547 concentration of AAV8 and 9 vector to titers ranging from 1 6 x1014 vgml: feasibility assessment for volume limited dosing

547 Concentration of AAV8 and 9 Vector to Titers Ranging from 1 6 x1014 vg/mL Feasibility Assessment for Volume Limited Dosing Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The Ameri[.]

AAV VECTORS III in the heart of Balb/c mice However, detailed protein and mRNA data demonstrated that these promoters also showed expression in the liver To further restrict transgene expression, with the goal of eliminating liver expression, we employed a microRNA-binding site from mir122 This new data demonstrates that the transcriptional and post-transcriptional manipulation facilitates cardiac targeting Three copies of the binding site for liver specific mir122 were inserted into the 3’UTR of AAV9-CMV-EF1a-Luc and injected into Balb/c mice Bioluminescence imaging showed decreased luciferase signal in the upper abdominal region and after homogenization liver tissue revealed a 30 folds decrease in luciferase activity versus the same gene without mir122 binding sites When mir122 were used in the AAV9aCARD-EF1a-Luc, exclusive heart luciferase expression and none liver expression were observed in Balb/c mice with bioluminescent imaging and in vitro luciferase assay, while genome copy number ratio remained unchanged The same result was confirmed in S100A1 knock-out mice with C57/B6 background To apply this targeting strategy in gene therapy of heart failure, mir122 binding sites were inserted into the 3’UTR of AAV9-aCARD-EF1a-S100A1, and therapeutic effect of S100A1 will be studied in S100A1 knock-out mice post systematic delivery in a myocardial infarct model In conclusion, we have successfully restricted transgene expression to heart in both Balb/c and C57/B6 mice by using cardiac biased promoter aCARD and mir122 binding sites without altering the tropism of AAV9 This vector is promising for gene therapy of heart failure via systematic gene delivery 545 Reprogramming Adipose Tissue Derived Mesenchymal Stem Cells (AT-MSC) into Pluripotent Stem Cells by a Mutant AAV Vector Mong-Jen Chen,1 Yuanqing Lu,1 Takashi Hamazaki,1 Hsin-Yin Tsai,1 Kirsten Erger,1 Thomas Conlon,1 Arun Srivastava,1 Mark Brantly,1 Vince Chiodo,1 William Hauswirth,1 Naohiro Terada,1 Sihong Song.1 Departments of Pharmaceutics, Pathology, Pediatrics and Molecular Genetics and Microbiology, University of Floida, Gainesville, FL Induced pluripotent stem (iPS) cells have great potential for personalized regenerative medicine Although several different methods for generating iPS cells have been reported, improvement of its safety and efficiency is imperative In this study, we tested the feasibility of using recently established triple tyrosine-mutant AAV2 (Y444+500+730F) vector, designated as AAV 2.3m, to generate iPS cells We developed a polycistronic rAAV2.3m vector expressing reprogramming factors, Klf4, Oct4, and Sox2, and then used this vector to infect mouse adipose derived mesenchymal stem cell (ATMSC) to induce the generation of iPS cells We demonstrated that (i) triple tyrosine mutant AAV2 vector is able to reprogram mouse adult adipose tissue-derived stem cells into pluripotent state Those rAAV2.3m derived iPS (rAAV2.3m-iPS) cells express endogenous pluripotency associated genes including Oct4, Sox2, and SSEA1, and form teratomas containing multiple tissues in vivo; (ii) c-Myc, an oncogene, is dispensable in rAAV2.3m mediated cellular reprogramming; (iii) transgene expressions are undetectable after reprogramming, while vector DNA is detectable, indicating transgenes are silenced; and (iv) rAAV2.3m-iPS cells can be further differentiated into hepatocyte-like cells in vitro and produce liver specific proteins These results indicated the rAAV vector may have some advantages in generating iPS cells S210 546 Optimized Kidney-Targeted Gene Delivery Using AAV in a Mouse Model of Cystinosis Celine J Rocca,1 Sarah M Ur,1 Corinne Antignac,2 Jude R Samulski,3 Stephanie Cherqui.1 Pediatrics, UCSD, La Jolla, CA; 2INSERM U983, hôpital NeckerEnfants Malades, Paris, France; 3UNC Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC A wide range of monogenic kidney disorders have been identified and thus far no gene therapy approach has been developed to specifically target the kidney, even though renal transplantation is associated with significant morbidity and mortality Moreover, due to the severe shortage of donor organs, patients may wait three to six years for transplantation The main goal of our project is to develop an efficient and minimally invasive kidney-targeted gene delivery system using recombinant Adeno-Associated Viruses (rAAV) As a proof of concept, we are using the mouse model of cystinosis, which develops chronic renal disease similar to the patients Cystinosis is an autosomal recessive metabolic disease characterized by intracellular accumulation of cystine The defective gene is CTNS encoding the lysosomal cystine transporter, cystinosin Affected individuals typically present with proximal tubulopathy before one year of age and eventually progress to end-stage renal failure Our goal is to optimize kidney-targeted gene delivery via retrograde renal vein injection by testing several rAAV serotypes that have the potential of transducing a wide range of renal cells We performed injections of rAAV5, 6, and coding for either the luciferase or the green fluorescent protein (GFP) Our preliminary results showed that rAAV9 is the most suitable serotype to efficiently target the kidney Indeed, using the IVIS live imaging system and the luciferase assay, we observed that only rAAV9 allows a transduction of both the cortex and the medulla while the other serotypes transduced primarily the medulla This has been confirmed by confocal GFP observation and GFP-specific RTqPCR quantification We are also testing the Parathyroid Hormone Receptor (PTHR) promoter as a strategy to improve kidney-specific gene delivery Once the optimal conditions are defined, we propose to test this approach based on renal vein injection of rAAV-CTNS as a minimally invasive procedure for treating the renal dysfunction in cystinosis If successful, this strategy may be used in many monogenic hereditary nephropathies 547 Concentration of AAV8 and Vector to Titers Ranging from 1-6 x1014 vg/mL: Feasibility Assessment for Volume-Limited Dosing J Fraser Wright,1,2 Bernd Hauck,1 Shang-Zhen Zhou,1 Olga Zelenaia,1 Marina Sumaroka,1 Katherine A High.1,2,3 Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA; 2Depts of Pathology and Hematology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA; 3Howard Hughes Medical Institute, The Children’s Hospital of Philadelphia, Philadelphia, PA For gene transfer translational research using recombinant AAV, certain clinical programs may require high doses to be administered in a relatively small volume Furthermore, toxicology studies required to assess investigational product safety at doses typically 10-fold above the anticipated clinical high dose may require vector preparations at very high titers Aggregation is the primary concern for concentrated AAV vectors It was previously reported that recombinant AAV2 undergoes aggregation in a concentrationdependent manner for titers exceeding 1x1013 vector genomes per milliliter (vg/mL; studies herein performed with empty capsid-free vector preparations) when formulated in physiologic ionic strength buffers Elevated ionic strength was found to prevent aggregation to titers up to 5x1013 (Mol Ther 12:171, 2005); however slightly elevated ionic strength formulations compatible with direct parenteral Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy AAV VECTORS III injection to sites such as CNS / ocular tissues limit AAV2 vector titers to approximately 2x1013 In the current study the outcome following concentration of purified recombinant AAV8 and AAV9 by tangential flow filtration (TFF), a process step compatible with clinical scale vector processing, was determined Highly purified AAV vectors recovered following cesium chloride banding were diafiltered into 10mM NaPhos, 180mM NaCl, pH7.3 and then concentrated to the smallest achievable volume by TFF (100kDa MW cutoff) The titers of the vector at the following stages: 1) before TFF; 2) following concentration but prior to 0.2micron filtration; and 3) after filtration were measured by qPCR and spectrophotometry ( = 260 and 280nm) (Mol Ther 7:122, 2003) Aggregation was assessed by visual inspection, dynamic light scattering, and spectrophotometry ( = 320nm) For AAV8 and AAV9 vectors, post concentration titers of 1x1014 and 6x1014, respectively, were achieved, without evidence of aggregation These were the highest titers achievable, limited by the quantity of starting material and the minimum working volume of the TFF system i.e no aggregation was observed under all conditions examined For both vectors, mono-disperse, non-aggregated vector was documented immediately following concentration, as well as after storage at 2-8 °C, and following freeze (-80 °C) / thaw cycling Together these data support that, while AAV2 vectors are prone to aggregation at concentrations exceeding the range 1-2 x1013 vg/mL in parenteral compatible buffers, recombinant AAV8 and -based vectors can be prepared in neutral physiological buffers at much higher concentrations, facilitating gene transfer applications requiring high dose administration in limited volumes 548 Enhanced Fracture Healing Utilizing Directed Evolution of Adeno-Associated Virus Seth G Levy,1 Damien Marsic,3 Shawn Gilbert,2 Sergei Zolotukhin,3 Selvarangan Ponnazhagan.1 Pathology, The University of Alabama at Birmingham, Birmingham, AL; 2Surgery, The University of Alabama at Birmingham, Birmingham, AL; 3Pediatrics, University of Florida, Gainsville, FL Recombinant adeno-associated virus (rAAV) has gained much attention for its lack of pathogenicity as a gene transfer vector With up to 10% of the million fractures each year in the US resulting in a delayed healing or failure to form bony union (non-union), rAAV can be exploited to potentially deliver therapeutic stimuli to the fracture site through gene transfer Nonetheless, targeted enrichment of rAAV at fracture sites in particular of bone progenitor cells has been a limitation to this concept To overcome this and develop rAAV therapy for non-union fractures, we have employed an in vitro selection process for generating bone progenitor cell-specific AAV capsids using a novel capsid library, consisting of random peptide insertion constructed through isothermal DNA assembly The library containing heterogenome capsid mutants were selected for in in vitro evolution in mouse mesenchymal stem cells Capsid mutants recovered from several rounds of biopanning were analyzed by sequencing The novel mutants will be further characterized for osteoprogenitor cell transduction in vitro and for efficacy of therapeutic gene delivery in vivo for non-union fractures in an immunocompetent mouse model Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy 549 Targeted Bio-Engineering of Adeno Associated Virus Serotype rh.10 Capsid Improves Its Gene Delivery In Vitro and In Vivo Ruchita Selot,1 Nishanth Gabriel,2 Monika Kumari,1 Giridhara R Jayandharan.1,2 Center for Stem Cell Research, Christian Medical College, Vellore, India; 2Hematology, Christian Medical College, Vellore, India AAVrh.10, a Clade E primate AAV has favorable biological properties as a vector for neonatal liver-directed gene transfer, including higher levels of transgene expression and vector copynumber persistence during the period of rapid neonatal growth In addition this isolate has the ability to transverse blood-brain-barrier and presumably has the lowest level of circulating neutralizing antibodies against any AAV serotype in humans In the present study, we investigated if targeted mutagenesis of AAVrh.10 viral capsid residues in a group of lysine (K>R), serine (S>A) and threonine (T>A) residues can improve AAVrh.10 mediated gene delivery This is based on our previous observation in AAV2 vectors, where modifications within the phospho S, T residues and ubiquitinable K residues that reside in close vicinity (9-13 residues in spatial proximity) and form phosphodegron motifs recognized by ubiquitin ligases was beneficial We first introduced single mutations on the AAVrh.10 capsid at sites (K84R, K137R, K333R, S498A, S501A, S671A, T108A, T252A, T674A) Highly purified stocks of selfcomplementary wild-type (WT) AAVrh.10 or the mutant AAVrh.10 vectors containing the enhanced firefly luciferase gene driven by the chicken -actin promoter were generated by polyethyleneimine based triple transfection of AAV-293 cells The packaging titers of all these vectors were comparable (1x1011vg/ml to 3.2x1012vg/ml) The transduction efficiency of these mutant AAVrh.10 vectors was then tested in vitro in human cervical carcinoma cell line (HeLa) at an MOI of 5000 Forty-eight hours post infection, the transgene expression was determined Amongst all mutant vectors tested, the AAVrh.10- K333R, T674A and S671A vectors demonstrated 4-6 fold higher transduction efficiency in comparison to AAVrh.10- WT vector (Fig 1) To further determine their efficiency in vivo, groups of C57BL6/J mice were injected with 5x10^10 vgs per animal of either the AAVrh.10- WT or the AAVrh.10S671A vector Our results show that two weeks post gene transfer, the AAVrh.10-S671 vectors exhibit (>2-fold) higher transgene expression in comparison to AAVrh.10-WT vectors Long-term follow up data from these animals will further demonstrate the efficiency, safety and bio-distribution profile of these novel AAV rh.10 vectors S211 ... light scattering, and spectrophotometry ( = 320nm) For AAV8 and AAV9 vectors, post concentration titers of 1x1 014 and 6x1 014 , respectively, were achieved, without evidence of aggregation These... triple transfection of AAV- 293 cells The packaging titers of all these vectors were comparable (1x1 011 vg/ml to 3.2x1 012 vg/ml) The transduction efficiency of these mutant AAVrh .10 vectors was then tested... comparison to AAVrh .10 - WT vector (Fig 1) To further determine their efficiency in vivo, groups of C57BL6/J mice were injected with 5x10 ^10 vgs per animal of either the AAVrh .10 - WT or the AAVrh .10 S671A