement of therapeutic engineered cells. This long-term gene therapy strategy is hampered by a major safety concern: random integration of therapeutic DNA. However, promising site-specific integration vectors based on AAV rep or phage phiC31 integrase are at the pre-clinical stage. Unlike these approaches, gene targeting is driven by homologous recombination and has thus target flexibility. It mediates DNA exchanges between chromosomal and transfecting/transducing DNA, thereby providing the means to modify at will the sequence of target chromosomal DNA. Gene targeting stands thus as the ultimate process for gene repair/alteration and targeted (site-specific) transgene integration. It is however overwhelmed by random integration and is highly inefficient unless a double-strand break (DSB) hits target chromosomal DNA.