279. Phase II Clinical Trial of Gene Therapy for Adenosine Deaminase-Deficient Severe Combined Immune Deficiency (ADA-SCID)

RESULTS OF PRECLINICAL AND CLINICAL DEVELOPMENT FOR HEMATOLOGICAL AND IMMUNOLOGICAL DISEASES engraftment is often restricted to T cells with lifelong dependence on IgG supplementation. Over the 1st 2 decades of life the haplodonor T cells may decrease in number and function, and patients may have: Recurrent infections with progressive bronchiectasis; Warts and molluscum; Auto/allo-immune disease including rashes, alopecia, and gastrointestinal inflammation with malabsorption and protein-losing enteropathy; Impaired growth and nutritional status. Gene therapy of SCID-X1 infants mediated by murine retrovirus vector encoding gc without conditioning restored T cell immunity, without long term B cell gene marking or NK cell production. Similar gene therapy without conditioning to treat post-haplotransplant older children with SCID-X1 with waning immunity and clinical morbidity resulted in no significant gene marking or significant clinical benefit. We have combined marrow conditioning with use of a self-inactivating (short EF1α internal promoter) gc lentivector (Cl20-i4-EF1α hgc OPT) generated from a novel stable producer line for ex vivo transduction of autologous hematopoietic stem cells (HSC) to treat 2 IVIG-dependent young adults (P1, P2) aged 23 & 22 years with SCID-X1. P1 & P2 had waning immunity and significant clinical morbidity (including severe protein-losing enteropathy in P1) despite parental haploidentical transplant as infants and subsequent booster transplants. P1 & P2 received busulfan 6mg/kg (3mg x 2d) conditioning before receiving 20x10^6/kg (27% CFU marked) and 16x10^6/kg (17% CFU marked) transduced HSC, respectively. At 15 & 9 months post treatment, myeloid gene marking stabilized at 17% & 13%, respectively. B cell gene marking increased early in both patients to 20%, but varied from 13% to 47% thereafter. Gene marked NK cells were observed in both subjects, with significant increases in NK cell numbers from 2 to 28/uL and 3 to 17/uL (normal 109-607/ul), respectively. Slowly increasing T cell gene marking started after 6 months, reaching 15% & 3%, respectively. The presence of haplodonor T cells may be a barrier to a robust increase in rate of emergence of gene corrected autologous T cells. Of note, PI & P2 achieved normalization of serum IgG (739mg/dl and 811mg/dl, respectively) despite prolonged time interval between IVIG to 8-10 weeks. This is a particularly profound change in P1, who prior to treatment experienced trough IgG levels below 200mg/dl at 3 wks post IVIG due to protein losing enteropathy. P1 also demonstrated specific B-cell response to influenza vaccination. Thus, non-myeloablative busulfan conditioning followed by infusion of gc lentivector transduced autologous HSC in two young adults with XSCID has achieved unprecedented gene marking of B cells with evidence of significant restoration of Ig production and B-cell function, increasing gene marked NK cells, and associated improvement in clinical status manifested by weight gain, reduced infections and increased nutritional status.

279. Phase II Clinical Trial of Gene Therapy for Adenosine Deaminase-Deficient Severe Combined Immune Deficiency (ADA-SCID)

Kit L. Shaw,1 Robert Sokolic,2 Alejandra Davila,1 Christopher Silvin,2 Elizabeth Garabedian,2 Satiro de Oliveira,1 Provaboti Barman,1 Berkley Brown,1 Denise Carbonaro,1 Sabine Geiger,1 Suparna Mishra,1 Monika Smogorzewska,3 Jayashree Jagadeesh,2 Michael S. Hershfield,4 Alan Wayne,2 Gay M. Crooks,1 Theodore Moore,1 Fabio Candotti,2 Donald B. Kohn.1 1 University of California Los Angeles, Los Angeles, CA; 2National Institutes of Health, Bethesda, MD; 3Bone Marrow Transplant, Children’s Hospital Los Angeles, Los Angeles, CA; 4Biochemistry, Duke University, Durham, NC.

We report follow-up of subjects treated in a Phase II study of gene therapy for ADA-SCID. Between 2009 and 2012, ten ADA-deficient SCID patients were treated by γ-retroviral-mediated gene transfer (MND-ADA) to their bone marrow CD34+ cells. The subjects were given non-myelo-ablative chemotherapy (busulfan, 90mg/m2) and were withdrawn from PEG-ADA enzyme replacement therapy (ERT) Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy

prior to infusion of autologous gene-modified cells. Subject age at the time of treatment ranged from 3 months to 15 years (median=11.5 months). Follow-up times ranged from 12 to 54 months. One subject, who was 15-years old at the time of treatment, was re-started on PEGADA ERT at 5 months after the procedure because of lack of efficacy; all other subjects remain off ERT. Red blood cell deoxynucleotide levels have remained low, and ADA enzymatic activity in peripheral blood mononuclear cells (PBMC) has normalized in all subjects, except in the oldest subject. In the subjects who have discontinued ERT, T-cell counts have been sustained (range 400-1000 cells/mcL) and show normalization of proliferative responses to mitogen in all but one subject. Three subjects were able to discontinue intravenous immunoglobulin, and prophylactic antibiotics have been discontinued in five subjects. Vector marking in peripheral blood leukocytes continue to remain detectable and ranged from 0.002 to 2.6 vector copies per cell in PBMC (0.2 to 100% of cells containing at least 1 vector copy) and 0.0007 to 0.5 vector copies per cell in granulocytes (0.07 to 50% of cells containing at least 1 vector copy). The most robust immune reconstitution has occurred in the subjects treated during infancy. Surveillance of insertional oncogenesis is on-going and has not detected any monoclonal proliferative events. Our data continue to support gene therapy for ADA-deficient SCID as an efficacious option with low risk of developing lymphoproliferative disease.

280. Clonal Tracking of T-Cell Composition, Fate and Activity In Vivo in Humans

Luca Biasco,1 Serena Scala,1,2 Nicoletta Cieri,3 Luca BassoRicci,1 Francesca Dionisio,1 Cristina Baricordi,1 Andrea Calabria,1 Stefania Giannelli,1 Caterina Cancrini,4 Alessia Scarselli,4 Luigi Naldini,1 Eugenio Montini,1 Chiara Bonini,3 Alessandro Aiuti.1,5 1 San Raffaele Telethon Institute for Gene Therapy (TIGET), Milan, Italy; 2Università Vita-Salute San Raffaele, Milan, Italy; 3San Raffaele Scientific Institute, Milan, Italy; 4Ospedale Pediatrico Bambin Gesù, Rome, Italy; 5University of Rome “Tor Vergata”, Rome, Italy.

A deeper understanding of T lymphocytes survival and differentiation potential in humans is paramount for the development of effective gene/cell therapies based on T-cell engineering. We performed a comprehensive study of T-cells dynamics and plasticity in humans by a novel combination of phenotypic/functional studies and high-throughput integration sites (IS) analyses. We analyzed samples from hematopoietic stem cells (HSC) (n=12) or lymphocytes (PBL) gene therapy (GT) (n=4) treated ADA (adenosine deaminase) deficient-SCID patients. For comparative analyses, we also collected data from pediatric (n=19) and adult (n=52) healthy donors (HD), and from bone marrow transplanted patients (BMT) with primary immunodeficiencies (n=10, 4 of which had ADA-SCID). We showed that vector-positive putative naïve cells were detected for more than 11 years in peripheral blood of PBL-GT patients without the support of gene-corrected precursors. We strikingly found that the vast majority of these CD62L+/CD45RA+ cells (80.3%) in PBL-GT patients were actually the recently discovered T memory stem cell (Tscm), that we characterized through phenotypic (CD95, IL2Rb and IL7Rα expression) and functional (IFNg production and aCD3/ aCD28 in vitro differentiation) assays. By reproducing the original PBL-GT transduction protocol and through additional experiments on sorted subpopulations we showed that Tscm were enriched in vitro upon PHA+IL-2 culture condition prior to re-infusion. We further correlated, in the different groups of individuals, Tscm contribution and T-cell composition with age, conditioning regimens, disease backgrounds, GT approaches, thymic output and long term T-cell reconstitution after transplant. To track individual T-cell clones we sorted naïve, Tscm and memory subtypes and collected by LAM-PCR+Illumina-Miseq sequencing 677.802 sequence reads S107