- Email: [email protected]
292. Mini-Intronic Plasmid (MIP) Vector Sequences Enhance AAV-Mediated Transgene Expression In Vitro and In Vivo
AAV VECTOR DEVELOPMENT 290. A Rationally Engineered Novel Capsid Variant of AAV9 for Peripheral Tissue-Detargeted and CNS-Directed Systemic Gene Delivery
Li Zhong, Shaoyong Li, Kim Van Vliet, Mengxin Li, Jun Xie,1 Jia Li,1 Qin Su,1 Ran He,1 Yu Zhang,1 Huapeng Li,1 Dan Wang,1 Jason Goetzmann,5 Terence R. Flotte,1,2 Mavis AgbandjeMcKenna,4 Guangping Gao.1,3 1 Gene Therapy Center, UMass Medical School, Worcester; 2Dept of Pediatrics, UMass Medical School, Worcester; 3Dept of Microbio & Physiol Systems, Umass Medical School, Worcester; 4Dept of Biochemistry and Mol Biology, University of Florida, Gainesville; 5 New Iberia Research Center, University of Louisiana at Lafayette, New Iberia. 1,2
1
4
1
Intravenous (i.v.) gene delivery to the CNS by rAAV is an attractive approach for treating central nervous system (CNS) disorders that affect large areas of the CNS. Among the rAAVs that can cross the blood-brain-barrier (BBB) and achieve global CNS transduction, rAAV9 is the first reported vector with such a propensity. However, one caveat with systemic rAAV9-mediated CNS transduction is its strong inadvertent transduction of all major peripheral tissues and possible vector and transgene-related toxicity. Here, we report a rationally engineered novel capsid variant of AAV9 for peripheral tissue-detargeted and CNS-directed systemic gene delivery. We previously reported the isolation of a novel natural variant of AAV9 called AAVClvD8 with a significantly reduced transcytosis property. Furthermore, we also made and tested the vectors with single and combinatory mutations for each of the a.a. on the AAV9 capsid that are mutated in AAVClvD8. We created a novel capsid mutant of AAV9 called AAV9HR with lowered luciferase expression and vector genome abundance (VGA) in liver by all three routes of delivery (i.v., i.m. and i.n.) and local tissue-restricted expression and genome persistence by i.m. and i.n. delivery. AAV9.HR also displayed a blood clearance pattern similar to that of rAAV9wt by all three-route administration, suggesting that its ability to cross vascular barrier from tissue to vessel was not impaired. Therefore, we investigated AAV9HR for systemic gene delivery for CNS targeting. Following i.v. delivery of rAAV9wt and rAAV9HR.EGFP to both neonatal and adult C57BL/6 mice, rAAVHR vector produced comparable EGFP transduction in the neurons but remarkably reduced EGFP expression in glial cells in the brain and spinal cord as compared to rAAV9wt. Moreover, rAAV9HR led to significantly diminished EGFP expression in liver, pancreas, skeletal muscle and heart as compared to rAAV9wt. Importantly, similar to the wt AAV9, following i.v. delivery of rAAV9HR.ASAP in Day 1 successfully rescued the early lethality of ASPA-/- mice with significantly reduced AspA expression in peripheral tissues, suggesting its potential clinical utility in treating the neurological diseases with global CNS pathology. Our study represents a significant advance in understanding vector biology of AAV9 and developing novel vectors with the CNS-restricted tropism but improved safety profile for i.v. gene therapy of CNS disorders. a, Equally contributed authors; b, Co-corresponding authors.
