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677. Stabilizing the rAAV Vector Genome Increases AAV Vector Transduction Efficiency
AAV Vector Development, Production and Application II AAV Vector Development, Production and Application II 676. DNA Sequences Encoding shRNAs Can Replace Mutant ITR in scAAV Genome for Efficient Replication and Packaging and Transcribe shRNAs by pol III Promoter Activity of wt ITR for Efficient Gene Silencing
Jun Xie,1,2 Qin Mao,1 Ran He,1 Ye Zhu,1,3 Jia Li,1 Qin Su,1 Mengxin Li,1 Li Zhong,1 Chris Muller,1 Heather Zhou,4 Guangping Gao.1,2 1 Gene Therapy Center, Worcester; 2Dept. of Microbiol & Physiol. Syst, UMASS, Worcester; 3West China Hospital, Sichuan University, Chengdu, China; 4Merck & Co, Whitehouse Station.
The two inverted terminal repeats (ITRs) at each end of recombinant adeno-associated virus (rAAV) genome are the only sequences inherited from wild type (Wt) viral genome. They contribute to rAAV genome replication, packaging, intracellular processing and stabilization, and integration into host genome. Previously we showed DNA sequences encoding shRNAs (shDNA) are the barriers in self-complementary AAV (scAAV) genome replication, resulting in vector genome truncations and heterogeneity. We now observe the same phenomena in the genome replication and packaging of single-stranded AAV vectors harboring shDNA. When detected on a denaturing gel, these truncated genomes were doubled in size as seen for scAAV, indicating they are intra-molecular double-stranded DNA molecules. We hypothesized DNA with hairpin structures could serve as the mutant ITR during rAAV genome replication and packaging. To test this, we replaced the mutant ITR in scAAV genome with the DNA encoding shRNA against Apob or Firefly luciferase gene. Southern blot of Hirt’s DNAs revealed that vector genomes flanked with shDNA and wtITR, but not with shDNA and mutant ITR, were efficiently rescued and replicated when co-transfected with adenohelper plasmid and Rep/Cap trans-plasmid into HEK293 cells. Large scale AAV production and analyses of CsCl gradient purified rAAV genomes in both native and denaturing gels further demonstrated that hybrid shDNA-wtITR vector genomes were efficiently packaged into AAV capsid as double-stranded genomes. To test their functionalities in vivo, we intravenously injected 2x1011 GCs of scAAV9 containing shApoB-wtITR genome with an EGFP gene into adult C57 mice and harvested liver tissues 3 weeks later. Compared to regular scAAVEGFP vector, the shApoB-wtITR AAV vectors achieved comparable EGFP transduction efficacy in the liver. Interestingly, without a H1 promoter the shApob produced abundant small antisense RNA and silenced mouse ApoB gene as efficienct as conventional scAAVshRNA vectors. Southern blots of mouse liver detected linear and circularized forms of AAV genomes, thus implying that wtITR in the circularized genome may serve as a strong pol III promoter to drive shRNA expression. Currently, we are in the process of replacing Wt ITR with an artificial chimeric ITR with only rep binding element, terminal resolution sites and D sequence of authentic ITR retained. In summary, hairpin structure sequences can function as an alternative mutant ITR for production of functional scAAV. Our findings may have significant impact on AAV biology and vectorology, potentially enabling us to create artificially intelligent and programmable vector genomes with improved packaging efficacy, expanded genome sizes, modulated tissue tropism and propensity of integration, regulatable in vivo persistence and potency, etc.
