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50 integration site analysis in a clinical trial of lentiviral vector based HSC gene therapy for MLD shows high levels of polyclonal hematopoietic reconstitution and no signs of genotoxicity at one year after transplant

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50 Integration Site Analysis in a Clinical Trial of Lentiviral Vector Based HSC Gene Therapy for MLD Shows High Levels of Polyclonal Hematopoietic Reconstitution and No Signs of Genotoxicity at One Ye[.]

CLINICAL GENE & CELL THERAPY ORAL ABSTRACT SESSION in vivo selection drug resistance transgene mutant methylguanine methyltransferase P140K, anti-HIV, and fluorescent reporters We are now able and plan to test the engraftment of MniPSC derived hematopoietic cells transplanted at different stages of differentiation in an autologous transplantation setting for their ability to give us to the elusive HSC as well as to use this system as a model to evaluate the efficacy of stem cell therapies for the treatment of HIV and hematologic genetic diseases 48 Derivation of “Semi-Universal Donor Stem Cells” That Express a Specific Class I Human Leukocyte Antigen (HLA) A0201 German G Gornalusse,1 Roli K Hirata,1 Laura Riolobos,1 David W Russell.1,3 Department of Medicine, University of Washington, Seattle, WA; Department of Biochemistry, University of Washington, Seattle, WA The clinical use of Human Embryonic Stem Cells (hESCs) is restricted because of immunological rejection reactions, mostly due to genetic disparities in the Human Leukocyte Antigen (HLA) locus One way to circumvent this problem is to create HLA-deficient, universal donor stem cells by targeted disruption of the β2-Microglobulin (B2M) gene, which encodes the common subunit of all HLA class I heterodimers However, this strategy may result in immunological rejection mediated by Natural Killer (NK) cells, which can eliminate HLA class I-negative cells, and could also pose problems if the transplanted cells harbor viral infections or transform into cancer cells To overcome this problem, we demonstrate that it is feasible to express a specific HLA class I allele (HLA-A0201) as a single chain dimer on the surface of B2M knock-out hESCs by employing an integrating foamy virus vector ΔΨ-PPE-HLA(bBA0201) This vector includes a puromycin resistance gene driven by the ubiquitously expressed PGK promoter, and a separate expression cassette with the EF1α promoter driving the expression of the HLA single chain construct This novel engineered protein is a fusion polypeptide in which the β2Microglobulin subunit is covalently attached to the coding sequence of HLA-A0201 We transduced independent β2M-/- hESC clones with the ΔΨ-PPE-HLA(bBA0201) foamy vector and obtained puromycin resistant colonies (at MOI=10, we obtained 40 puroR clones/1500 unselected CFU, which is ∼3% resistant CFU) Southern blot analysis demonstrated that all the clones analyzed (n=21) carried the intact provirus, as evidenced by the presence of a unique ∼3.9 kb band, and the majority of them were single copy integrants By flow cytometry analysis, we showed that the B2M-HLA-A0201 fusion protein was robustly expressed on the cell surface of B2M-/- hESCs, and we are currently analyzing the immunological reactions to these cells and their differentiated derivatives In addition to these overexpression experiments, in which we ectopically express the B2M-HLA-A0201 fusion protein from an EF1a promoter, we are also employing a gene targeting protocol to knock-in an HLA-A0201 gene as a fusion construct at the B2M locus and confer properly regulated gene expression Both approaches ultimately generate hESC lines that express a single, unique, HLA class I protein on the cell surface In the case of HLA-A0201, this allele is particularly common in the United States where it exists in 48% of Caucasians, 46% of Hispanics, and 24% of African-Americans Therefore this single cell line should be compatible with a large percentage of the US population, and can be considered a semi-universal donor cell A similar approach could also be used to