291. Regional-CNS MiniPromoters for AAV Are Identified in a High Through-Put Pipeline
Elizabeth M. Simpson,1 Charles N. de Leeuw,1 Siu Ling Lam,1 Andrea J. Korecki,1 Russell J. Bonaguro,1 Kaelan Wong,1 Michelle Zhou,1 Garrett E. Berry,2 Tess C. Lengyell,1 Olga Kaspieva,1 Stéphanie Laprise,1 Lisa Borretta,1 Simone C. McInerny,1 Alice Y. Chou,1 Elodie Portales-Casamar,1 Cletus A. D’Souza,3 Magdalena I. Swanson,1 George S. Yang,3 Steven J. M. Jones,3 Robert A. Holt,3 Aravind Asokan,2 Wyeth W. Wasserman,1 Daniel Goldowitz.1 1 Centre for Molecular Medicine and Therapeutics, The University of British Columbia, Vancouver, BC, Canada; 2Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC; 3Canada’s Michael Smith Genome Sciences Centre, Vancouver, BC, Canada. Small human promoters, capable of driving cell-type restricted gene expression, will be critical for the success of many gene therapies. Such promoters will restrict expression and thus limit off-target side effects and immunogenicity, show a more physiological expression than the often used viral promoters, and contain exclusively human sequences that will be less susceptible to inactivation and more acceptable to regulatory agencies. However, the conversion of typically large human promoters into MiniPromoters for use in such space-limited vectors as adeno-associated virus (AAV) presents a substantial challenge. To address this challenge, we established a high through-put AAV pipeline to test MiniPromoters designed for expression in the brain, eye, and spinal cord. Candidate promoters were chosen from: 1) our previous work developing Pleiades (Ple) MiniPromoters for use single-copy site-specific in the mouse genome (Portales-Casamar et al., PNAS, 2010; de Leeuw et al., MTM, 2014); 2) new bioinformaticsdriven designs; and 3) from the work of others (e.g. Vista Enhancer Browser, 2012). The pipeline employed MiniPromoters driving icre injected intravenously at 5e11 genome copies of AAV9 into newborn B6129F1-Gt(ROSA)26Sortm1Sor mice. This is an “historical” reporter system, in which areas of promoter function were detected by the cre-induced activation of lacZ expression from the mouse genome. A subset of promoters was also tested using “current” reporters; emGFP and MiniSOG. Results of this work include new human MiniPromoters for use in targeting AAV expression to the cells of the: raphe nuclei (Ple67, FEV regulatory regions (RRs)), striatum (Ple94 (GPR88 RRs)), and blood brain barrier (Ple34 and Ple261, CLDN5 RRs). All Pleiades MiniPromoters are available to the gene therapy community. Additional information can be found at www.pleiades. org or www.caneucre.org. Published materials are available through Addgene (www.addgene.org), and unpublished materials are available by contacting Elizabeth M. Simpson ([email protected]).
292. Mini-Intronic Plasmid (MIP) Vector Sequences Enhance AAV-Mediated Transgene Expression In Vitro and In Vivo
Jiamiao Lu,1 Jim Williams,2 Jeremy Luke,2 Feijie Zhang,1 Mark A. Kay.1 1 Departments of Pediatrics and Genetics, Stanford University, Palo Alto, CA; 2Nature Technology Corporation, Lincoln, NE. In quiescent tissues, minicircle DNA vectors can provide 10 times higher sustained levels of transgene expression compared to that achieved with a canonical plasmid containing the same expression cassette (Chen et al., Molecular Therapy 2003; Chen et al., Molecular Therapy 2005). We recently established that transgene silencing occurs when > 1kb of spacer DNA exists between the 5’ end of promoter and 3’ end of polyA site (Lu et al., Molecular Therapy 2012). Based on these findings, we constructed a mini-intronic plasmid (MIP) in which the components required for plasmid propagation S112
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
AAV VECTOR DEVELOPMENT in bacteria (bacterial replication origin and selectable marker) are positioned within an intron of a non-coding exon contained within the expression cassette ((Lu et al., Molecular Therapy 2013). MIP vectors are not subject to transgene silencing and in addition provide 2-10 times or higher levels of transgene expression compared with minicircle vectors in vivo and in vitro. We placed the 1.4 kb MIP intron containing an expression cassette designed to express human alpha 1 antitrypsin, MIP.RSV.hAAT.bpA into a rAAV8 vector. We found that this vector resulted in 40-50 times higher transgene expression compared to an equivalent dose of vector containing the same expression cassette minus the MIP intron after infusion into mice. However because of the packaging size limitation of an AAV vector, when we tried to minimize the size or replace the sequences within the intron, we lost its ability to enhance transgene expression. Next we replaced the shorter introns with mini-bacterial origins of replication (140-320bp). These sequences with the nonantibiotic selectable marker, RNA-OUT were less than 500bp in length. MIP DNA vectors with these new introns retain the abilities of enhancing transgene expression in vivo. The size and the transgene expression enhancing features make these miniorigin containing MIP introns are now being tested in AAV vectors. The use of these novel mini-intronic DNA expression cassettes has great potential for enhancing transgene expression not only from DNA plasmid vectors but rAAV vectors.