677. Stabilizing the rAAV Vector Genome Increases AAV Vector Transduction Efficiency
Katie A. Pokiniewski,1 Andrea R. Moore,1 Biao Dong,1 Qizhao Wang,1 Weidong Xiao.1 1 Microbiology and Immunology, Temple University, Philadelphia, PA. It has been determined that AAV vector efficiency decreases due to the following mechanisms: ineffective endocytosis, endosomal degradation, inefficient trafficking, and the need to convert from a single-stranded AAV genome to a transcriptional active doublestranded form. Administering rAAV vectors in conjunction with a drug that can enhance vector transduction may overcome some of these cellular barriers encountered during infection as well as help stabilize the genome. Here we discovered that Cidofovir(CDV), a monophosphate nucleotide analogue that competitively inhibits the incorporation of deoxycytidine triphosphate into viral DNA via viral DNA polymerase, is able to increase single-stranded rAAV transgene expression in the nucleus by 2 to 9 fold, depending on the cell type and concentration. This increase has been consistently observed in 16095, HelaS3, and Hek293 cells with little toxicity to the cells. However, there is less than a two-fold increase with Cidofovir treatment when using self-complementary AAV vectors(scAAV), suggesting that CDV might enhance conversion of the AAV genome from single to double stranded form. These observations were confirmed using two different serotypes, AAV2 and AAV8, in vitro indicating that the mechanism of enhancement is not serotype dependent. Southern Blot analysis along with real-time PCR demonstrated that Cidofovir treatment in HelaS3 cells increases viral DNA accumulation 24 hours post-infection compared to PBS treated cells. Cells pretreated with CDV then infected with rAAV revealed no difference in the amount of vector present between 0 and 2 hrs post infection, suggesting CDV does not enhance viral entry. However, cytoplasmic and nuclear fractions of HelaS3 cells pretreated with CDV or PBS demonstrated that CDV pretreatment enhances viral accumulation of rAAV vectors in both the cytoplasm and nucleus 24 hours post-infection. In vivo, Hemophilia A BALB/c(BALB/c-HA) mice were treated with CDV prior to infection with a rAAV containing the fVIII Human Light chain(LC) gene. It showed that CDV treatment enhanced the amount of LC antigen when compared to control group. Approximately two fold increase was observed up to fourteen weeks post-injection. These results demonstrate that CDV treatment can enhance and sustain rAAV levels in vivo. CDV has already been approved by the Food and Drug Administration (FDA) for the treatment of cytomegalovirus retinitis in patients with acquired immunodeficiency syndrome (AIDS). The effects of CDV on small DNA viruses that lack their own viral DNA polymerase, like AAV, have not been documented. The use of drugs like Cidofovir can greatly contribute to the understanding of rAAV trafficking and eventually lead the development of novel strategies to increase overall efficiency of AAV transduction.
678. Increasing AAV Vector Yield By RiboswitchMediated Attenuation of Toxic Transgene Effects in HEK-293 Producer Cells
Benjamin Strobel,1 Benedikt Klauser,2 Thorsten Lamla,1 Florian Gantner,1 Sebastian Kreuz.1 1 Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany; 2University of Konstanz, Konstanz, Germany.
Adeno-associated virus (AAV) vectors are among the most promising viral vector systems for human gene therapy and have also been used extensively to investigate gene function in preclinical research. Achievement of high-titer vector yields regardless of the packaged transgene hence is a key goal of process optimization. However, transgenes expressed by constitutive, ubiquitously active Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
S269
Jun Xie,1,2 Qin Mao,1 Ran He,1 Ye Zhu,1,3 Jia Li,1 Qin Su,1 Mengxin Li,1 Li Zhong,1 Chris Muller,1 Heather Zhou,4 Guangping Gao.1,2 1 Gene Therapy Center, Worcester; 2Dept. of Microbiol & Physiol. Syst, UMASS, Worcester; 3West China Hospital, Sichuan University, Chengdu, China; 4Merck & Co, Whitehouse Station.
The two inverted terminal repeats (ITRs) at each end of recombinant adeno-associated virus (rAAV) genome are the only sequences inherited from wild type (Wt) viral genome. They contribute to rAAV genome replication, packaging, intracellular processing and stabilization, and integration into host genome. Previously we showed DNA sequences encoding shRNAs (shDNA) are the barriers in self-complementary AAV (scAAV) genome replication, resulting in vector genome truncations and heterogeneity. We now observe the same phenomena in the genome replication and packaging of single-stranded AAV vectors harboring shDNA. When detected on a denaturing gel, these truncated genomes were doubled in size as seen for scAAV, indicating they are intra-molecular double-stranded DNA molecules. We hypothesized DNA with hairpin structures could serve as the mutant ITR during rAAV genome replication and packaging. To test this, we replaced the mutant ITR in scAAV genome with the DNA encoding shRNA against Apob or Firefly luciferase gene. Southern blot of Hirt’s DNAs revealed that vector genomes flanked with shDNA and wtITR, but not with shDNA and mutant ITR, were efficiently rescued and replicated when co-transfected with adenohelper plasmid and Rep/Cap trans-plasmid into HEK293 cells. Large scale AAV production and analyses of CsCl gradient purified rAAV genomes in both native and denaturing gels further demonstrated that hybrid shDNA-wtITR vector genomes were efficiently packaged into AAV capsid as double-stranded genomes. To test their functionalities in vivo, we intravenously injected 2x1011 GCs of scAAV9 containing shApoB-wtITR genome with an EGFP gene into adult C57 mice and harvested liver tissues 3 weeks later. Compared to regular scAAVEGFP vector, the shApoB-wtITR AAV vectors achieved comparable EGFP transduction efficacy in the liver. Interestingly, without a H1 promoter the shApob produced abundant small antisense RNA and silenced mouse ApoB gene as efficienct as conventional scAAVshRNA vectors. Southern blots of mouse liver detected linear and circularized forms of AAV genomes, thus implying that wtITR in the circularized genome may serve as a strong pol III promoter to drive shRNA expression. Currently, we are in the process of replacing Wt ITR with an artificial chimeric ITR with only rep binding element, terminal resolution sites and D sequence of authentic ITR retained. In summary, hairpin structure sequences can function as an alternative mutant ITR for production of functional scAAV. Our findings may have significant impact on AAV biology and vectorology, potentially enabling us to create artificially intelligent and programmable vector genomes with improved packaging efficacy, expanded genome sizes, modulated tissue tropism and propensity of integration, regulatable in vivo persistence and potency, etc.
