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539. Transcriptional Repression with Zinc-Finger and Tale Protein Scaffold
AAV VECTORS III PCR analysis (TAQMAN®) of EGFP transcripts and (D) Vector copy number from hepatocyes 4 weeks post administration of AAV5 vectors in C57BL/6 mice.
538. Adeno Associated Virus Purification Independent of Gradient Centrifugation and Chromatography
Erik Arden,1 Joseph M. Metzger.1 1 Integrative Biology and Physiology, University of Minnesota, Minneapolis, MN. Recombinant Adeno Associated Virus is a valuable and often used gene therapy vector. With increased demand for highly purified virus comes the need for a standardized purification procedure that is applicable across many serotypes. Currently cesium chloride banding or affinity chromatography are the predominate forms of purification. These approaches expose the final purified virus to toxic contaminants or are highly capsid dependent and may require significant optimizations to isolate AAV. These methods may also limit crude viral lysate volume resulting in a significant loss of titer. To circumvent these issues, we have developed an AAV purification protocol independent of toxic compounds, supernatant volume and capsid moiety. This purification method standardizes virus purification across wild type, mosaic and custom designed capsids. Briefly, both viral supernatant and 293 cells are harvested post CaPO4 transfection. Crude viral lysate is then processed using a microfluidizer, filter clarified and treated with endonucleases to digest genomic and plasmid DNA. AAV virions are then precipitated using a combination of polyethylene glycol and sodium chloride and low speed centrifugation. The resulting pellet is resuspended in a physiological buffer at a 50x volume, and a second short low speed spin is performed to clarify the viral suspension. Lastly, a high-speed centrifuge spin is used to pellet viral capsids while endogenous cellular proteins remain in suspension. rAAV6 was used in a direct comparison of affinity chromatography to our PEG/Spin protocol. This procedure demonstrated equivalent purities on Agarose gel and SDS-PAGE analysis. We also captured up to 10-fold more virions and increased vector genome yields 3-fold. AAV41, an engineered mosaic capsid containing AAV1, 6, 7 and 8, was also purified using our PEG/ Spin method and displayed similar yields to AAV6. Both vectors contained a Luciferase expression cassette and were systemically delivered to C57/Bl6 mice. Quantified luciferase expression of striated muscle in these mice displayed up to 3.0x10e7 RFU/mg protein.
539. Transcriptional Repression with ZincFinger and Tale Protein Scaffold
Salvatore Botta,1 Elena Marrocco,1 Nicola de Prisco,1 Toni Cathomen,2 Claudio Mussolino,2 Enrico Maria Surace.1 1 Tigem, Telethon Institute of Genetics and Medicine, Naples, Italy; 2Laboratory of Cell and Gene Therapy, Center for Chronic Immunodeficiency, University Medical Center, Freiburg, Germany. We recently demonstrated that adeno-associated viral (AAV) vector somatic gene transfer of zinc-finger based transcriptional repressors results in the generation of robust transcriptional downregulation of one of the most expressed gene in mammals, the photoreceptor-specific Rhodopsin gene (RHO), irrespectively to wild-type or mutated alleles (mutational-independent approach). In principle this approach enables the correction of any photoreceptor disease associated with RHO mutation (both dominant and recessive forms). We showed that a selected artificial transcriptional repressor (named Zinc-Finger 6 repressor, ZF6R) delivered by AAV2/8 vector down regulates selectively mutated human RHO expression levels ameliorating the retinal phenotype in a mouse model of autosomal dominant Retinitis Pigmentosa (adRP). To further validate the transcriptional repression via synthetic design of DNA binding S208
proteins strategy to generate therapeutics, we designed RHO targeted transcriptional repressors with the novel Transcription Activator Like Effector (TALEs) platform. We generated two different TALEbased DNA binding domains, targeted to conserved regions of the human and mouse Rhodopsin promoter, that differ in the repeat variable diresidue (RVD) that recognizes the guanine DNA base with distinct affinity and specificity. TALE1 carries “NN” RVDs as the guanine-recognizing domain, while TALE2 was generated with “NK” RVDs. To test the ability of these two TALEs to modulate expression from the Rhodopsin promoter, we fused them to either the VP16 activator or KRAB repressor domains. These TALEs showed robust transcriptional activation or repression at the RHO promoter in in vitro assays to a similar extent as ZF6R. Currently, we are testing and comparing the TALE and Zinc-finger-based artificial transcription factors in vivo.
