- Email: [email protected]
304. Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design
RNA VIRUS VECTORS 302. Transient and Highly Efcient Delivery of Sleeping Beauty Transposase and Flp Recombinase mRNA in Target Cells Via RTDecient Retroviral Particles
303. Erasable Non-Integrating RNA Replicon Vectors for Generation of Immaculate iPS Cells Free from Transgenes and Sequence-Interference
Melanie Galla, Axel Schambach, Tobias Maetzig, Niels Heinz, Zsuzsanna Izsvak,2 Zoltan Ivics,2 Christopher Baum.1 1 Experimental Hematology, Hannover Medical School, Hannover, Germany; 2Mobile DNA, Max Delbrueck Center, Berlin, Germany. 1
1
1
1
DNA modifying enzymes such as transposases and recombinases are interesting tools for gene and cell therapy. To avoid cytotoxic and more subtle genotoxic off-target effects, their transient and fully reversible expression within cells is desirable. However, conventional transient expression methods such as physicochemical approaches or the delivery of retroviral episomes into the nuclei of cells are still limited in their efcacy and tend to suffer from residual integration events. We have previously shown that gammaretroviral vector mutants that are unable to initiate reverse transcription (RT) of their plus-stranded mRNA genomes mediate receptor-mediated and transient delivery of the site specic recombinase Cre (Galla et al., Mol Cell 2004). More recent studies showed that the active principle of this new approach of retrovirus particle mediated mRNA transfer (RMT) is the genomic mRNA that is released from the incoming particle and serves as ribosomal translation template (Galla et al., J. Virol 2008). The Sleeping Beauty (SB) transposon (Ivics et al., Cell 1997) is an alternative to viral gene delivery vehicles. To prevent remobilization of the therapeutic gene in target cells the SB transposase is supplied in trans (separated from the transposable element containing the transposase recognition sites) by transient expression. Our initial attempts to deliver two versions of the SB transposase (conventional SB and hyperactive SB100X transposase (Mates et al., Nat. Genet. 2009)) via RMT were promising but required further improvement. To elevate the expression of the genomic RNA of the RMT vector and improve post-transcriptional mRNA processing, we modied the 5’LTR promoter, added the woodchuck hepatitis virus posttranscriptional regulatory element (wPRE) and codonoptimized the hyperactive SB100X transposase cDNA (SB100Xco). Individually and even more collectively, these modications greatly enhanced RMT mediated DNA transposition. Combining RMT of SB100Xco with transfection of a transposable element encoding for EGFP resulted in transposition frequencies up to 100% in Hela cells. Thus, a single vector production from a 10 cm plate of producer cells provided RMT vectors in quantities sufcient for 60 experiments. In addition cotransduction of Hela cells with RMT-SB100Xco particles and integration defective lentiviral particles delivering the transposable element resulted in ∼20% transposition. We also obtained evidence that RMT of SB100Xco avoids cytotoxicity related to its permanent ectopic expression. Furthermore, we validated our RMT vector improvements in the setting of the site-specic recombinase Flp derived from Saccharomyces cerevisiae. With a single transduction we could achieve highly efficient (>90%) recombination of human and mouse broblasts carrying 2 or 3 copies of an Flp indicator allele, respectively. In conclusion, optimized vector and transgene design clearly improves RMT. The remarkable efciency underlines the potential of this method for transient and fully reversible protein expression.