677. Stabilizing the rAAV Vector Genome Increases AAV Vector Transduction Efficiency
Katie A. Pokiniewski,1 Andrea R. Moore,1 Biao Dong,1 Qizhao Wang,1 Weidong Xiao.1 1 Microbiology and Immunology, Temple University, Philadelphia, PA. It has been determined that AAV vector efficiency decreases due to the following mechanisms: ineffective endocytosis, endosomal degradation, inefficient trafficking, and the need to convert from a single-stranded AAV genome to a transcriptional active doublestranded form. Administering rAAV vectors in conjunction with a drug that can enhance vector transduction may overcome some of these cellular barriers encountered during infection as well as help stabilize the genome. Here we discovered that Cidofovir(CDV), a monophosphate nucleotide analogue that competitively inhibits the incorporation of deoxycytidine triphosphate into viral DNA via viral DNA polymerase, is able to increase single-stranded rAAV transgene expression in the nucleus by 2 to 9 fold, depending on the cell type and concentration. This increase has been consistently observed in 16095, HelaS3, and Hek293 cells with little toxicity to the cells. However, there is less than a two-fold increase with Cidofovir treatment when using self-complementary AAV vectors(scAAV), suggesting that CDV might enhance conversion of the AAV genome from single to double stranded form. These observations were confirmed using two different serotypes, AAV2 and AAV8, in vitro indicating that the mechanism of enhancement is not serotype dependent. Southern Blot analysis along with real-time PCR demonstrated that Cidofovir treatment in HelaS3 cells increases viral DNA accumulation 24 hours post-infection compared to PBS treated cells. Cells pretreated with CDV then infected with rAAV revealed no difference in the amount of vector present between 0 and 2 hrs post infection, suggesting CDV does not enhance viral entry. However, cytoplasmic and nuclear fractions of HelaS3 cells pretreated with CDV or PBS demonstrated that CDV pretreatment enhances viral accumulation of rAAV vectors in both the cytoplasm and nucleus 24 hours post-infection. In vivo, Hemophilia A BALB/c(BALB/c-HA) mice were treated with CDV prior to infection with a rAAV containing the fVIII Human Light chain(LC) gene. It showed that CDV treatment enhanced the amount of LC antigen when compared to control group. Approximately two fold increase was observed up to fourteen weeks post-injection. These results demonstrate that CDV treatment can enhance and sustain rAAV levels in vivo. CDV has already been approved by the Food and Drug Administration (FDA) for the treatment of cytomegalovirus retinitis in patients with acquired immunodeficiency syndrome (AIDS). The effects of CDV on small DNA viruses that lack their own viral DNA polymerase, like AAV, have not been documented. The use of drugs like Cidofovir can greatly contribute to the understanding of rAAV trafficking and eventually lead the development of novel strategies to increase overall efficiency of AAV transduction.
678. Increasing AAV Vector Yield By RiboswitchMediated Attenuation of Toxic Transgene Effects in HEK-293 Producer Cells
Benjamin Strobel,1 Benedikt Klauser,2 Thorsten Lamla,1 Florian Gantner,1 Sebastian Kreuz.1 1 Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany; 2University of Konstanz, Konstanz, Germany.
Adeno-associated virus (AAV) vectors are among the most promising viral vector systems for human gene therapy and have also been used extensively to investigate gene function in preclinical research. Achievement of high-titer vector yields regardless of the packaged transgene hence is a key goal of process optimization. However, transgenes expressed by constitutive, ubiquitously active Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
S269