283. In Vivo Selection of MGMT(P140K) Gene Modified Hematopoietic Cells in the Nonhuman Primate Unmasks a Dormant Pool of Repopulating Clones with Progenitor Cell Ontology

hemAtologic & immuNologic diseAses i

282. Circumventing Hematopoietic Toxicities and Reversing Sickle Phenotype by LineageSpecifi c BCL11A Knock Down and Γ-Globin Induction

Christian Brendel1, Swaroopa Guda1, Raffaele Renella2, Daniel E. Bauer2, Matthew C. Canver3, Young-Jo Kim4, Matthew M. Heeney5, Denise Klatt6, Jonathan Fogel4, Michael D. Milsom7, Stuart H. Orkin8, Richard I. Gregory9, David A. Williams10 1 Boston Children’s Hospital, Harvard Medical School, Boston, MA, 2Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 3Harvard Medical School, Boston, MA, 4Boston Children’s Hospital, Boston, MA, 5Boston Children’s Hospital, Dana-Farber Cancer Institute, Boston, MA, 6Hannover Medical School, Hannover, Germany, 7German Cancer Research Center, Heidelberg, Germany, 8Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Boston, MA, 9Boston Children’s Hospital, Harvard Stem Cell Institute, Boston, MA, 10Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Harvard Stem Cell Institute, Boston, MA The fetal hemoglobin repressor BCL11A represents a therapeutic target for β-hemoglobinopathies as reduced expression of BCL11A leads to simultaneous increased γ-globin and reduced β-globin expression. Reversing this hemoglobin switch is particularly relevant in sickle cell disease to reduce the βS concentration and increase expression of the protective fetal hemoglobin (HbF, α2γ2). Here we show that despite use of optimized shRNAs embedded into a miRNA (shRNAmiR) architecture to reduce non-specific cellular toxicities (Guda et al. MT, 2015), the knockdown of BCL11A profoundly and specifically impaired long-term engraftment of both human and mouse hematopoietic stem cells (HSCs). In competitive transplantation assays cells transduced with shRNAmiRs targeting the BCL11A mRNA were consistently underrepresented after hematopoietic reconstitution with gene modified HSCs. Although this effect was Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy

particularly pronounced in the B-cell compartment, it was also the case for all other assessed hematopoietic lineages. Mechanistically, while knock-down of BCL11A did not lead to a detectable phenotype in terms of apoptosis, growth or differentiation in human or mouse HSCs in vitro, a significant increase in S/G2-phase human HSCs after engraftment into NSG mice was found, a phenotype associated with stem cell exhaustion. To avoid this BCL11A-specific HSC toxicity, we suppressed BCL11A in erythroid cells in a lineagespecific fashion by using transcriptional regulatory elements derived from the β-globin locus. Utilizing this approach for the expression of the optimized shRNAmiRs led to stable long-term engraftment of mouse and human gene modified cells in congenic or NSG-mice, respectively. Transduced primary normal or sickle cell disease (SCD) human HSCs gave rise to erythroid cells with up to 90% reduction of BCL11A protein. These erythrocytes demonstrated 60-70% γ-chain expression and a corresponding increase in HbF at low vector copy numbers per cell (VCN<1.5). Similar results were obtained after in vitro differentiation of CD34+ cells harvested 16 weeks following engraftment in NSG mice. Transplantation of gene modified murine HSCs from BERK sickle cell mice led to a substantial improvement of sickle-associated hemolytic anemia and reticulocytosis, key phenotypes of SCD. In summary, we have shown an unexpected and severe toxicity of BCL11A knockdown in repopulating HSCs that has direct and important consequences for translation into human gene therapy trials. By utilizing erythroid lineage-specific and microRNA embedded expression of targeting shRNAs we demonstrate the capacity of lentivirus vectors to effect significant γ-globin induction leading to clinically relevant increases in HbF while obviating HSC toxicity.

283. In Vivo Selection of MGMT(P140K) Gene Modifi ed Hematopoietic Cells in the Nonhuman Primate Unmasks a Dormant Pool of Repopulating Clones with Progenitor Cell Ontology Jennifer E. Adair1,2, Lauren Schefter1, Daniel Humphries1, Kevin G. Haworth1, Hans-Peter Kiem1,3 1 Clinical Research Division, Fred Hutch, Seattle, WA, 2Department of Medical Oncology, University of Washington, Seattle, WA, 3 Department of Medicine, University of Washington, Seattle, WA

Previously published gene modified cell transplantation studies in nonhuman primate models have described several key features of hematopoietic reconstitution (Kim, S. et al., 2014 and Wu, C. et al., 2014). These studies applied either retrovirus integration site analysis (ISA) or DNA barcode sequencing (DBS) to identify hematopoietic clones in bone marrow or peripheral blood cells. However, these distinct methods detected long-term clones at different time points (1 year after transplant by ISA; ~3 months after transplant by DBS), and neither evaluated reconstitution in the context of selective advantage, which is often the case in gene therapy. Here, we tracked tens of thousands of unique clones in 8 pigtail macaques for up to 10 years following myeloablative transplantation with autologous, lentivirus (LV) gene-modified CD34+ cells. Seven animals received cells gene modified with the P140K mutant methylguanine methyltransferase transgene, conferring resistance to O6- benzylguanine (O6BG) and bis-chloroethylnitrosourea (BCNU) chemotherapy. In two animals, MGMT(P140K)-expressing LVs were DNA barcoded, permitting simultaneous ISA and DBS tracking. Gene marking in peripheral blood cells ranged from 2.3% to 66%. Direct comparison of ISA and DBS demonstrated that abundant clones (> 1% of sequence reads) are readily detected by both methods, but DBS captures up to 2-fold more lower abundance clones. Before in vivo selection with O6BG/ BCNU, we observed a cell dose-dependent, successive pattern of hematopoietic reconstitution by ISA analysis, with short-term clones declining within 100 days after transplantation. Long-term clones S113

Hematologic & Immunologic Diseases I were observed as early as 1 month after transplant. In the first year after transplant, persistent clones ranged from 8% to 54% of clones detected at a > 1% frequency, and remained stable in the absence of selective pressure. Importantly, when O6BG/BCNU was administered we observed novel clonal patterns, which directly correlated with transplanted cell dose and time of chemotherapy administration after transplant. In all animals, chemotherapy induced emergence of previously undetected clones. In animals receiving cell doses exceeding 35x106 CD34+ cells/kg (n = 2), chemotherapy more than 1 year after transplant induced a completely novel clonal repertoire. Gene ontology analysis of integration loci among early, long-term and dormant clonal populations identified the greatest functional overlap between early and dormant pools. These data suggest that some short-term repopulating clones revert to a dormant phase within the first year after transplant. Additionally, these data indicate that transplant of excess CD34+ cell numbers results in early dormancy of a large proportion of early repopulating clones. Together, these findings suggest that previous estimates of short- and long-term clonal frequency are an underestimate of true graft repopulation potential.

284. Long-Term Therapeutic Immune Reconstitution in XSCID Canine Model via In Vivo Foamy Virus Delivery of Common Gamma Chain

Frieda Chan1, Olivier Humbert1, Christopher Burtner1, Daniel Humphrys1, Jennifer Adair1, Grant Trobridge2, Patricia O’Donnell3, Nicholas Hubbard4, Troy Torgerson4, Andrew Scharenberg4, David Rawlings4, Peter J. Felsburg3, Hans-Peter Kiem1 1 Fred Hutchinson Cancer Research Center, Seattle, WA, 2 Washington State University, Spokane, WA, 3University of Pennsylvania, Philadelphia, PA, 4Seattle Children’s Research Institute, Seattle, WA X-linked combined immunodeficiency disease (XSCID) is caused by mutation in the common gamma chain, γC (interleukin-2 receptor subunit gamma, IL2RG) in both humans and canines. It is characterized by the inability of T-cell development leading to absence of T-cells in peripheral blood, lack of T-cell mediated immune response, low IgA and IgG levels, and early infant mortality. In the 1990s, human XSCID clinical trials utilizing gamma-retroviral vectors to deliver the IL2RG gene caused leukemia in 5 out of 20 patients due to vector integration in or near proto-oncogenes. Recent studies showed Foamy virus based vectors as an excellent alternative for in vivo gene-therapy because it is non-pathogenic in humans while exhibiting increased serum stability and favorable integration pattern. Previously, we have demonstrated CD3+ T-cell reconstitution in the canine model via intravenous injection of foamy virus expressing human elongation factor-1 alpha promoter (Ef1α)γC. Unfortunately, the treated animals contained a low number of gene corrected progenitors at a sub-therapeutic level. Here, we achieved long-term therapeutic immune-reconstitution by intravenous delivery of a human phosphoglycerate kinase promoter (Pgk)-mediated γC foamy viral vector into XSCID neonatal canines. Long-term (2 years) post-injection follow-up demonstrated therapeutic levels of CD3+ T-cell expansion. Within the T-cell population, gene correction with Pgk-γC stabilized at ~80%. We validated T-cell functionality by using spectratyping analysis, which exhibited a diverse repertoire of receptor gene rearrangement. Retroviral integration site analysis (RIS) indicated polyclonal contribution to the reconstituted T-cells. Immunoglobulin ELISA assays showed that IgA and IgG levels in peripheral blood are comparable to normal healthy controls. We immunized the gene-corrected canine recipients with bacteriophage ϕx174 and confirmed production of specific IgG antibodies, showing the ability for isotype switching in B-lymphocytes. Currently, the gene-corrected canines exhibit comparable health and physical attributes to normal controls. Furthermore, semen from the geneS114