540. Mouse Gender Influences Brain Expression of a Reporter Gene by IntravascularlyAdministered AAV9
Casey A. Maguire,1,2 Matheus H. W. Crommentuijn,1,3 Dakai Mu,2,4 Eloise Hudry,2,4 Alberto Serrano-Pozo,2,4 Bradley Hyman,2,4 Bakhos A. Tannous.1,2,5 1 Experimental Therapeutics and Molecular Imaging Laboratory, Department of Neurology, The Massachusetts General Hospital, Charlestown, MA; 2Program in Neuroscience, Harvard Medical School, Boston, MA; 3Neuro-oncology Research Group, Department of Neurosurgery, Cancer Center Amsterdam, VU University Medical Center, Amterstam, Netherlands; 4Neurology, The Massachusetts General Hospital, Charlestown, MA; 5Center for Molecular Imaging Research, Department of Radiology, The Massachusetts General Hospital, Boston, MA. Vectors based on adeno-associated virus serotype 9 (AAV9) cross the blood-brain barrier (BBB) and mediate efficient gene expression in the mammalian brain. We observed that intravenous injection of AAV9 (1.5x1012 g.c./kg) encoding the reporter gene (firefly luciferase) yielded higher (3-40 fold) transgene expression in the brain of female mice compared to male mice as assessed by whole body bioluminescence imaging as well as luciferase activity in tissue homogenates. This observation was consistent in two strains of mice (nude, C57BL/6) and was correlated with an increased number of AAV genomes in female whole brain compared to males. Currently we are determining the identity and quantity of cell types transduced by AAV9-GFP after systemic injection of male and female mice. These observations stress the importance of carefully matching groups for gender and may also have implications for improving gene expression in males.
541. Site-Directed Mutagenesis of SurfaceExposed Lysine Residues Leads to Improved Transduction by Recombinant AAV2 and AAV8 Vectors in Murine Hepatocytes In Vivo
Baozheng Li,1 Wenqin Ma,1 George Aslanidi,1 Chen Ling,1 Kim Van Vliet,2 Mavis Agbandje-McKenna,2 Arun Srivastava.1 1 Pediatrics, University of Florida, Gainesville; 2Biochemistry & Molecular Biology, University of Florida, Gainesville. The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of recombinant AAV2 vectors, which negatively impacts the transduction efficiency of these vectors. We have previously reported that the primary signal for ubiquitination is phosphorylation of specific surface-exposed tyrosine (Y), serine (S), and threonine (T) residues on the AAV2 capsids, and that the removal of some of these residues significantly increases the transduction efficiency of wild-type (WT) AAV2 vectors. We and others have also reported that Y-, S-, or T-mutant AAV2 vectors can be used to Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
538. Adeno Associated Virus Purification Independent of Gradient Centrifugation and Chromatography
Erik Arden,1 Joseph M. Metzger.1 1 Integrative Biology and Physiology, University of Minnesota, Minneapolis, MN. Recombinant Adeno Associated Virus is a valuable and often used gene therapy vector. With increased demand for highly purified virus comes the need for a standardized purification procedure that is applicable across many serotypes. Currently cesium chloride banding or affinity chromatography are the predominate forms of purification. These approaches expose the final purified virus to toxic contaminants or are highly capsid dependent and may require significant optimizations to isolate AAV. These methods may also limit crude viral lysate volume resulting in a significant loss of titer. To circumvent these issues, we have developed an AAV purification protocol independent of toxic compounds, supernatant volume and capsid moiety. This purification method standardizes virus purification across wild type, mosaic and custom designed capsids. Briefly, both viral supernatant and 293 cells are harvested post CaPO4 transfection. Crude viral lysate is then processed using a microfluidizer, filter clarified and treated with endonucleases to digest genomic and plasmid DNA. AAV virions are then precipitated using a combination of polyethylene glycol and sodium chloride and low speed centrifugation. The resulting pellet is resuspended in a physiological buffer at a 50x volume, and a second short low speed spin is performed to clarify the viral suspension. Lastly, a high-speed centrifuge spin is used to pellet viral capsids while endogenous cellular proteins remain in suspension. rAAV6 was used in a direct comparison of affinity chromatography to our PEG/Spin protocol. This procedure demonstrated equivalent purities on Agarose gel and SDS-PAGE analysis. We also captured up to 10-fold more virions and increased vector genome yields 3-fold. AAV41, an engineered mosaic capsid containing AAV1, 6, 7 and 8, was also purified using our PEG/ Spin method and displayed similar yields to AAV6. Both vectors contained a Luciferase expression cassette and were systemically delivered to C57/Bl6 mice. Quantified luciferase expression of striated muscle in these mice displayed up to 3.0x10e7 RFU/mg protein.