Hiroshi Ban,1 Noemi Fusaki,1,2 Toshiaki Tabata,1 Koichi Saeki,1 Akihiro Iida,1 Makoto Inoue,1 Mamoru Hasegawa.1 1 DNAVEC Corporation, Tsukuba, Ibaraki, Japan; 2PRESTO, JST, Kawaguchi, Saitama, Japan. This is the first report of “erasable” technology of a vector system derived from the RNA replicon of Sendai virus of the family Paramixoviridae. Induced pluripotent stem cell (iPSC) is expected to be a powerful tool in the elds of regenerative medicine and drug development, and generated from somatic cells by co-introduction of several reprogramming factors such as Oct3/4, Sox2, Klf-4 and c-Myc. Therefore, the efcient delivery system for these factors is very critical for generation of iPSCs. Some viral vectors such as retrovirus and adenovirus vectors have been used to transduce genes for generating iPSCs. However, several problems remain such as the difculty of standardization of the cells because of the viral vector insertion into a host genome and the low efciency of iPSC generation. We have developed an advanced method of iPSC generation by use of Sendai virus (SeV) vector (PlasmExTM). The PlasmExTM bears some unique characteristics good for iPSC generation as follows. 1) The vector is the cytoplasmic RNA vector, which dose not integrate into a host genome, 2) transduces transgene(s) to almost all mammalian cell types at high efciency and 3) its expression level of transgene(s) is high and adjustable. Therefore, we tried to generate iPSCs from human somatic cells (BJ cells from neonate broblast and HDF cells from adult broblast) by using four PlasmExTM carrying human Oct3/4, Sox2, Klf-4 and c-Myc gene, respectively, and succeeded in the generation at high frequency of 1 to 3%. Vector-free-iPSC clones were isolated spontaneously, or negatively selected using antibody to SeV HN. Such vector-free-iPSC clones exhibited the characteristics of iPSC previously established by using other vectors. As expected, no integration of transgenes or vectors into the host genome was occurred. Next, we tried to develop an efcient and easy method to remove PlasmExTM from transduced cells, such as iPSCs. We constructed a series of temperature-sensitive (TS) mutants of PlasmExTM which carry mutation(s) in P and/or L genes that are responsible for viral transcription and replication. Candidate PlasmExTM were able to express transgenes at 37 °C, but not at above 38 °C. LLC-MK2 cells that were transduced with various TS PlasmExTM mutants were cultured at permissive- and non-permissive- temperatures. Some TS vector mutants were completely disappeared by culturing at nonpermissive temperature. This improved PlasmExTM system is able to transduce and express transgenes very efciently and disappear rapidly when required. This system will be benecially applied for the generation of iPSCs with intact genome sequence and without residual transgenes and vector.
304. Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design
Julia D. Sürth,1 Tobias Mätzig,1 Melanie Galla,1 Ute Modlich,1 Christopher Baum,1 Axel Schambach.1 1 Experimental Hematology, Hannover Medical School, Hannover, Germany. Insertional genotoxicity in the form of accidental insertional activation of proto-oncogenes or disruption of tumor suppressor genes poses serious challenges for human gene therapy using retroviral vectors (e. g. Hacein-Bey-Abina et al. 2003). Comparative analyses of integration sites of different retroviruses have elucidated distinct target site preferences highlighting alpharetroviral vectors as the ones with the most neutral integration spectrum (Mitchell et al. 2004; Hu
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Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
AAV VECTORS: VECTOR BIOLOGY et al., 2008). Usage of alpharetroviral vectors for human gene therapy may thus reduce the risk of genotoxic adverse events. We have modied the alpharetroviral vector system to meet the requirements for clinical applications. Based on the Rous Sarcoma Virus (RSV), we have set up a split-packaging design, encoding transfer vector, gag/pol and env on different plasmids, to minimize the likelihood of formation of recombination competent retroviruses (RCR) and to avoid the potentially immunogenic expression of viral proteins in transduced cells. By deleting all alpharetroviral coding sequences we have generated a gene transfer vector with the shortest leader sequence among all retroviral constructs we are aware of (cf. Alpha 371 vs. MLV 634 vs. HIV 1717 bp). As an additional safety measure, we have introduced a self-inactivation (SIN) deletion, removing the strong enhancer and transcriptional elements within the 3’ U3 region of RSV, to reduce the risk of activation of cellular genes, and to enable controlled transgene expression by internal promoters of choice. The importance of this modications could be underlined by our experiments conrming that the wildtype U3 region has a signicantly strong transcriptional activity in human cells and that the U3 deletion used in this study reverted this to background levels of an introduced spacer. By modifying the alpharetroviral gag/pol expression construct, we were able to generate unconcentrated viral particles from human 293T cells with titers in the range of 1x10e7 t.u./mL. This is remarkable because wt-RSV cannot replicate in mammalian cells. Efcient pseudotyping of alpharetroviral vectors was possible using the ecotropic MLV envelope, VSVg or a modied RD114 envelope, the latter efciently transducing human CD34-positive cells at low multiplicities of infection. In transduced broblasts or hematopoietic cells, the alpharetroviral vectors mediated slightly lower expression levels than HIV-1-based lentiviral or gammaretroviral vectors. Nevertheless, our newly developed alpharetroviral SIN vectors allowed for efcient expression of clinically relevant transgenes, such as interleukin-2 receptor common γ-chain (IL2RG). Finally, preliminary results from In Vitro Immortalization Assays suggest that alpharetroviral SIN vectors have a lower insertional genotoxicity than HIV-1-based lentiviral vectors. In summary we have generated an alpharetroviral vector system with promising safety and efciency, potentially fullling stringent requirements for use in clinical trials.
AAV Vectors: Vector Biology 305. Dissecting rAAV Integration Frequency in Non-Human Primates by Deep Sequencing
Ali Nowrouzi,1 Magalie Penaud-Budloo,2 Christine Kaeppel,1 Anne Arens,2 Caroline Le Guiner,1 Adrien Leger,2 Karim Zaoui,1 Christian Weber,1 Richard O. Snyder,2,3,4 Christof von Kalle,1 Philippe Moullier,2 Manfred Schmidt.1 1 Department of Translational Oncology, German Cancer Research Center, Heidelberg, Germany; 2INSERM UMR649, IRT 1, Université de Nantes, Nantes, France; 3Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL; 4Center of Excellence for Regenerative Health Biotechnology, University of Florida, Gainesville, FL. Long-term transgene expression can be achieved following recombinant adeno-associated virus (rAAV) vector-mediated gene transfer to skeletal muscle and liver. We have previously shown that intramuscular injection of rAAV1 and rAAV8 based vectors in nonhuman primates results in maintenance of rAAV genomes as episomal monomeric and concatemeric circles. Molecular analyses of rAAV integration using LAM-PCR proved that rAAV integrates inefciently in the genome of myocytes in vivo and is maintained in highly structured concatemeric congurations. However, evaluation of rAAV integration frequency in primate and canine large animal models is still hampered by the excess of episomal forms limiting detection of Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
rare integration events. To reach a precise and reliable quantication of rAAV integration frequency in different tissues and (pre)clinical settings, we have successfully adapted rAAV directed LAM-PCR to next generation sequencing platforms allowing simultaneous and efcient identication of complex AAV concatemeric forms and proviruses. We report that pyrosequencing technologies prove feasible in comprehensive sequencing of complete inverted terminal repeats (ITR). Sequencing of >100000 rAAV specic LAM-PCR amplicons reveals substantial quantitative differences of individual ITR breakpoints in primate muscle and liver up to 22 months post injection. We show in high resolution that sequence coverage of the ITR reveals preferential breakpoint sites present in distinct loops of the ITR hairpin structure in vivo. Further, we demonstrate that the retrieval frequency of rare insertional events and concatemers following pyrosequencing is feasible and highly valuable for strategies addressing and quantifying integration frequency of rAAV in muscle and liver under different dose conditions at a sensitivity of <0,1%. Our current bioinformatical analyses of sequenced amplicons suggest that rAAV integration frequency varies in liver and muscle but integration events do not exceed 1 in 1000 cells in vivo. These insights are relevant for further vector biosafety assessment of rAAV serotypes in preclinical models and upcoming rAAV clinical trials.
306. Non-Canonical Integrating AAV Vectors as a Safe Tool for the Future of Gene Therapy
Leszek Lisowski,1 Zhongya Wang,2 Ashley Lau,1 Feijie Zhang,1 Markus Grompe,2 Mark A. Kay.1 1 Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA; 2Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR.
In light of the problems caused by retro- and lentiviral vectors in recent clinical trials, the future of the gene therapy depends on the ability to generate safer vector systems. The ideal vector would allow for transgene delivery to a wide range of target cells and safe and persistent expression in both dividing and non-dividing cells. Recombinant AAV vectors have signicant potential due to the low toxicity and the availability of a large number of capsids, which extends the array of cells and tissues susceptible to gene transfer. The main limitation of rAAV is that their episomal proviral genomes are lost with cell division, rendering gene transfer transient in most tissues. We have developed a novel rAAV system capable of robust targeted DNA integration into the host genome. An hFIX cassette was anked by ribosomal DNA(rDNA), which resulted in targeted vector integration into the mouse liver rDNA loci. After surgically-induced liver regeneration, the serum hFIX levels in AAV-rDNA-hFIXtreated mice were 30% of what was observed in the rst few weeks post-treatment (∼400ng FIX/ml after PH/CCl4) or 40% of non-liver regeneration controls followed for the same period of time. In contrast to the results observed with AAV-rDNA-hFIX vectors, mice treated with control vectors, in which the hFIX cassette was anked with mammalian stuffer DNA, had only 0.45% of their original serum hFIX after liver regeneration. LAM-PCR analysis conrmed that over 90% of analyzed clones in the AAV-rDNA-hFIX group were integrated in the rDNA genomic loci, while the remaining 9% could not be annotated. In the AAV-stuffer-hFIX group, over 40% of analyzed integration sites were found to be in random genomic locations, while the remaining 60% were episomal or integrated in a genomic region that could not be annotated. Furthermore, we undertook a systematic and detailed analysis of rAAV-rDNA vector integration by using a similar set of vectors in large animal cohorts. To minimize the anking sequences and maximize the size of the transgene payload, we produced vectors
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303. Erasable Non-Integrating RNA Replicon Vectors for Generation of Immaculate iPS Cells Free from Transgenes and Sequence-Interference
Melanie Galla, Axel Schambach, Tobias Maetzig, Niels Heinz, Zsuzsanna Izsvak,2 Zoltan Ivics,2 Christopher Baum.1 1 Experimental Hematology, Hannover Medical School, Hannover, Germany; 2Mobile DNA, Max Delbrueck Center, Berlin, Germany. 1
1
1
1
DNA modifying enzymes such as transposases and recombinases are interesting tools for gene and cell therapy. To avoid cytotoxic and more subtle genotoxic off-target effects, their transient and fully reversible expression within cells is desirable. However, conventional transient expression methods such as physicochemical approaches or the delivery of retroviral episomes into the nuclei of cells are still limited in their efcacy and tend to suffer from residual integration events. We have previously shown that gammaretroviral vector mutants that are unable to initiate reverse transcription (RT) of their plus-stranded mRNA genomes mediate receptor-mediated and transient delivery of the site specic recombinase Cre (Galla et al., Mol Cell 2004). More recent studies showed that the active principle of this new approach of retrovirus particle mediated mRNA transfer (RMT) is the genomic mRNA that is released from the incoming particle and serves as ribosomal translation template (Galla et al., J. Virol 2008). The Sleeping Beauty (SB) transposon (Ivics et al., Cell 1997) is an alternative to viral gene delivery vehicles. To prevent remobilization of the therapeutic gene in target cells the SB transposase is supplied in trans (separated from the transposable element containing the transposase recognition sites) by transient expression. Our initial attempts to deliver two versions of the SB transposase (conventional SB and hyperactive SB100X transposase (Mates et al., Nat. Genet. 2009)) via RMT were promising but required further improvement. To elevate the expression of the genomic RNA of the RMT vector and improve post-transcriptional mRNA processing, we modied the 5’LTR promoter, added the woodchuck hepatitis virus posttranscriptional regulatory element (wPRE) and codonoptimized the hyperactive SB100X transposase cDNA (SB100Xco). Individually and even more collectively, these modications greatly enhanced RMT mediated DNA transposition. Combining RMT of SB100Xco with transfection of a transposable element encoding for EGFP resulted in transposition frequencies up to 100% in Hela cells. Thus, a single vector production from a 10 cm plate of producer cells provided RMT vectors in quantities sufcient for 60 experiments. In addition cotransduction of Hela cells with RMT-SB100Xco particles and integration defective lentiviral particles delivering the transposable element resulted in ∼20% transposition. We also obtained evidence that RMT of SB100Xco avoids cytotoxicity related to its permanent ectopic expression. Furthermore, we validated our RMT vector improvements in the setting of the site-specic recombinase Flp derived from Saccharomyces cerevisiae. With a single transduction we could achieve highly efficient (>90%) recombination of human and mouse broblasts carrying 2 or 3 copies of an Flp indicator allele, respectively. In conclusion, optimized vector and transgene design clearly improves RMT. The remarkable efciency underlines the potential of this method for transient and fully reversible protein expression.
Hiroshi Ban,1 Noemi Fusaki,1,2 Toshiaki Tabata,1 Koichi Saeki,1 Akihiro Iida,1 Makoto Inoue,1 Mamoru Hasegawa.1 1 DNAVEC Corporation, Tsukuba, Ibaraki, Japan; 2PRESTO, JST, Kawaguchi, Saitama, Japan. This is the first report of “erasable” technology of a vector system derived from the RNA replicon of Sendai virus of the family Paramixoviridae. Induced pluripotent stem cell (iPSC) is expected to be a powerful tool in the elds of regenerative medicine and drug development, and generated from somatic cells by co-introduction of several reprogramming factors such as Oct3/4, Sox2, Klf-4 and c-Myc. Therefore, the efcient delivery system for these factors is very critical for generation of iPSCs. Some viral vectors such as retrovirus and adenovirus vectors have been used to transduce genes for generating iPSCs. However, several problems remain such as the difculty of standardization of the cells because of the viral vector insertion into a host genome and the low efciency of iPSC generation. We have developed an advanced method of iPSC generation by use of Sendai virus (SeV) vector (PlasmExTM). The PlasmExTM bears some unique characteristics good for iPSC generation as follows. 1) The vector is the cytoplasmic RNA vector, which dose not integrate into a host genome, 2) transduces transgene(s) to almost all mammalian cell types at high efciency and 3) its expression level of transgene(s) is high and adjustable. Therefore, we tried to generate iPSCs from human somatic cells (BJ cells from neonate broblast and HDF cells from adult broblast) by using four PlasmExTM carrying human Oct3/4, Sox2, Klf-4 and c-Myc gene, respectively, and succeeded in the generation at high frequency of 1 to 3%. Vector-free-iPSC clones were isolated spontaneously, or negatively selected using antibody to SeV HN. Such vector-free-iPSC clones exhibited the characteristics of iPSC previously established by using other vectors. As expected, no integration of transgenes or vectors into the host genome was occurred. Next, we tried to develop an efcient and easy method to remove PlasmExTM from transduced cells, such as iPSCs. We constructed a series of temperature-sensitive (TS) mutants of PlasmExTM which carry mutation(s) in P and/or L genes that are responsible for viral transcription and replication. Candidate PlasmExTM were able to express transgenes at 37 °C, but not at above 38 °C. LLC-MK2 cells that were transduced with various TS PlasmExTM mutants were cultured at permissive- and non-permissive- temperatures. Some TS vector mutants were completely disappeared by culturing at nonpermissive temperature. This improved PlasmExTM system is able to transduce and express transgenes very efciently and disappear rapidly when required. This system will be benecially applied for the generation of iPSCs with intact genome sequence and without residual transgenes and vector.
304. Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design
Julia D. Sürth,1 Tobias Mätzig,1 Melanie Galla,1 Ute Modlich,1 Christopher Baum,1 Axel Schambach.1 1 Experimental Hematology, Hannover Medical School, Hannover, Germany. Insertional genotoxicity in the form of accidental insertional activation of proto-oncogenes or disruption of tumor suppressor genes poses serious challenges for human gene therapy using retroviral vectors (e. g. Hacein-Bey-Abina et al. 2003). Comparative analyses of integration sites of different retroviruses have elucidated distinct target site preferences highlighting alpharetroviral vectors as the ones with the most neutral integration spectrum (Mitchell et al. 2004; Hu
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Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
AAV VECTORS: VECTOR BIOLOGY et al., 2008). Usage of alpharetroviral vectors for human gene therapy may thus reduce the risk of genotoxic adverse events. We have modied the alpharetroviral vector system to meet the requirements for clinical applications. Based on the Rous Sarcoma Virus (RSV), we have set up a split-packaging design, encoding transfer vector, gag/pol and env on different plasmids, to minimize the likelihood of formation of recombination competent retroviruses (RCR) and to avoid the potentially immunogenic expression of viral proteins in transduced cells. By deleting all alpharetroviral coding sequences we have generated a gene transfer vector with the shortest leader sequence among all retroviral constructs we are aware of (cf. Alpha 371 vs. MLV 634 vs. HIV 1717 bp). As an additional safety measure, we have introduced a self-inactivation (SIN) deletion, removing the strong enhancer and transcriptional elements within the 3’ U3 region of RSV, to reduce the risk of activation of cellular genes, and to enable controlled transgene expression by internal promoters of choice. The importance of this modications could be underlined by our experiments conrming that the wildtype U3 region has a signicantly strong transcriptional activity in human cells and that the U3 deletion used in this study reverted this to background levels of an introduced spacer. By modifying the alpharetroviral gag/pol expression construct, we were able to generate unconcentrated viral particles from human 293T cells with titers in the range of 1x10e7 t.u./mL. This is remarkable because wt-RSV cannot replicate in mammalian cells. Efcient pseudotyping of alpharetroviral vectors was possible using the ecotropic MLV envelope, VSVg or a modied RD114 envelope, the latter efciently transducing human CD34-positive cells at low multiplicities of infection. In transduced broblasts or hematopoietic cells, the alpharetroviral vectors mediated slightly lower expression levels than HIV-1-based lentiviral or gammaretroviral vectors. Nevertheless, our newly developed alpharetroviral SIN vectors allowed for efcient expression of clinically relevant transgenes, such as interleukin-2 receptor common γ-chain (IL2RG). Finally, preliminary results from In Vitro Immortalization Assays suggest that alpharetroviral SIN vectors have a lower insertional genotoxicity than HIV-1-based lentiviral vectors. In summary we have generated an alpharetroviral vector system with promising safety and efciency, potentially fullling stringent requirements for use in clinical trials.