corrected male canine was used via artificial insemination to produce a litter of viable offsprings. In summary, our data demonstrate that Pgk-γC foamy viral vector delivered long-term therapeutic gene correction in a large-animal model for XSCID gene therapy. Most importantly, these results indicate that in vivo Pgk-γC foamy vector administration is a viable option for long-term immune reconstitution in future XSCID human clinical trials.

285. Foamy Viral Vector Expressing Human CD18 Results in High Levels of Transduction and Multilineage Engraftment with CD18+ LAD-1 Cells in NSG Mice

Richard H. Smith1, Md Nasimuzzaman2, Roop Dutta1, Gulbu Uzel3, Thomas R. Bauer4, Steven M. Holland3, David W. Russell5, Dennis D. Hickstein4, Punam Malik2, Johannes C. M. van der Loo6, Andre Larochelle1 1 Hematology Branch, National Institutes of Health, Bethesda, MD, 2Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 3National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 4National Cancer Institute, National Institutes of Health, Bethesda, MD, 5Division of Hematology, University of Washington, Seattle, WA, 6Center for Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia, Philadelphia, PA Compared to other classes of retroviral vectors used for HSPC gene therapy, foamy viral vectors (FVV) have distinct advantages, including their non-pathogenicity, a safer integration profile, and a high-efficiency transduction of quiescent cells. Proof-of principle of its therapeutic safety and efficacy in HSPCs was provided with the correction of canine Leukocyte Adhesion Deficiency (CLAD). Dogs with CLAD and individuals with LAD type 1 (LAD-1) suffer from recurrent and life-threatening infections caused by mutations in the β2 integrin CD18 subunit. In this study, G-CSF-mobilized CD34+ HSPCs were isolated from a 19 YO male with severe LAD-1 due to homozygous deletions in CD18. CD34+ cells were transduced for 16 hours at MOI of 0, 5, 10, and 20 with a FVV expressing a human codon optimized CD18 transgene. Flow cytometry of CD34+ cells cultured for 3 days after transduction demonstrated CD18+ cell surface expression in 40-45% of cells. Thirty NSG mice were transplanted with CD18-FVV-transduced human LAD-1 HSPCs (~1.2 x 105 cells/ mouse), and human cell engraftment was measured in murine BM 5 months after transplantation using flow cytometry. Human CD45+ cells were detected in all mice (average ~1%). Mice transplanted with mock-transduced (MOI=0) LAD-1 CD34+ cells showed a 3.4-fold, 2-fold and 1.4-fold lower engraftment compared to mice injected with CD34+ cells transduced with FVV at MOI 5 (p<0.01), 10 (p<0.05) and 20 (p>0.05), respectively, suggesting a selective homing/engraftment or survival/proliferative advantage of CD18+ cells. The inverse relationship between engraftment levels and MOI correlated with a gradual decrease in cell survival with increasing MOI, most likely due to toxicity from DMSO (required for FVV cryopreservation) during transduction; cell viabilities of 91%, 84%, 82% and 69% were obtained at MOI 0, 5, 10 and 20, respectively, indicating that further increase in MOI would lead to increasing toxicity. High-level, clinically relevant gene marking levels were obtained; the percentages of human cells expressing CD18 in the murine BM 5 months posttransplantation were 36.0 ± 3.9%, 33.9 ± 5.1%, and 44.5 ± 1.6% at MOI 5, 10 and 20, respectively. Quantitative PCR analysis of vector integrants within engrafted human cells indicated a single integration event occurred in the majority of long-term repopulating HSPCs at all MOI tested. Flow cytometry-based lineage analysis of bone marrow from mice transplanted with FVV-transduced LAD-1 CD34+ cells revealed human CD18+ cells in all lineages, with a predominance Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy