Expression of Normal Dystrophin Following Myoblast Transplantation to Duchenne Muscular Dystrophy Patients

CLINICAL GENE THERAPY the absence of enzyme replacement therapy. All the children are presently healthy, they did not experience any severe infections or adverse effect, with the longest follow up being of 40 months after gene therapy. A systematic analyses to identify and map vector integration sites by inverse-PCR and LM-PCR was undertaken. Cloned integrations were selected for analyses only if contained the correct retroviral and primer sequences and yielded a unique best hit with ≥95% identity to the human genome (NCBI34). Collective data from 160 mapped integrations showed a frequency of insertions inside RefSeq genes of 33.4% (n=53), similar to the one reported in cell lines infected in vitro with retroviral vectors. Genomic regions within 5 Kb upstream from transcription start site were favored sites of integrations (15.1%). There was no preferential target locus, with the exception that two independent integrations were found outside a gene on chromosome 3p13, within a distance of 120 Kb. Integrations in circulating T cells were highly polyclonal (>50 in Pt1 and Pt3), as shown by the random cloning of PCR products from peripheral blood T cells as well as by PCR analyses in T-cell clones generated ex vivo. Fewer integrations (3-15) were detected in myeloid cells from bone marrow and peripheral blood. Common integrants were identified in various lineages during the follow-up of patients, demonstrating the engraftment of transduced HSC with multilineage potential. Collectively, these data show the efficacy of gene therapy in correcting the immune and metabolic defect of ADA-SCID. In addition, these studies are providing new information on the safety and biology of retroviral vectors as well as on the in vivo dynamics of HSC and their progeny. *MGR and CB equally contributed to the work

1005. Expression of Normal Dystrophin Following Myoblast Transplantation to Duchenne Muscular Dystrophy Patients Jacques P. Tremblay,1 Daniel Skuk,1 Bouchard Jean-Pierre,2 Michel Sylvain,1 Roy Raynald,1 Goulet Marilyne,1 Roy Brigitte,1 Pierre Chapdelaine,1 Dugré Francine,1 Jean-Guy Lachance,1 Louise Deschènes,1 Hélène Senay.1 1 Centre de Recherche du CHUQ, Centre Hospitalier Universitaire de Québec, Québec, QC, Canada; 2Neurologie, Centre Hospitalier Affilier de Québec, Québec, QC, Canada. Three Duchenne muscular dystrophy (DMD) patients received injections of myogenic cells obtained from skeletal muscle biopsies of normal donors. Cells were injected in 1 cm3 of the Tibialis anterior by 25 parallel injections. We performed similar patterns of saline injections in the contralateral muscles as controls. The patients received tacrolimus for immunosuppression. Muscle biopsies were performed at the injected sites 4 weeks later. We observed dystrophin-positive myofibers in the cell-grafted sites: 9 % (patient 1), 6.8 % (patient 2) and 11 % (patient 3). Since patients 1 and 2 had identified dystrophin-gene deletions these results were obtained using mAbs specifically to epitopes coded by the deleted exons. Donor-dystrophin was absent in the control sites. Patient 3 had exon duplication and thus specific donor-dystrophin detection was not possible. However there was 4-fold more dystrophin-positive myofibers in the cell-grafted than in the control site. Donordystrophin transcripts were detected by RT-PCR (using primers reacting with a sequence in the deleted exons) only in the cell-grafted sites in patients 1 and 2. Dystrophin transcripts were more abundant in the cell-grafted than in the control site in patient 3. Therefore, significant dystrophin expression can be obtained in the skeletal muscles of DMD patients following specific conditions of cell delivery and immunosuppression. I am president and part owner of CellGene inc.

Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright  The American Society of Gene Therapy

1006. Preliminary Results of a Phase I Trial Using Retroviral Gene Transfer of G156A MGMT To Protect Hematopoiesis during BG and BCNU Therapy of Advanced Malignancies Jane Reese,1 Karen Lingas,1 Pamela Ksenich,1 Colin Sweeney,1 Omer Koc,1 Stanton Gerson.1 1 Hematology/Oncology, Center for Stem Cell and Regenerative Medicine, Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH. We have extensively studied the G156A MGMT mutation which confers O6alkylguanine DNA alkyltransferase (AGT) that is resistant to O6benzylguanine (BG). In a Phase I clinical trial at our institution, BG was shown to inhibit tumor AGT at 120mg/m2 and the maximum tolerated dose of BCNU was 40 mg/m2. In the laboratory, we have shown that the retrovirus MFG-G156A-MGMT confers BG and BCNU resistance ex vivo into human CD34+ cells. We have also reported that G156A-MGMT transduced murine hematopoietic progenitors repopulate the bone marrow and can be enriched up to 1000-fold after 2 cycles of BG and BCNU. These studies demonstrate the strong capacity of this model to confer BG and BCNU resistance and a selection advantage in vivo. Based on these data, we initiated a clinical trial using gene transfer into autologous CD34+ cells in patients with advanced malignancies. Patients undergo CD34+ mobilization with G-CSF and GM-CSF. CD34+ enriched cells (CliniMACS, Miltenyi) are transduced with MFG-G156A MGMT (produced in PG13 cells by the NGVL, K. Cornetta, Director) in the presence of the fibronectin fragment CH-296 (provided by Takara Bio Inc) and the cytokine combination SCF, Tpo, and Flt-3 ligand for 86 hours. The cell culture is harvested on day 4 and the entire culture is re-infused into the patient who received BG and BCNU 48 hours prior. Patients receive subsequent cycles of BG and BCNU every 6 weeks with analysis of marrow and blood cells for evidence of gene transfer prior to each cycle. In the first three patients, the frequency of gene transfer measured by proviral containing CFU in the post transduction culture was 20 ± 7% and 6.3 ± 3 % of the cells expressed G156A-AGT by flow cytometry. An average of 4.4 ± 2 x 107viable mononuclear cells with a CD34 purity of 93 ± 5% were infused. In one patient, 10% of bone marrow CFU showed presence of the provirus 5 weeks after infusion but prior to the second cycle of BG and BCNU. In this same patient, insertion site analysis by LAM-PCR showed a similar banding pattern in both the lymphocyte-monocyte and granulocyte fractions, indicating multilineage reconsitituion by a single transduced stem cell. Our preliminary results demonstrate efficient gene transfer into cytokine stimulated CD34+ cells using the MFG-G156A-MGMT vector and these cells can be infused without toxicity. Given the strong degree of selection observed in pre-clinical models, we anticipate enrichment for transduced cells following each cycle of chemotherapy.

1007. Safety of Adenoviral Vectors: Results of Clinical Investigations in 445 Cancer Patients Treated with Advexin® (Adenoviral p53) Gene Therapy Louis A. Zumstein,1 Donna Call,1 James Merritt,1 Robert E. Sobol,1 Kerstin Menander.1 1 Introgen Therapeutics, Inc, Houston, TX. Safety data were collected from 445 patients treated with Advexin® (adenoviral p53) gene therapy for cancer in 14 clinical trials (Phase I, II and III). Advexin gene therapy was used as monotherapy in 11 trials and in combination with chemotherapy or radiation in 3 clinical studies. The treated patients had advanced cancers, primarily lung and squamous cell head and neck cancers, but also included patients with prostate cancer, colorectal cancer, and other solid tumors. Most patients were treated with multiple S385