539. Transcriptional Repression with ZincFinger and Tale Protein Scaffold
Salvatore Botta,1 Elena Marrocco,1 Nicola de Prisco,1 Toni Cathomen,2 Claudio Mussolino,2 Enrico Maria Surace.1 1 Tigem, Telethon Institute of Genetics and Medicine, Naples, Italy; 2Laboratory of Cell and Gene Therapy, Center for Chronic Immunodeficiency, University Medical Center, Freiburg, Germany. We recently demonstrated that adeno-associated viral (AAV) vector somatic gene transfer of zinc-finger based transcriptional repressors results in the generation of robust transcriptional downregulation of one of the most expressed gene in mammals, the photoreceptor-specific Rhodopsin gene (RHO), irrespectively to wild-type or mutated alleles (mutational-independent approach). In principle this approach enables the correction of any photoreceptor disease associated with RHO mutation (both dominant and recessive forms). We showed that a selected artificial transcriptional repressor (named Zinc-Finger 6 repressor, ZF6R) delivered by AAV2/8 vector down regulates selectively mutated human RHO expression levels ameliorating the retinal phenotype in a mouse model of autosomal dominant Retinitis Pigmentosa (adRP). To further validate the transcriptional repression via synthetic design of DNA binding S208
proteins strategy to generate therapeutics, we designed RHO targeted transcriptional repressors with the novel Transcription Activator Like Effector (TALEs) platform. We generated two different TALEbased DNA binding domains, targeted to conserved regions of the human and mouse Rhodopsin promoter, that differ in the repeat variable diresidue (RVD) that recognizes the guanine DNA base with distinct affinity and specificity. TALE1 carries “NN” RVDs as the guanine-recognizing domain, while TALE2 was generated with “NK” RVDs. To test the ability of these two TALEs to modulate expression from the Rhodopsin promoter, we fused them to either the VP16 activator or KRAB repressor domains. These TALEs showed robust transcriptional activation or repression at the RHO promoter in in vitro assays to a similar extent as ZF6R. Currently, we are testing and comparing the TALE and Zinc-finger-based artificial transcription factors in vivo.
540. Mouse Gender Influences Brain Expression of a Reporter Gene by IntravascularlyAdministered AAV9
Casey A. Maguire,1,2 Matheus H. W. Crommentuijn,1,3 Dakai Mu,2,4 Eloise Hudry,2,4 Alberto Serrano-Pozo,2,4 Bradley Hyman,2,4 Bakhos A. Tannous.1,2,5 1 Experimental Therapeutics and Molecular Imaging Laboratory, Department of Neurology, The Massachusetts General Hospital, Charlestown, MA; 2Program in Neuroscience, Harvard Medical School, Boston, MA; 3Neuro-oncology Research Group, Department of Neurosurgery, Cancer Center Amsterdam, VU University Medical Center, Amterstam, Netherlands; 4Neurology, The Massachusetts General Hospital, Charlestown, MA; 5Center for Molecular Imaging Research, Department of Radiology, The Massachusetts General Hospital, Boston, MA. Vectors based on adeno-associated virus serotype 9 (AAV9) cross the blood-brain barrier (BBB) and mediate efficient gene expression in the mammalian brain. We observed that intravenous injection of AAV9 (1.5x1012 g.c./kg) encoding the reporter gene (firefly luciferase) yielded higher (3-40 fold) transgene expression in the brain of female mice compared to male mice as assessed by whole body bioluminescence imaging as well as luciferase activity in tissue homogenates. This observation was consistent in two strains of mice (nude, C57BL/6) and was correlated with an increased number of AAV genomes in female whole brain compared to males. Currently we are determining the identity and quantity of cell types transduced by AAV9-GFP after systemic injection of male and female mice. These observations stress the importance of carefully matching groups for gender and may also have implications for improving gene expression in males.
541. Site-Directed Mutagenesis of SurfaceExposed Lysine Residues Leads to Improved Transduction by Recombinant AAV2 and AAV8 Vectors in Murine Hepatocytes In Vivo
Baozheng Li,1 Wenqin Ma,1 George Aslanidi,1 Chen Ling,1 Kim Van Vliet,2 Mavis Agbandje-McKenna,2 Arun Srivastava.1 1 Pediatrics, University of Florida, Gainesville; 2Biochemistry & Molecular Biology, University of Florida, Gainesville. The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of recombinant AAV2 vectors, which negatively impacts the transduction efficiency of these vectors. We have previously reported that the primary signal for ubiquitination is phosphorylation of specific surface-exposed tyrosine (Y), serine (S), and threonine (T) residues on the AAV2 capsids, and that the removal of some of these residues significantly increases the transduction efficiency of wild-type (WT) AAV2 vectors. We and others have also reported that Y-, S-, or T-mutant AAV2 vectors can be used to Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy