477 Three Dimensional Imaging of Multiple AAV Serotype/Mutant Vector Transductions in the Non Human Primate Brain By Massively Parallel Sequencing Molecular Therapy Volume 23, Supplement 1, May 2015 C[.]
Presidential Symposium mutation in pts), pts (29%) had prior allogeneic stem cell transplant (allo-SCT), and 22 pts (78%) had either primary refractory disease or CR1 duration of 5% blasts in BM) and 13 pts had minimal residual disease (MRD) 24 of 27 pts (89%) were in complete remission (CR) following the 19-28z CAR T cell infusion; 11 of these pts had morphologic disease at the time of T cell infusion, and 13 pts had MRD 21 of 24 CR (88%) was MRD negative A median time to CR was 22.5 days (range, 9-33) 10 pts successfully underwent allo-HSCT following the 19-28z CAR T cell therapy The median follow-up is months (range, 1-38 months) with 15 pts having at least months of follow-up Responses appear durable with 12 pts remaining disease-free, including pts without subsequent allo-SCT Median OS of all pts is 8.5 months While none of the 13 pts with MRD at the time of T cell infusion developed significant cytokine release syndrome (CRS), of 15 pts with morphologic disease developed severe CRS that required either vasopressors or mechanical ventilation for hypoxia, successfully managed with corticosteroid and/or IL-6R inhibitor Conclusion: Treatment with 19-28z CAR T cells can induce a high CR rate of 89% in these heavily pretreated adult pts with R/R ALL regardless of the disease risk characteristics including Ph+ status, age, pre-T cell disease burden, prior allo-SCT, and CR1 duration While CRS can develop following T cell infusion, the risk of CRS predictably correlates with the disease burden and can successfully be managed with corticosteroid and/or IL-6R inhibitor These findings support the use of 19-28z CAR T cells in adult pts with R/R ALL and warrant a further investigation in a larger phase trial 476 Clonal Tracking of Engineered Hematopoiesis In Vivo in Humans By Insertional Barcoding Luca Biasco,1 Francesca Dionisio,1 Danilo Pellin,2 Serena Scala,1,3 Luca Basso Ricci,1 Samantha Scaramuzza,1 Cristina Baricordi,1 Andrea Calabria,1 Stefania Giannelli,1 Victor Neduva,4 David J Dow,4 Eugenio Montini,1 Clelia Di Serio,2,3 Luigi Naldini,1 Alessandro Aiuti.1,3 San Raffaele Telethon Institute for Gene Therapy (TIGET), Milan, Italy; 2CUSSB, Univ Vita-Salute San Raffaele, Milan, Italy; Univ Vita-Salute San Raffaele, Milan, Italy; 4Molecular and Cellular Technologies, GlaxoSmithKline, Stevenage Herts, United Kingdom; 5Univ degli studi Roma Tre, Rome, Italy Upon gene therapy (GT) for adenosine deaminase (ADA) deficientSCID and Wiskott-Aldrich Syndrome (WAS), gene-corrected hematopoietic stem/progenitor cells (HSPC) generated a stable genetically engineered hematopoietic system where each vectormarked cell is univocally barcoded by a vector integration site (IS) To study the dynamics of human hematopoietic system, we collected by LAM-PCR+Illumina sequencing 28.539.414 sequence reads corresponding to 89.373 IS tagging clones belonging to 13 different cell types purified from the bone marrow and the peripheral blood of WAS patients up to 48 months after GT We firstly identified identical IS shared among CD34+ progenitors, and mature Myeloid/ Lymphoid cells as markers of the real-time clonal output of vectormarked HSPC clones in vivo We unraveled the nature and timing of short, intermediate and long term HSPC output showing that CD34+ clones active at 3-6 months after GT are not detectable at later follow up Distinct waves of HSPC diversity were observed during the first 6-9 months after GT reaching a homeostatic equilibrium only by 12 months By unsupervised clustering of IS similarities among lineages we unveiled diverse HSPC output towards lymphoid, myeloid and megakaryo-erythroid cells showing hints of a NK cells origin distinct from T and B cells We exploited IS similarities to Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy infere and test hematopoietic hierarchies by combining conditional probability distributions and static/dynamic graphical models of dependencies We also estimated by mark-recapture approaches that just few thousands clones (1185-2884) are responsible for the longterm maintenance of the whole genetically engineered hematopoietic system The ongoing analyses on IS collected from distinct CD34+ subtypes isolated from GT patients will further increase HSPC tracking resolution To evaluate the preservation of activity by transplanted HSPC we exploited data derived from the IS-based tracking of 4.845 clones in ADA-SCID patients performed for up to years after GT We showed that identical IS are consistently detected at multiple lineages level even several years after GT Strikingly, by semi-quantitative PCRs on specific vector-genome junctions we tracked a fluctuating but consistent output of marked HSPC over a period of years without the manifestation of clonal quiescence phases Since gamma-retroviral vector used in this GT trial actively transduce only replicating cells, this provide the first evidence that HSPC, awaken from dormancy in vitro, can still retain in vivo longterm activity Overall our work constitute the first molecular tracking of hematopoietic system in humans The information gathered are crucial for the design of therapeutic approaches for a broad spectrum of hematological diseases and tumors 477 Three-Dimensional Imaging of Multiple AAV Serotype/Mutant Vector Transductions in the Non-Human Primate Brain By Massively Parallel Sequencing Kei Adachi,1 Zheng Liu,1,2 Christopher D Kroenke,1,2 Gregory A Dissen,2 Sergio R Ojeda,2 Hiroyuki Nakai.1 Oregon Health and Science University, Portland, OR; 2Oregon Health and Science University, Oregon National Primate Research Center, Beaverton, OR Global therapeutic gene delivery to the central nervous system (CNS) offers a promising approach to treat CNS diseases and preclinical studies using rodents have shown AAV9 as a potential candidate to achieve this goal However, recent work has highlighted the existence of nonhuman primate (NHP)-rodent differences in AAV vector biology and transduction profiles in the CNS after gene delivery, making imperative a more rigorous evaluation of various AAV serotype and capsid mutant vectors in NHPs It is of particular importance to understand how each AAV strain (serotype or mutant) is distributed and transduces cells in the brain of a primate species closely related to humans when injected into the bloodstream or cerebrospinal fluids (CSF) Here we report a novel approach to generate three-dimensional (3D) vector transduction heat maps of more than one hundred of AAV strains in the rhesus monkey brain when vectors are delivered via the cisterna magna In this approach, we integrate the vector transduction data obtained by AAV Barcode-Seq (an Illumina sequencing-based high-throughput vector transduction analysis) into an MRI image To show proofof-principle of the method, a barcoded-AAV library containing 145 different AAV strains was directly injected into CSF via the cisterna magna of three rhesus macaques (4x1012 vg/kg) and their brains were subjected to MRI analysis before collection of the organ weeks postinjection Six mm-thick coronal sections of the brain were made and photographed, and the right half of each section slice was diced into x x mm3 cubes using a matrix, generating more than 100 cubes from a cerebral hemisphere Total DNA was then extracted from each cube using an automated magnetic particle processor and subjected to AAV Barcode-Seq analysis to determine transduction efficiency of each AAV strain in each cube by multiplexed Illumina sequencing The coordinates of each cube were determined by aligning the photo images of each coronal section to the corresponding MRI images using an image alignment algorithm This strategy allowed us to S189 Gene Editing and Gene Regulation II create 3D heat maps showing distribution and transduction of each AAV strain in the entire NHP brain A preliminary analysis using one of the three monkeys revealed that AAV1 vector spreads globally and transduces all brain regions including the cerebral cortex while the distribution of AAV2 was limited locally Unexpectedly, we found that many other serotypes and mutants transduced the NHP brain better than AAV9 when delivered into CSF Thus, imaging of AAV vector distribution and transduction throughout the NHP brain by AAV Barcode-Seq combined with MRI provides a powerful approach to comprehensively study AAV vector-mediated CNS gene delivery in clinically relevant animal models Our results emphasize the large difference in AAV distribution and transduction efficiency that exists in the brain of higher primates compared to rodent models 478 Highly Efficient Targeted Gene Addition in CD34+ Hematopoietic Stem/Progenitor Cells by Combining ZFN mRNA and AAV6 Donor Delivery Jianbin Wang,1 Colin M Exline,2 Josh DeClercq,1 Patrick Li,1 Philip D Gregory,1 Paula M Cannon,2 Michael C Holmes.1 Sangamo Biosciences Inc., Richmond, CA; 2Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, CA Zinc-finger nucleases (ZFNs) drive highly efficient genome editing by generating a site-specific DNA double-strand break (DSB) at a predetermined site in the genome Subsequent repair of this break via the non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways results in targeted gene disruption or gene correction/addition, respectively Hematopoietic stem/progenitor cells (HSPC) are an important clinical target for gene therapy These cells can be readily harvested from the subject, engineered using viral vectors, and re-introduced back into the patient where they give rise to all of the mature cells of the blood and immune system Thus the ability to edit the genome of HSPCs could provide a corrective therapy for a number of monogenic diseases However, achieving high levels of editing by HDR may be significantly more challenging than gene knockout by NHEJ Here we report that the combination of delivering the ZFNs as mRNA and corrective donor DNA via a rAAV6 vector can induce highly efficient targeted gene addition in CD34+ HSPCs Using CCR5-specific ZFNs and donor DNA templates as a model system we show that targeted addition of a novel restriction fragment length polymorphism (RFLP) was consistently achieved at >15%, while insertion of an eGFP expression cassette was achieved at >10% of the CCR5 loci in mobilized CD34+ cells Furthermore, targeting the AAVS1 “safe harbor” locus with a pair of AAVS1-specific ZFNs (mRNA) and a RFLP donor (rAAV6) resulted in >30% of AAVS1 alleles specifically being edited to include the RFLP, suggesting this genome editing approach to achieve highly efficient targeted gene addition in CD34+ cells can be applied to different genetic loci Edited HSPC undergo normal hematopoiesis in both in vitro differentiation assays and during engraftment of NSG mice Using fetal liver derived cells, editing of primitive and more committed CD34+ HSPC subsets was observed at similar levels in HSPC CFU cultures, and by analysis of sorted populations of treated HSPC, including LT-HSC, and through the secondary transplantation of NSG mice The use of ZFNs in combination with rAAV6 delivery of the donor DNA provides a significant advance in genome editing that has great potential for targeted correction of human disease-causing mutations and permanent transgene addition in human CD34+ HSPC S190 Gene Editing and Gene Regulation II 479 ZFN-Mediated In Vivo Genome Editing Results in Supraphysiological Levels of Lysosomal Enzymes Deficient in Hunter and Hurler Syndrome and Gaucher Disease Thomas Wechsler,1 Russel DeKelver,1 Michelle Rohde,1 Susan Tom,1 Scott Sproul,1 Leo Ou,2 Kanut Laoharawee,2 Kelly M Podetz-Pedersen,2 Chester B Whitley,2 Scott R McIvor,2 Philip D Gregory,1 Michael C Holmes.1 Sangamo BioSciences Inc., Richmond, CA; 2Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN Lysosomal storage diseases (LSDs) represent a group of inherited metabolic disorders associated with mutations in genes encoding lysosomal enzymes leading to systemic accumulation of toxic storage materials Manifestations include organomegaly, skeletal dysplasias, cardiopulmonary obstruction, and severe neurologic impairment, often leading to death by age 10 Current LSD therapies include enzyme replacement therapy, substrate reduction therapy and hematopoietic stem cell transplant (HSCT) However, all of these therapies are expensive, incompletely effective, and HSCT is associated with significant risk of morbidity and mortality Due to the unmet need genome editing strategies are proposed to permanently modify patient cells by genetically complementing the LSD defect This is achieved by utilizing engineered zinc finger nucleases (ZFNs) to introduce a DNA cut at the target locus, mediating integration of a therapeutic LSD donor cDNA To ensure long-term expression of the transgenes in vivo we target the albumin locus as a “safe harbor” in hepatocytes via co-delivery of the albumin ZFNs and LSD donors by adeno-associated virus (AAV) This system exploits the high transcriptional activity of the native albumin enhancer/promoter; uses stably modified hepatocytes to potentially allow long-term expression of the inserted transgene; and utilizes an endogenous promoter obviating this requirement in the AAV payload We have previously exploited AAV-mediated in vivo targeting of the murine albumin “safe harbor” locus for the synthesis of therapeutic levels of FVIII and FIX to overcome the clotting defect in hemophilic mice Using the same approach, we co-delivered mouse albumin ZFNs with donor constructs encoding either human iduronate-2-sulfatase (IDS) deficient in Hunter syndrome, α-L-iduronidase (IDUA) for Hurler syndrome, or Glucocerebrosidase (GBA) for Gaucher disease via AAV in WT mice We show stable integration of the LSD donors at the albumin locus, resulting in liver-specific expression and secretion of these proteins into plasma This led to a 4-fold (GBA), 10-fold (IDUA) or 100-fold (IDS) increase in enzymatic activity in the plasma, demonstrating that the secreted proteins are functional Importantly, increased activity was also detected in secondary tissues (spleen) showing efficient uptake and activity in distal tissues Moreover, preliminary data in MPSI and MPSII mice suggests these levels of IDS and IDUA expression may lead to correction of the enzyme deficiency IDS, IDUA and GBA expression remained stable throughout the study (up to months), while expression of FIX has been stable for >1 yr suggesting this process results in long-term protein expression In summary, our data provide proof of concept for ZFN-mediated targeting of the albumin locus in hepatocytes as an in vivo protein replacement platform to express different proteins associated with lysosomal storage diseases Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy ... mutants transduced the NHP brain better than AAV9 when delivered into CSF Thus, imaging of AAV vector distribution and transduction throughout the NHP brain by AAV Barcode-Seq combined with MRI provides... obviating this requirement in the AAV payload We have previously exploited AAV- mediated in vivo targeting of the murine albumin “safe harbor” locus for the synthesis of therapeutic levels of FVIII... cells by genetically complementing the LSD defect This is achieved by utilizing engineered zinc finger nucleases (ZFNs) to introduce a DNA cut at the target locus, mediating integration of a therapeutic