AAV Vectors: Vector Biology 305. Dissecting rAAV Integration Frequency in Non-Human Primates by Deep Sequencing
Ali Nowrouzi,1 Magalie Penaud-Budloo,2 Christine Kaeppel,1 Anne Arens,2 Caroline Le Guiner,1 Adrien Leger,2 Karim Zaoui,1 Christian Weber,1 Richard O. Snyder,2,3,4 Christof von Kalle,1 Philippe Moullier,2 Manfred Schmidt.1 1 Department of Translational Oncology, German Cancer Research Center, Heidelberg, Germany; 2INSERM UMR649, IRT 1, Université de Nantes, Nantes, France; 3Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL; 4Center of Excellence for Regenerative Health Biotechnology, University of Florida, Gainesville, FL. Long-term transgene expression can be achieved following recombinant adeno-associated virus (rAAV) vector-mediated gene transfer to skeletal muscle and liver. We have previously shown that intramuscular injection of rAAV1 and rAAV8 based vectors in nonhuman primates results in maintenance of rAAV genomes as episomal monomeric and concatemeric circles. Molecular analyses of rAAV integration using LAM-PCR proved that rAAV integrates inefciently in the genome of myocytes in vivo and is maintained in highly structured concatemeric congurations. However, evaluation of rAAV integration frequency in primate and canine large animal models is still hampered by the excess of episomal forms limiting detection of Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
rare integration events. To reach a precise and reliable quantication of rAAV integration frequency in different tissues and (pre)clinical settings, we have successfully adapted rAAV directed LAM-PCR to next generation sequencing platforms allowing simultaneous and efcient identication of complex AAV concatemeric forms and proviruses. We report that pyrosequencing technologies prove feasible in comprehensive sequencing of complete inverted terminal repeats (ITR). Sequencing of >100000 rAAV specic LAM-PCR amplicons reveals substantial quantitative differences of individual ITR breakpoints in primate muscle and liver up to 22 months post injection. We show in high resolution that sequence coverage of the ITR reveals preferential breakpoint sites present in distinct loops of the ITR hairpin structure in vivo. Further, we demonstrate that the retrieval frequency of rare insertional events and concatemers following pyrosequencing is feasible and highly valuable for strategies addressing and quantifying integration frequency of rAAV in muscle and liver under different dose conditions at a sensitivity of <0,1%. Our current bioinformatical analyses of sequenced amplicons suggest that rAAV integration frequency varies in liver and muscle but integration events do not exceed 1 in 1000 cells in vivo. These insights are relevant for further vector biosafety assessment of rAAV serotypes in preclinical models and upcoming rAAV clinical trials.
306. Non-Canonical Integrating AAV Vectors as a Safe Tool for the Future of Gene Therapy
Leszek Lisowski,1 Zhongya Wang,2 Ashley Lau,1 Feijie Zhang,1 Markus Grompe,2 Mark A. Kay.1 1 Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA; 2Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR.
In light of the problems caused by retro- and lentiviral vectors in recent clinical trials, the future of the gene therapy depends on the ability to generate safer vector systems. The ideal vector would allow for transgene delivery to a wide range of target cells and safe and persistent expression in both dividing and non-dividing cells. Recombinant AAV vectors have signicant potential due to the low toxicity and the availability of a large number of capsids, which extends the array of cells and tissues susceptible to gene transfer. The main limitation of rAAV is that their episomal proviral genomes are lost with cell division, rendering gene transfer transient in most tissues. We have developed a novel rAAV system capable of robust targeted DNA integration into the host genome. An hFIX cassette was anked by ribosomal DNA(rDNA), which resulted in targeted vector integration into the mouse liver rDNA loci. After surgically-induced liver regeneration, the serum hFIX levels in AAV-rDNA-hFIXtreated mice were 30% of what was observed in the rst few weeks post-treatment (∼400ng FIX/ml after PH/CCl4) or 40% of non-liver regeneration controls followed for the same period of time. In contrast to the results observed with AAV-rDNA-hFIX vectors, mice treated with control vectors, in which the hFIX cassette was anked with mammalian stuffer DNA, had only 0.45% of their original serum hFIX after liver regeneration. LAM-PCR analysis conrmed that over 90% of analyzed clones in the AAV-rDNA-hFIX group were integrated in the rDNA genomic loci, while the remaining 9% could not be annotated. In the AAV-stuffer-hFIX group, over 40% of analyzed integration sites were found to be in random genomic locations, while the remaining 60% were episomal or integrated in a genomic region that could not be annotated. Furthermore, we undertook a systematic and detailed analysis of rAAV-rDNA vector integration by using a similar set of vectors in large animal cohorts. To minimize the anking sequences and maximize the size of the transgene payload, we produced vectors
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