293. Optimizing rAAV Vector for CFTR Expression Using Synthetic Promoter and Enhancers
Ziying Yan,1 Xingshen Sun,1 Guiying Li,2 Zoe A. Stewart,2 Stephen J. Elledge,3 John F. Engelhardt.1 1 Department of Anatomy and Cell Biology, The University of Iowa, Iowa City; 2Department of Surgery, The University of Iowa, Iowa City; 3Department of Genetics, Howard Hughes Medical Institute, Brigham and Women’s Hospital, Boston. The use of recombinant adeno-associated viral (rAAV) vectors to express the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been under development for two decades as a gene therapy for cystic fibrosis (CF). However, due to the size (4.43kb) of the CFTR cDNA, CFTR expression from rAAV vectors has been suboptimal because it necessities the use of short and less active promoters that will package into the 4.67kb rAAV genome. Despite the fact that rAAV vector can package oversized genomes, we observe that pushing the package limits of rAAV-CFTR vectors above 5kb dramatically jeopardizes the vector functionality. We find that the CFTR expression cassette must be less than 4.6kb for effective CFTR expression; this leaves <200bp for all the essential transcriptional elements for the full-length CFTR cDNA. Previously, we developed a rAAV-CFTR vector (rAAV-CFtg83) that uses an 83-bp synthetic promoter and 60bp synthetic polyadenylation signal for full length human CFTR cDNA. To develop a ferret CFTR expressing AAV vector for our recently established CF ferret model, we attempted to incorporate short enhancer element to strengthen the function of the tg83 promoter. In the previous screening of an oligonucleotide library of 100-mer synthetic sequences, several sequences were identified that significantly enhance the CMV IE minimal promoter in the different cell lines tested. We further tested these enhancers for transcriptional activation of the tg83 promoter in different airway cell lines and polarized airway epithelium culture grown at an airliquid interface (ALI). One of these enhancer sequences promoted significant transcription of the tg83 promoter-luciferase transgene in both polarized human and ferret airway ALI cultures, as well as in ferret airway in vivo. However, this 183bp enh/tg83 combination and 4.43kb CFTR cDNA exceeded efficient packaging capacity for rAAV. Thus, we utilized a shortened ferret CFTR minigene fCFTRΔR, for which 159bp sequence in the R domain is deleted. We constructed Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
a rAAV vector (AV2.enhtg83-fCFTRΔR) and tested its ability to functionally correct CFTR-mediated Cl- transport in CF ALI culture by short circuit current. Compared with the enhancer-less AV2.tg83fCFTRΔR, we found that the CF ALI cultures infected with AV2. enhtg83-fCFTRΔR acquired 77% CFTR-mediated Cl- transport of that seen in non-CF cultures, while only 22% correction was observed following infection with AV2.tg83-fCFTRΔR. Analysis of virusderived ferret CFTR mRNA levels by TaqMan PCR demonstrated that there was a 6.4-fold and 17.1-fold higher production of fCFTR mRNA following AV2.enhtg83-fCFTRΔR infection than AV2.tg83fCFTRΔR infection at the time points of 3- and 10-day post-infection, respectively. These studies have identified a small enhancer/promoter combination that may have unique utility for rAAV-mediated gene therapy to the airway.
294. Effective Delivery of Large Genes To the Murine Retina By Dual AAV Vectors
Ivana Trapani,1 Pasqualina Colella,1 Andrea Sommella,1 Carolina Iodice,1 Giulia Cesi,1 Sonia de Simone,1 Elena Marrocco,1 Settimio Rossi,2 Massimo Giunti,3 Arpad Palfi,4 G. Jane Farrar,4 Roman Polishchuk,1 Alberto Auricchio.1,5 1 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy; 2Department of Ophthalmology, Second University of Naples, Naples, Italy; 3Department of Veterinary Morphophysiology and Animal Production, University of Bologna, Bologna, Italy; 4The School of Genetics & Microbiology, Trinity College Dublin, Dublin, Ireland; 5Medical Genetics, Department of Translational Medicine, Federico II University, Naples, Italy. Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV’s limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt’s disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on “forced” packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size, which raise safety concerns. Taking advantage of AAV’s ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We compared their transduction efficiency in vitro and in the murine retina. Finally we assessed the efficacy of the best performing strategies in the retina of Abca4-/- and sh1 murine models of STGD and USH1B, respectively. We found that dual AAV vectors are more efficient than oversized vectors, both in vitro and in the murine retina. While dual AAV OV vectors efficiently transduce only RPE, dual AAV trans-splicing and hybrid approaches transduce efficiently photoreceptors, a major target for retinal gene therapy, in addition to RPE. Although the levels of murine photoreceptor transduction mediated by dual AAV trans-splicing and hybrid vectors are lower than those achieved with a single AAV, they resulted in significant improvement of the retinal phenotype of the mouse models of STGD and USH1B. In conclusion we found that dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.
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Li Zhong, Shaoyong Li, Kim Van Vliet, Mengxin Li, Jun Xie,1 Jia Li,1 Qin Su,1 Ran He,1 Yu Zhang,1 Huapeng Li,1 Dan Wang,1 Jason Goetzmann,5 Terence R. Flotte,1,2 Mavis AgbandjeMcKenna,4 Guangping Gao.1,3 1 Gene Therapy Center, UMass Medical School, Worcester; 2Dept of Pediatrics, UMass Medical School, Worcester; 3Dept of Microbio & Physiol Systems, Umass Medical School, Worcester; 4Dept of Biochemistry and Mol Biology, University of Florida, Gainesville; 5 New Iberia Research Center, University of Louisiana at Lafayette, New Iberia. 1,2
1
4
1
Intravenous (i.v.) gene delivery to the CNS by rAAV is an attractive approach for treating central nervous system (CNS) disorders that affect large areas of the CNS. Among the rAAVs that can cross the blood-brain-barrier (BBB) and achieve global CNS transduction, rAAV9 is the first reported vector with such a propensity. However, one caveat with systemic rAAV9-mediated CNS transduction is its strong inadvertent transduction of all major peripheral tissues and possible vector and transgene-related toxicity. Here, we report a rationally engineered novel capsid variant of AAV9 for peripheral tissue-detargeted and CNS-directed systemic gene delivery. We previously reported the isolation of a novel natural variant of AAV9 called AAVClvD8 with a significantly reduced transcytosis property. Furthermore, we also made and tested the vectors with single and combinatory mutations for each of the a.a. on the AAV9 capsid that are mutated in AAVClvD8. We created a novel capsid mutant of AAV9 called AAV9HR with lowered luciferase expression and vector genome abundance (VGA) in liver by all three routes of delivery (i.v., i.m. and i.n.) and local tissue-restricted expression and genome persistence by i.m. and i.n. delivery. AAV9.HR also displayed a blood clearance pattern similar to that of rAAV9wt by all three-route administration, suggesting that its ability to cross vascular barrier from tissue to vessel was not impaired. Therefore, we investigated AAV9HR for systemic gene delivery for CNS targeting. Following i.v. delivery of rAAV9wt and rAAV9HR.EGFP to both neonatal and adult C57BL/6 mice, rAAVHR vector produced comparable EGFP transduction in the neurons but remarkably reduced EGFP expression in glial cells in the brain and spinal cord as compared to rAAV9wt. Moreover, rAAV9HR led to significantly diminished EGFP expression in liver, pancreas, skeletal muscle and heart as compared to rAAV9wt. Importantly, similar to the wt AAV9, following i.v. delivery of rAAV9HR.ASAP in Day 1 successfully rescued the early lethality of ASPA-/- mice with significantly reduced AspA expression in peripheral tissues, suggesting its potential clinical utility in treating the neurological diseases with global CNS pathology. Our study represents a significant advance in understanding vector biology of AAV9 and developing novel vectors with the CNS-restricted tropism but improved safety profile for i.v. gene therapy of CNS disorders. a, Equally contributed authors; b, Co-corresponding authors.
291. Regional-CNS MiniPromoters for AAV Are Identified in a High Through-Put Pipeline
Elizabeth M. Simpson,1 Charles N. de Leeuw,1 Siu Ling Lam,1 Andrea J. Korecki,1 Russell J. Bonaguro,1 Kaelan Wong,1 Michelle Zhou,1 Garrett E. Berry,2 Tess C. Lengyell,1 Olga Kaspieva,1 Stéphanie Laprise,1 Lisa Borretta,1 Simone C. McInerny,1 Alice Y. Chou,1 Elodie Portales-Casamar,1 Cletus A. D’Souza,3 Magdalena I. Swanson,1 George S. Yang,3 Steven J. M. Jones,3 Robert A. Holt,3 Aravind Asokan,2 Wyeth W. Wasserman,1 Daniel Goldowitz.1 1 Centre for Molecular Medicine and Therapeutics, The University of British Columbia, Vancouver, BC, Canada; 2Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC; 3Canada’s Michael Smith Genome Sciences Centre, Vancouver, BC, Canada. Small human promoters, capable of driving cell-type restricted gene expression, will be critical for the success of many gene therapies. Such promoters will restrict expression and thus limit off-target side effects and immunogenicity, show a more physiological expression than the often used viral promoters, and contain exclusively human sequences that will be less susceptible to inactivation and more acceptable to regulatory agencies. However, the conversion of typically large human promoters into MiniPromoters for use in such space-limited vectors as adeno-associated virus (AAV) presents a substantial challenge. To address this challenge, we established a high through-put AAV pipeline to test MiniPromoters designed for expression in the brain, eye, and spinal cord. Candidate promoters were chosen from: 1) our previous work developing Pleiades (Ple) MiniPromoters for use single-copy site-specific in the mouse genome (Portales-Casamar et al., PNAS, 2010; de Leeuw et al., MTM, 2014); 2) new bioinformaticsdriven designs; and 3) from the work of others (e.g. Vista Enhancer Browser, 2012). The pipeline employed MiniPromoters driving icre injected intravenously at 5e11 genome copies of AAV9 into newborn B6129F1-Gt(ROSA)26Sortm1Sor mice. This is an “historical” reporter system, in which areas of promoter function were detected by the cre-induced activation of lacZ expression from the mouse genome. A subset of promoters was also tested using “current” reporters; emGFP and MiniSOG. Results of this work include new human MiniPromoters for use in targeting AAV expression to the cells of the: raphe nuclei (Ple67, FEV regulatory regions (RRs)), striatum (Ple94 (GPR88 RRs)), and blood brain barrier (Ple34 and Ple261, CLDN5 RRs). All Pleiades MiniPromoters are available to the gene therapy community. Additional information can be found at www.pleiades. org or www.caneucre.org. Published materials are available through Addgene (www.addgene.org), and unpublished materials are available by contacting Elizabeth M. Simpson ([email protected]).
292. Mini-Intronic Plasmid (MIP) Vector Sequences Enhance AAV-Mediated Transgene Expression In Vitro and In Vivo
Jiamiao Lu,1 Jim Williams,2 Jeremy Luke,2 Feijie Zhang,1 Mark A. Kay.1 1 Departments of Pediatrics and Genetics, Stanford University, Palo Alto, CA; 2Nature Technology Corporation, Lincoln, NE. In quiescent tissues, minicircle DNA vectors can provide 10 times higher sustained levels of transgene expression compared to that achieved with a canonical plasmid containing the same expression cassette (Chen et al., Molecular Therapy 2003; Chen et al., Molecular Therapy 2005). We recently established that transgene silencing occurs when > 1kb of spacer DNA exists between the 5’ end of promoter and 3’ end of polyA site (Lu et al., Molecular Therapy 2012). Based on these findings, we constructed a mini-intronic plasmid (MIP) in which the components required for plasmid propagation S112
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
AAV VECTOR DEVELOPMENT in bacteria (bacterial replication origin and selectable marker) are positioned within an intron of a non-coding exon contained within the expression cassette ((Lu et al., Molecular Therapy 2013). MIP vectors are not subject to transgene silencing and in addition provide 2-10 times or higher levels of transgene expression compared with minicircle vectors in vivo and in vitro. We placed the 1.4 kb MIP intron containing an expression cassette designed to express human alpha 1 antitrypsin, MIP.RSV.hAAT.bpA into a rAAV8 vector. We found that this vector resulted in 40-50 times higher transgene expression compared to an equivalent dose of vector containing the same expression cassette minus the MIP intron after infusion into mice. However because of the packaging size limitation of an AAV vector, when we tried to minimize the size or replace the sequences within the intron, we lost its ability to enhance transgene expression. Next we replaced the shorter introns with mini-bacterial origins of replication (140-320bp). These sequences with the nonantibiotic selectable marker, RNA-OUT were less than 500bp in length. MIP DNA vectors with these new introns retain the abilities of enhancing transgene expression in vivo. The size and the transgene expression enhancing features make these miniorigin containing MIP introns are now being tested in AAV vectors. The use of these novel mini-intronic DNA expression cassettes has great potential for enhancing transgene expression not only from DNA plasmid vectors but rAAV vectors.
