309 Optimization of Production of AAVrh 10 Viral Vectors AAV VectoRs ii Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy S124 307 Hitting the[.]
AAV Vectors II 307 Hitting the Kidney from All Angles: Using Direct Intrarenal Pelvis Injection Combined with Serotype and Promoter Targeting to Achieve Renal AAV Gene Delivery Claire A Schreiber, Sara Holditch, Yasuhiro Ikeda Molecular Medicine, Mayo Clinic, Rochester, MN Using adeno-associated virus (AAV)-based gene therapy to treat renal diseases is an underdeveloped area Renal anatomy and physiology make gene delivery to this organ especially difficult Multiple AAV serotypes and routes of entry in rodents have been tried, with the majority of combinations targeting the renal tubules To improve upon the current methods of AAV renal transduction for future use in the clinical setting, we employed direct intrarenal pelvis injections of AAV2, 6, and with the luciferase (Luc) or GFP transgene under the control of the cytomegalovirus (CMV) or putative kidney-specific promoters from myo-inositol oxygenase(MIOX), acyl-CoA synthetase medium-chain family member (Acsm2), uromodulin (UMOD), kidney androgen-regulated protein (Kap) and solute carrier family 34 (sodium phosphate), member (Slc34a1) mouse genes in C57Bl/6J mice Renal luciferase transgene expression was strongest with AAV6, and 9-CMV-Luc AAV6 and 8-CMVLuc resulted in stronger expression in the injected kidney, whereas AAV9-CMV-Luc showed equal, bilateral renal expression AAV9 with the UMOD promoter gave the strongest and most highly renalspecific luciferase expression among all serotype and kidney promoter combinations Using the GFP transgene we further established AAV transduction of multiple cell types in the kidney AAV8-CMV-GFP transduced both glomerular and tubular renal cells, while AAV9CMV-GFP most strikingly transduced periglomerular tubule cells Both AAV8 and AAV9-UMOD-GFP solely transduced renal tubule cells We are currently testing AAV variants isolated from clinical urine samples to determine their ability to transduce renal cells in vivo Our results demonstrate efficient renal transduction by direct intrarenal pelvis injection of AAV vectors Coupling specific renal cell tropisms of AAV serotypes and renal cell-type-specific promoters, along with the intrarenal pelvis injection strategy would pave the way for a renal-targeted AV gene therapy 308 Ultracentrifugation-Free ChromatographyMediated Large-Scale Purification of Recombinant Adeno-Associated Virus Serotype (rAAV1) and rAAV9 from the Serum-Free Culture Supernatant Taro Tomono , Yukihiko Hirai , Hironori Okada , Kumi Adachi , Akiko Ishii3, Takashi Shimada2, Masafumi Onodera4, Akira Tamaoka3, Takashi Okada2 Nippon Medical School, Tokyo, National Center for Child Health and Development, Tokyo, University of Tsukuba, Ibaraki, Japan, 2Nippon Medical School, Tokyo, Japan, 3University of Tsukuba, Ibaraki, Japan, 4National Center for Child Health and Development, Tokyo, Japan 2 [Background] The current production of rAAV from the transfected cell lysate and purification based on CsCl or iodixanol density ultracentrifugation are not suitable for large-scale processing Although rAAV1 and rAAV9 are promising therapeutic vectors for genetic neuromuscular disorders, the large-scale purification method for those vectors has not yet been established In this study, we elaborate the novel chromatographymediated methods for purification of rAAV1 and rAAV9 from the serum-free culture supernatant with ultracentrifugation-free technique towards large-scale and GMP production [Methods] rAAV1 (scAAV1-CBA-EGFP) and rAAV9 (scAAV9-CBA-EGFP) were produced by the triple-transfection to HEK293 or HEK293EB S124 cells in serum-free medium with polyethyleneimine (PEI) Five days later, the culture supernatant was tangential flow-filtrated (TFF) and concentrated by the Hollow Fiber system After reducing protein debris by 1/3-saturated ammonium sulfate (1/3 AS) precipitation, rAAV1 or rAAV9 was precipitated in 1/2 AS solution Subsequently, the precipitated rAAV1 was dissolved in 10 ml of PBS containing mM MgCl2 After the sample was dialyzed against a buffer containing mM NaCl, the dialysate was diluted with dH2O and loaded to tandem quintet cation-exchange membranes (Mustang SXT) and quintet anion-exchange membranes (Mustang QXT) with a column volume of 0.86 ml After rAAV1 was eluted from Mustang QXT, it was finally purified by gel-filtrated chromatography (Superdex 200 10/300 GL) The precipitated rAAV9 was dissolved in 22 ml of 3.3 mM MES, 3.3 mM HEPES, 3.3 mM sodium acetate (MHN) buffer (pH8.0) containing 50 mM NaCl and 0.01% Pluronic F-68 After the resultant sample was diluted with MHN buffer, it was loaded to tertiary amine charged anion-exchange column The passed through fraction containing rAAV9 was finally purified by gel-filtrated chromatography The physiological and biological properties of the purified rAAV1 and rAAV9 were characterized by qPCR, electron micrograph, FACS, western blot and SDS-PAGE [Results] The purified rAAV1 and rAAV9 displayed three major bands (VP1, VP2, and VP3) on SDS-PAGE and more than 90% of rAAV1 particles or 95% of rAAV9 particles was contained fully packaged viral genomes according to electron micrographic analysis We confirmed increasing infectivity of rAAV1 products depending on chromatographic purification step and that of rAAV9 is under evaluation The resultant genomic titer of the purified rAAV1 was 4.17 x 1013 v.g (3.63 x 1013 v.g./ml) from the x 109 HEK293 cells and the purified rAAV9 was 1.49 x 1015 v.g (1.14 x 1014 v.g./ml) from the 5.1 x 109 HEK293EB cells [Conclusion] Chromatography-mediated purification from the culture supernatant is a major breakthrough, especially for the production of rAAV9 We obtained rAAV1 and rAAV9 by this protocol with high titer, high purity and minimum contamination of empty particles This novel chromatography-based method would be consistent with GMP production and facilitate clinical studies in the future 309 Optimization of Production of AAVrh.10 Viral Vectors Hyunmi Lee, Lucy Quach, Fan Fan, Sara Cram, Dolan Sondhi, Ronald Crystal, Stephen Kaminsky Weill Cornell Medical College, New York, NY With the increasing interest in the use of adeno-associated virus (AAV) vectors to treat human disorders, there is increasing focus on optimizing AAV production to improve yield, purity and potency The production of AAV vectors can be conceptualized in two stages: upstream cell culture and downstream purification and formulation As an example of how AAV production can be optimized, we assessed upstream (cell density at transfection, plasmid and transfection reagent concentration, cell culture nutrients and harvest timing) and downstream (virus recovery, purification methods, potency) steps in the production of AAVrh.10, a nonhuman primate vector, using adherent 293T cells and a plasmid transfection system using polyethylenimine (PEI) Upstream, vector yield, as measured by quantitative PCR in the cell harvest from transfections at each of several different cell densities peaked at x 104 cell/cm2 Total vector yield as a function of time after transfection peaked at day posttransfection with diminishing returns and greater partitioning into the cell supernatant at longer times Higher vector yield correlated with a greater total quantity of plasmid and PEI, but changing the Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy Targeted Genome Editing II PEI:plasmid ratio from 1:1 diminished productivity Fetal bovine serum concentration had minimal effect Downstream, freeze-thaw methods were not reproducible Physically lysing the cells was not as effective for AAV recovery as the detergents, triton or tween, which were equally effective, with the lowest effective detergent concentration important to minimize the residual detergent in the final product While iodixanol gradients are the traditional AAV purification systems, depth filtration followed by tangential flow filtration worked well for small scale production (>90%), but for large scale was less efficient (>70%) Affinity chromatography using AVB (GE Healthcare Life Sciences), provided an effective purification with maximum vector loads of up to x 1012 vector genomes per ml of packed column Finally, concentration of the final bulk AAV product was critically dependent on a formulation that stabilizes the solubility of the AAV monomer, with 0.01% pluronic acid providing AAV recovery of nearly 80% Together, the optimized methods for the best yield, purity and potency relied on transfection with equal amounts of plasmid and PEI and a cell density of x 104 cells/cm2 combined with the use of detergent to disrupt and recover AAV and sequential purification by depth filtration, tangential flow filtration and affinity chromatography This process improved the yield from harvest to final product from 9% to 40% and achieved final purified AAV at x 109 vector genomes/cm2 of adherent cell culture These systematic refinements to the individual steps of AAV production demonstrate improved yield and facilitates transfer of a method for GMP production for clinical use 310 Analysis of AAV5 Biodistribution and Viral Shedding in the Presence or Absence of Neutralizing Antibodies David Salas1, Anna Majowicz2, Nerea Zabaleta1, Estefania Rodríguez-Garcia1, Harald Petry2, Valerie Ferreira2, Gloria Gonzalez Aseguinolaza1 Gene Therapy and Regulation of Gene Expression Program, FIMA, Pamplona, Spain, 2Research Department, uniQure B.V., Amsterdam, Netherlands Neutralizing antibodies (NAbs) are the main obstacle that must be overcome to achieve a successful delivery of a viral vector to the target organ NAbs acquisition can be induced naturally after infection with wild type AAV or after the first administration of AAV vector in the course of gene therapy treatment Therefore, the objective of this project was to evaluate the efficacy and presence of the vector in the organism, as well as the response and vector elimination in the presence and absence of NAbs We analyzed the presence of the reporter transgene (hFIX), humoral and cellular immune responses against the vector, viral shedding and biodistribution The administration of AAV2/5 was successful in absence of NAbs In contrast, vector administration in presence of specific NAbs was unsuccessful, probably due to the vector being completely depleted by anti-AAV2/5 NAbs Our data demonstrate that while in the absence of antibodies the vector can be detected in serum and in other body fluids for 70 days after vector administration, the presence of antibodies immediately clears the vector from the organism 24 hours after the AAV-hFIX administration 1X1008 gc/mL are detected in the serum in the absence of NAbs but in the presence of NAbs the concentration is reduced 1000-fold In both groups the presence of NAbs increased upon infection without a cellular immune response against the vector Finally, the biodistribution studies showed remarkable differences between the two groups: in the absence of NAbs vector genomes are detected mainly in liver, heart, spleen and inguinal LN, while in the presence of NAbs vector genomes are almost undetectable in all tissues analyzed Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy Targeted Genome Editing II 311 In Vivo Zinc Finger Nuclease-Mediated Targeted Integration of a Glucose-6-Phosphatase Transgene Enhances Biochemical Correction in Mice with Glycogen Storage Disease Type IA Dustin J Landau, Elizabeth D Brooks, Pablo Pablo Perez-Pinera, Hiruni Amarasekara, Adam Mefferd, Songtao Li, Andrew Bird, Charles A Gersbach, Dwight D Koeberl Duke University, Durham, NC Glycogen storage disease type Ia (GSD Ia) is caused by glucose6-phosphatase (G6Pase) deficiency in association with severe, lifethreatening hypoglycemia that necessitates lifelong dietary therapy Here we show that use of a zinc-finger nuclease (ZFN) targeted to the ROSA26 safe harbor locus and a ROSA26-targeting vector containing a G6PC donor transgene, both delivered with adeno-associated virus (AAV) vectors, markedly improved survival of G6Pase knockout (G6Pase-KO) mice compared with mice receiving the donor vector alone (p