Kick-Off / Plenary Session: Presented Sunday, 28 October 2007 at ESGCT 2007

Preliminary Data from the First Hematopoietic Stem Cell Gene Therapy Trial with Lentiviral Vector Demonstrate Expression of the Therapeutic Protein in High Percentage of Lymphocytes and Monocytes in Two Patients with X-Linked Adrenoleukodystrophy

Nathalie Cartier1,2,3; Salima Hacein-Bey-Abina4,5; Gabor Veres6; Michel Vidaud1,7; Liliane Dal-Cortivo4,5; Laure Caccavelli4,5; Nizar Malhaoui8; Veronique Kiermer9,*; Denice Mittelstaedt9,; Andrew Simmons10; Cline Bellesme3; Francoise Audat4,5; Stephane Blanche8; Pierre Charnaud10; Muriel Audit11; Bruno Lohomme1,2,13; Jing- Chao Zhao- Emmonet1,2,13; Serge Fichelson14; Francoise Pflumio14; Anne Dubart-Kupperschmitt14; Rachel Salzman15; Amber Salzman16; Pierre Bougnores3; Alain Fischer5,8; Marina Cavazzana-Calvo4,5; Patrick Aubourg1,2,3 1

INSERM U745, 2Faculty of Pharmacy, University Ren Descartes, and 3Department of Pediatric Endocrinology and Neurology, 13Hopital Saint-Vincent de Paul, Paris, France; 4Department of Biotherapies, 5U768, and 8Unit dImmunologie et Hematologie Pediatrique, Hopital Necker- Enfants Malades, Paris, France; 6Applied Genetic Technology Corporation, Alachua, FL, USA; 10R&D Development, 9Cell Genesys, Inc., South San Francisco, CA, USA; 11Institut Pasteur, CNRS-URA 3015, Paris, France; 12Gnthon, Evry, France; 14Department of Hematology,
U567, Institut Cochin, Paris, France; 15The StopALD Foundation, Houston, TX, USA; 16The StopALD Foundation and GlaxoSmithKline, Philadelphia, PA, USA; *Present address: Nature Methods, New York, NY, USA; Present address: San Diego, CA, USA

We report preliminary results in two children with cerebral X-linked adrenoleukodystrophy (ALD) who received in September 2006 and January 2007 hematopoietic stem cell (HSC) gene therapy using a HIV1-derived lentiviral vector. We have previously shown that the cerebral demyelination associated with cerebral ALD can be stopped or reversed within 18 months by allogeneic HSC transplantation. For the current HSC gene therapy procedure, mobilized peripheral blood CD34_ cells were transduced ex vivo for 18 hr with a non-replicative HIV1- derived lentiviral vector (provided by Cell Genesys, Inc.) expressing the ALD cDNA under the control of the MND promoter, and in the presence of Il-3, SCF, Flt3-ligand, MGDF, and CH-296 retro nectine. Transduced cells were frozen to perform replication-competent lentivirus (RCL) assays. After thawing and prior to reinjection, 50% and 30%, respectively, of transduced CD34_ cells expressed the ALD protein with a mean of 0.7 and 0.6 copies of integrated provirus/cell. Transduced CD34_ cells were infused to ALD patients after full myeloablation with cyclophosphamide and busulfan.

Hematopoietic recovery occurred at day 15 post-transplant, and the procedure was uneventful. The percentage of corrected lymphocytes and monocytes in the peripheral blood of treated patients remained stable from day 30 to the last follow-ups. From 25% to 30% (Patient P1, 9 months after transplant) and 20% (Patient P2, 41/2 months after transplant) of CD14_, CD3_, CD19_, and CD3_CD56_ cells expressed the ALD protein (0.4 integrated provirus copy/cell). Tests assessing vector-derived RCL and vector mobilization were negative up to the last follow-ups. These early results support that: (1) ex vivo HSC gene therapy using HIV1-derived lentiviral vector is not associated with the emergence of RCL and vector mobilization; (2) a high percentage of hematopoietic progenitors were transduced expressing ALD protein in the short term; (3) no early evidence of selective advantage of the transduced ALD cells or clonal expansion was observed; and (4) HSC gene therapy appears to have short-term neurological effects comparable with allogeneic HSC transplantation.

Sponsored by INSERM and conducted under an R&D collaboration with Cell Genesys, Inc., South San Francisco, CA.

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