uniquely express other common HLA class I proteins on B2M-/- hESCs Clinical Gene & Cell Therapy Oral Abstract Session 49 Transgene T Cell Response in a Phase Clinical Trial of rAAV1.Alpha-1 Antitrypsin Gene Therapy Vector: CD8+ Mediated and HLA C*05 Restricted Roberto Calcedo,1 Suryanarayan Somanathan,1 Qiuyue Qin,1 Terence Flotte,2 Jeffrey Chulay,3 James M Wilson.1 Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2University of Massachusetts Medical School, Worcester, MA; Applied Genetic Technologies, Alachua, FL T cell responses to A1AT transgene product was analyzed in subjects with A1AT PI*Z mutations enrolled in a Phase clinical trial of a recombinant adeno-associated virus (rAAV) alpha-1 antitrypsin (A1AT) gene therapy vector None of the subjects had detectable T cells to the A1AT transgene product before vector administration After vector administration, subjects developed a T cell response to A1AT measured by IFN-γ ELISPOT The magnitude of the response in one of the subjects was high enough for epitope mapping and intracellular cytokine staining (ICS) analyses The mapping analysis revealed an immuodominant epitope located at position 202-216 of the mature protein and distant from the site of the PI*Z mutation; which is located at position 342 of the mature protein The ICS analysis revealed an IFN-γ and TNF-α CD8+ T cell mediated response with a CD45RO- CD27- effector phenotype We also evaluated differential antigen processing and presentation of the mutant PI*Z A1AT using an ex vivo mouse model of antigen presentation where mouse fibroblasts expressing PI*Z- or PI*M-A1AT were co-cultured with A1AT-specific T cells Both antigens stimulated a similar level of activated IFN-γ secreting cells Next, we HLA typed all subjects enrolled in the trial and found that HLA-C* alleles 04 and 05 were uniquely present in this subject In silico analysis using a HLA-peptide binding prediction algorithm showed that HLA-C*04/05 alleles bind 9-mer sequences in the dominant epitope more strongly than other alleles To evaluate antigen presentation of the A1AT dominant epitope by HLA-C*04/05 alleles, an ex-vivo antigen presentation model was developed by transfecting individual HLA alleles into K562 cells, loading them with the immunodominant peptide and using them as antigen presenting cells Only cells transfected with HLA-C*05 and loaded with the A1AT dominant epitope activated T cells from the subject These data demonstrated that the A1AT-specific T cell response observed in this subject was CD8+ mediated and restricted by HLA C*05 This importance of HLA haplotype in influencing the adaptive immune response to vector encoded transgene should be considered in the design of clinical trials 50 Integration Site Analysis in a Clinical Trial of Lentiviral Vector-Based HSC Gene Therapy for MLD Shows High Levels of Polyclonal Hematopoietic Reconstitution and No Signs of Genotoxicity at One Year after Transplant Eugenio Montini,1 Alessandra Biffi,1 Andrea Calabria,1 Martina Cesani,1 Fabrizio Benedicenti,1 Laura Lorioli,1 Tiziana Plati,1 Simone Leo,2 Gianluigi Zanetti,2 Maria Sessa,1 Christof von Kalle,3 Manfred Schmidt,3 Luigi Naldini.1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; 2CRS4, Pula, Italy; 3National Center for Tumor Diseases, Heidelberg, Germany A self-inactivating lentiviral vector (LV) has been used in an ongoing HSC-based clinical trial for metachromatic leukodystrophy (MLD) performed in our Institute in Milan High sustained levels of vector marking have been achieved in all treated patients up to now, S20 Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy CLINICAL GENE & CELL THERAPY ORAL ABSTRACT SESSION reaching up to 80% of the reconstituted multi-lineage hematopoiesis Vector integration site profiling in vector-marked cells from patients of HSC gene therapy enables to assess the safety and efficacy of gene transfer as well as the dynamics of hematopoietic reconstitution Here we report the integration site analysis in different cell lineages and time points after transplantation in the first MLD patients LAM PCR was used to retrieve LV/genomic DNA junctions from patients’ transduced CD34+ cells, and from bulk or lineage marker-sorted cell populations from bone marrow or peripheral blood (1, 3, 6, and 12 months after transplant) A highly polyclonal pattern of bands was observed in all cell compartments with exception of T-cells at the earliest time point This is an expected finding because the patient did not receive a T-cell ablative preconditioning treatment By high-throughput next-generation 454-pyrosequencing of LAM PCR products we obtained 6x10e6 sequencing reads and mapped >25000 unique integrations from the analyzed patients The LV integration profile in this trial is highly reminiscent to those previously described in the LV-based adrenoleukodystrophy clinical trial and our preclinical xenotransplantation mouse models, including the occurrence and identity of Common Insertion Sites (Biffi et al., Blood 2011) CIS clustering at specific mega-base wide chromosomal regions suggests that these CIS may be originating by an intrinsically benign viral integration bias No skewing from in vitro to in vivo in the frequency of CIS was found so far Gene Ontology analysis at different time points shows constantly similar gene classes such as transcription factors and chromatin remodeling genes Analysis of sequencing reads as a surrogate of the abundance of specific cell clones shows long-term variable contribution of multiple clones without evidence of clonal dominance Importantly, sequencing of the same samples multiple times provided important information on complexity and clonality in a dynamic fashion By this approach we show that the number of integrations shared between different sequencing runs of the same LAM PCR products is relatively small (about 50%), indicating that our retrieval and sequencing depth is far from saturation These findings further indicate that a highly polyclonal reconstitution has been achieved in this clinical trial Overall, our data show that despite the unprecedented high vector marking levels obtained in MLD treated patients no overt evidence of genotoxicity has been observed EM and AB contributed equally weeks for a total of ten infusions A 48-hour inpatient observation was required for the first infusion and all patients received prophylactic dexamethasone mg i.v hour prior to dosing, H1 and H2 blockers i.v 30 minutes prior, indocin 25 mg, p.o approximately 30 minutes prior to receiving SGT-53, and acetaminophen 650 mg p.o prior and subsequent to SGT-53 for pyretic reactions In patients with biopsies of tumor ± normal skin, p53 was detected using DNA PCR employing primers specific for the exogenous p53 gene Eight of 11 evaluable patients (73%) demonstrated SD at week assessment and three had PD The tumor in one patient (adenoid cystic carcinoma) underwent significant necrosis and was reclassified from inoperable to operable In another patient (leiomyosarcoma with metastases in liver and lung), post-treatment CT assessment showed development of necrotic centers in all the liver metastases DNA PCR analysis for exogenous p53 DNA in the tumors (metastases) from three patients and normal skin biopsies showed a tumor specific and DNA dose-dependent presence of the exogenous p53 (5+ tumor vs normal skin) One patient experienced a possibly-related grade adverse event (AE), fatigue (at 0.6 mg DNA, concurrent with tumor necrosis) and a second patient possibly-related grade chest pain and tachycardia (at 3.6 mg DNA) SGT-53-related grade 1, AE occurring in ≥5% of patients primarily consisted of transient fever starting 6-8 hours after infusion and lasting 12-16 hours (5 of 12; 42%) and transient hypotension over a similar time frame in (7 of 12; 58%) This is the first report of tumor-specific delivery to metastatic lesions in patients after systemic administration Integrating the results of the current study with data derived from pre-clinical p53 restoration models, a clinical Phase Ib study of SGT 53 combined with docetaxel, has been activated and is accruing patients 52 Increases in CD4 Counts and Effects on HIV in Aviremic HIV-Infected Subjects Infused with Zinc Finger Nuclease (ZFN) CCR5 Modified Autologous CD4 T-Cells (SB-728-T) 51 Results of a Phase I Trial of SGT-53: A Systemically Administered, Tumor-Targeting Immunoliposome Nanocomplex Incorporating a Plasmid Encoding wtp53 Winson Tang,1 Jay Lalezari,2 Carl June,3 Pablo Tebas,3 Gary Lee,1 Marty Giedlin,1 Shelley Wang,1 David Stein,5 Hiroyu Hatano,4 Elizabeth Sinclair,4 Joseph K Wong,4 Cindy Desmarais,6 Travis Wood,1 Steven G Deeks,4 Geoff M Nichol,1 Ronald Mitsuyasu,7 Dale Ando.1 Sangamo BioSciences, Inc, Richmond, CA; 2Quest Clinical Research, San Francisco, CA; 3University of Pennsylvania, Philadelphia, PA; 4UCSF, San Francisco, CA; 5Albert Einstein College of Medicine, Bronx, NY; 6Adaptive Biotechnologies, Seattle, WA; 7UCLA, Los Angeles, CA The effective oncologic application of gene therapy is predicated on the efficient and selective systemic delivery of therapeutic molecules to both primary and metastatic cancer cells throughout the body SGT53 is a therapeutic complex comprised of a DOTAP:DOPE cationic liposome encapsulating a plasmid encoding normal human wild type (wt) p53 cDNA and decorated with the anti-transferrin receptor single chain antibody fragment designed to target cancer cells by binding to the transferrin receptor (TfR) Eleven patients with solid tumors, having exhausted all standard and/or approved therapies, were enrolled in this sequential dose-escalating study ranging from 0.6 mg DNA per infusion through 1.2, 2.4 and 3.6 mg Pertinent inclusion criteria were ECOG 0-2, measurable biopsy-accessible disease, willingness to allow skin biopsy, and standard adequate organ function SGT-53 was administered twice weekly for five Background: ZFN modification of the CCR5 receptor in autologous CD4 T-cells may render a survival advantage to these cells in HIV-infected subjects We report data relating to safety, increases in CD4 T-cell counts, persistence and trafficking of SB-728-T, and effects on HIV-RNA (VL) and proviral DNA from two Phase studies of SB-728-T Methods: In the CA study, immunologic nonresponders (INR) with CD4= 200-500 cells/uL were enrolled into cohorts that received 1x, 2x, and 3x1010 total cells In the PENN study, immunologic responders (IR) with CD4 ≥450 cells/uL and INR with CD4 ≤500 cells/uL were infused with 1010 total cells At Wk 4, IR underwent a 12-week HAART Treatment Interruption (TI) The median duration of follow-up was 246 days (range 42-561) for all subjects T-cell profiling was performed with multiplex PCR on subjects to sequence TCR CDR3 chains to determine the composition of various T-cell clones in the peripheral blood, gut mucosa, and lymph nodes The method amplifies rearranged TCR CDR3 sequences to explore all Vbeta and Jbeta combinations Results: SB-728-T was well tolerated with only one SAE attributed to a transfusion reactionfever, chills, myalgia, and arthralgia All AEs were reversible and most were mild to moderate in severity The mean CD4 and SB-728-T count increased by 1533 cells/uL (range 216-3025) and 83 cells/uL, Neil Senzer,1,2,3 John Nemunaitis,1,2,3 Derek Nemunaitis,1 Cynthia Bedell,1 Gerald Edelman,1,3 Minal Barve,1,3 Robert Nunan,1 Kathleen F Pirollo,4 Antonina Rait,4 Esther H Chang.4,5 Mary Crowley Cancer Research Centers, Dallas; 2Texas Oncology, P.A., Dallas; 3Medical City Dallas Hospital, Dallas; Georgetown University Medical Center, Washington, DC; SynerGene Therapeutics, Inc., Potomac Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy S21 ... sequencing runs of the same LAM PCR products is relatively small (about 50% ), indicating that our retrieval and sequencing depth is far from saturation These findings further indicate that a highly... highly polyclonal reconstitution has been achieved in this clinical trial Overall, our data show that despite the unprecedented high vector marking levels obtained in MLD treated patients no overt... with data derived from pre -clinical p53 restoration models, a clinical Phase Ib study of SGT 53 combined with docetaxel, has been activated and is accruing patients 52 Increases in CD4 Counts and

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