Scholarly article on topic 'Integration Analyses in ADA-Deficient SCID Patients Successfully Treated with Stem Cell Gene Therapy'

Integration Analyses in ADA-Deficient SCID Patients Successfully Treated with Stem Cell Gene Therapy Academic research paper on "Clinical medicine"

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Academic research paper on topic "Integration Analyses in ADA-Deficient SCID Patients Successfully Treated with Stem Cell Gene Therapy"

supercoiled standard) showed expression of detectable levels of FIX, but expression was transient, with peak levels of 3% in one subject and 11.8% in the other. FIX levels had returned to baseline by 4 weeks and 10 weeks post vector infusion respectively. In the latter subject, the decline in circulating FIX levels was accompanied by a transient rise in serum transaminase levels. This subject had a low baseline neutralizing antibody titer (NAB) to AAV-2 (1:2), which rose to >1:1000 two weeks later. The other subject experienced no change in serum transaminases; his pre-treatment NAB to AAV-2 was 1:17, and increased to 1:96,000 two weeks later. To further assess the role of the immune response, PBMCs from these two subjects were incubated with peptides derived from the AAV-2 capsid sequence and from the wild-type hF.IX sequence. IFN-y secretion was measured in ELISpot assays, which showed, in the latter subject, a 10-fold elevation in IFN-y compared to media control at the 4 wk time point following incubation with one of the AAV peptide pools and with the F.IX peptide pool. When normalized to levels of maximal IFN-y secretion in response to a positive control, IFN-y response was >10-fold that of normal controls for the F.IX peptide pool, and >2-fold that of normal controls for the reactive AAV-2 peptide pool. We conclude that: 1) AAV-F.IX can transduce human hepatocytes in vivo, resulting in therapeutic circulating factor levels; 2) the vector dose required to achieve a therapeutic factor level was accurately predicted by animal models; 3) the transient nature of expression seen in subjects treated at 2x1012 vg/kg was not predicted by earlier studies in animal models; 4) T cell responses to AAV-2 and to F.IX may determine the outcome of AAV-2-mediated gene transfer in human subjects. More recently the clinical study has resumed with a planned target dose of 1.2x1012 vg/kg. The goal is to determine whether subjects can achieve therapeutic levels of expression at this dose, and whether specific immune responses to either AAV or F.IX will limit duration of expression in humans.

Some co-authors (Sommer, Couto, Pierce, Lessard, Luk) are employees of Avigen, or hold equity in Avigen (Kay).

1003. HIV-1-Derived Lentivirus Vector-Based Antisense Gene Therapy: Towards an Alternative Treatment for HIV/AIDS

Boro Dropulic,1,2 Laurent Humeau,1 Gwendolyn K. Binder,1 Xiaobin Lu,1 Vladimir Slepushkin,1 Randall Merling,1 Patricia Echeagaray,1 Mario Pereira,1 Tatiana Slepushkina,1 Scott Barnett,3 Lesia K. Dropulic,4 Richard Carroll,5 Bruce Levine,5 Rob Roy MacGregor,5 Carl H. June.5

'VIRxSYS Corporation, Gaithersburg, MD; 2Sydney Kimmel Comprehensive Cancer Center, The Johns Hopkins School of Medicine, Baltimore, MD; 3The Gary Lambert Research Center, The Johns Hopkins University, Baltimore, MD; 4Department of Medicine, The Johns Hopkins University Division of Infectious Disease, Baltimore, MD; 5Abramson Family Cancer Research Institute and School of Medicine, The University of Pennsylvania, Philadelphia, PA.

Despite the significant advancement in HIV/AIDS treatment with highly active antiretroviral therapy (HAART), HIV/AIDS remains incurable. Limitations of HAART include expense, disabling complications, and a stringent requirement for compliance. In the United States, where HAART has become the standard of care, approximately 20% of newly transmitted virus is already resistant to at least one antiretroviral drug. In anticipation of expanded HAART in Africa, there is concern regarding rapid emergence of drug resistant strains of HIV that brings into question HAART as a long-term solution. We have developed an alternative therapy for HIV/AIDS therapy using an HIV-1-based lentiviral vector expressing a 937-base antisense gene against the HIV envelope, VRX496, for autologous T cell therapy. T cells are transduced with vector in the presence of co-stimulatory CD3/CD28 beads, and subsequently

expanded in culture. Preclinical studies demonstrated the feasibility of this approach in normal CD4+ T cells and inCD4+ T cells isolated from 20 early and late stage patients, as defined by viral loads. An efficient gene transfer procedure was established, which demonstrated stable transfer rates of 94% +/- 5%. Antisense inhibited HIV in cultures by >93% over mock transduced controls, regardless of patient status or the tropism of the infecting virus. A Phase I open label non-randomized clinical trial was then initiated to investigate the safety and tolerability of autologous T cells modified with VRX496. In this study, 5 HAART resistant patients having CD4 T cells counts between 200-500 cells/mm3 are serially enrolled in the study that includes 3-week, and 3 and 6-month monitoring points. Each patient is given approximately 1 x 1010 modified autologous CD4 T cells in a single dose. Patients are monitored for viral load, CD4 count, emergence of any replication competent lentivirus (RCL), immunological parameters, and clinical indications. Adverse events are defined in part by a sustained 0.5 log increase in viral load, or sustained 0.5 log decrease in CD4 count within 3 weeks post dose. To date, 3 subjects have been infused, and the first and second subjects have completed the 6 and 3-month monitoring points, respectively. No adverse events have been noted, and all RCL assays have been negative. CD4 counts remained steady and engraftment of VRX496-modified cells was observed for at least 6 months in the first patient and 3 months in the second patient. At dosing, subject 1 had an average viral load of 188,500 and subject 2 had an average viral load of 40,428. After three and six months post dosing, subjects 1 and 2 respectively, had viral loads below baseline. This interim analysis supports the promise of lentiviral vectors for late stage HIV infection, and that VRX496 could become a viable treatment modality for individuals infected with HIV/AIDS.

The presenter and some of the authors are employees ov VIRxSYS Corporation, whose vector is presented in the data. However, these studies are a result of a collaborative effort between Johns Hopkins University, The University of Pennsylvania and VIRxSYS Corporation.

1004. Integration Analyses in ADA-Deficient SCID Patients Successfully Treated with Stem Cell Gene Therapy

A. Aiuti,1 B. Cassani,1 F. Cattaneo,1 G. Andolfi,1 M. Mirolo,1 A. Recchia,2 L. Callegaro,1 A. Tabucchi,3 F. Carlucci,3 S. Slavin,4 R. Miniero,5 F. Mavilio,2 M. G. Roncarolo,1 C. Bordignon.1 'San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Clinical Research Unit (HSR-TIGET), Milan, Italy; 2MolMed, Milan, Italy; Dip. MISEMB, Univ. of Siena, Italy; 4Dep. of BMT and Pediatrics, Hadassah Univ. Hospital, Jerusalem, Israel; 5Univ. of Turin, Italy.

Adenosine deaminase (ADA)-deficiency results in severe combined immunodeficiency (SCID) associated with systemic metabolic toxicity. Gene therapy with hematopoietic stem cells (HSC) may represent a definitive treatment for this disease, considering the current limitations of bone marrow transplantation from non-HLA identical donors or of enzyme replacement therapy (PEG-ADA). We treated four children with autologous bone marrow CD34+ cells engineered with a retroviral vector encoding ADA, combined with low-intensity conditioning with busulfan. Long-term engraftment of gene corrected HSC with differentiation into lymphoid and myeloid lineages, was sustained during the follow-up. All patients showed increased peripheral blood lymphocyte counts, restoration of thymopoiesis, and normalization of proliferative responses to mitogens and antigens. Serum immunoglobulin levels improved, allowing IVIG discontinuation in Pt1 and Pt3, with production of specific antibodies after antigen vaccination. Sustained ADA expression resulted in correction of the systemic toxicity and restoration of normal patients' growth, in

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

'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. Donor-dystrophin 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.

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

'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

'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

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