293. Optimizing rAAV Vector for CFTR Expression Using Synthetic Promoter and Enhancers
Ziying Yan,1 Xingshen Sun,1 Guiying Li,2 Zoe A. Stewart,2 Stephen J. Elledge,3 John F. Engelhardt.1 1 Department of Anatomy and Cell Biology, The University of Iowa, Iowa City; 2Department of Surgery, The University of Iowa, Iowa City; 3Department of Genetics, Howard Hughes Medical Institute, Brigham and Women’s Hospital, Boston. The use of recombinant adeno-associated viral (rAAV) vectors to express the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been under development for two decades as a gene therapy for cystic fibrosis (CF). However, due to the size (4.43kb) of the CFTR cDNA, CFTR expression from rAAV vectors has been suboptimal because it necessities the use of short and less active promoters that will package into the 4.67kb rAAV genome. Despite the fact that rAAV vector can package oversized genomes, we observe that pushing the package limits of rAAV-CFTR vectors above 5kb dramatically jeopardizes the vector functionality. We find that the CFTR expression cassette must be less than 4.6kb for effective CFTR expression; this leaves <200bp for all the essential transcriptional elements for the full-length CFTR cDNA. Previously, we developed a rAAV-CFTR vector (rAAV-CFtg83) that uses an 83-bp synthetic promoter and 60bp synthetic polyadenylation signal for full length human CFTR cDNA. To develop a ferret CFTR expressing AAV vector for our recently established CF ferret model, we attempted to incorporate short enhancer element to strengthen the function of the tg83 promoter. In the previous screening of an oligonucleotide library of 100-mer synthetic sequences, several sequences were identified that significantly enhance the CMV IE minimal promoter in the different cell lines tested. We further tested these enhancers for transcriptional activation of the tg83 promoter in different airway cell lines and polarized airway epithelium culture grown at an airliquid interface (ALI). One of these enhancer sequences promoted significant transcription of the tg83 promoter-luciferase transgene in both polarized human and ferret airway ALI cultures, as well as in ferret airway in vivo. However, this 183bp enh/tg83 combination and 4.43kb CFTR cDNA exceeded efficient packaging capacity for rAAV. Thus, we utilized a shortened ferret CFTR minigene fCFTRΔR, for which 159bp sequence in the R domain is deleted. We constructed Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
a rAAV vector (AV2.enhtg83-fCFTRΔR) and tested its ability to functionally correct CFTR-mediated Cl- transport in CF ALI culture by short circuit current. Compared with the enhancer-less AV2.tg83fCFTRΔR, we found that the CF ALI cultures infected with AV2. enhtg83-fCFTRΔR acquired 77% CFTR-mediated Cl- transport of that seen in non-CF cultures, while only 22% correction was observed following infection with AV2.tg83-fCFTRΔR. Analysis of virusderived ferret CFTR mRNA levels by TaqMan PCR demonstrated that there was a 6.4-fold and 17.1-fold higher production of fCFTR mRNA following AV2.enhtg83-fCFTRΔR infection than AV2.tg83fCFTRΔR infection at the time points of 3- and 10-day post-infection, respectively. These studies have identified a small enhancer/promoter combination that may have unique utility for rAAV-mediated gene therapy to the airway.
294. Effective Delivery of Large Genes To the Murine Retina By Dual AAV Vectors
Ivana Trapani,1 Pasqualina Colella,1 Andrea Sommella,1 Carolina Iodice,1 Giulia Cesi,1 Sonia de Simone,1 Elena Marrocco,1 Settimio Rossi,2 Massimo Giunti,3 Arpad Palfi,4 G. Jane Farrar,4 Roman Polishchuk,1 Alberto Auricchio.1,5 1 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy; 2Department of Ophthalmology, Second University of Naples, Naples, Italy; 3Department of Veterinary Morphophysiology and Animal Production, University of Bologna, Bologna, Italy; 4The School of Genetics & Microbiology, Trinity College Dublin, Dublin, Ireland; 5Medical Genetics, Department of Translational Medicine, Federico II University, Naples, Italy. Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV’s limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt’s disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on “forced” packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size, which raise safety concerns. Taking advantage of AAV’s ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We compared their transduction efficiency in vitro and in the murine retina. Finally we assessed the efficacy of the best performing strategies in the retina of Abca4-/- and sh1 murine models of STGD and USH1B, respectively. We found that dual AAV vectors are more efficient than oversized vectors, both in vitro and in the murine retina. While dual AAV OV vectors efficiently transduce only RPE, dual AAV trans-splicing and hybrid approaches transduce efficiently photoreceptors, a major target for retinal gene therapy, in addition to RPE. Although the levels of murine photoreceptor transduction mediated by dual AAV trans-splicing and hybrid vectors are lower than those achieved with a single AAV, they resulted in significant improvement of the retinal phenotype of the mouse models of STGD and USH1B. In conclusion we found that dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.
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