Successful gene therapy of canine leukocyte adhesion deficiency using foamy viral vectors

ABSTRACTS / Blood Cells, Molecules, and Diseases 40 (2008) 248–294

blood and bone marrow. A third patient, a 5 year old child, was treated in a similar way in Zurich. In this case only low levels of engraftment and gene correction were achieved. Although the treatment has been beneficial for all treated patients, the side effects observed in the two adults demand modifications in vector design for sustained gene expression and long term correction of the disease without side effects. doi:10.1016/j.bcmd.2007.10.045

36 Determination of the profile and site preference of lentiviral vector integration in primary astrocyte cultures C.V. Hacker1,2, G.S. Ralph1, S.M. Kingsman1, K.A. Mitrophanous1, J.E. Miskin1 1 Oxford BioMedica, The Oxford Science Park, Oxford, OX4 4GA, UK 2 [email protected] Oxford BioMedica has developed minimal lentiviral vectors based on Equine Infectious Anemia Virus (EIAV), and these are currently being developed for use in the clinic. One advantage of lentiviral vectors over other retroviral vector systems is that they can integrate in non-dividing cells such as neurons. An EIAV-based vector for the treatment of Parkinson’s disease ProSavin® has now been developed. ProSavin has demonstrated efficacy in industry standard models of Parkinson’s disease and it is hoped that ProSavin will enter clinical evaluation in the near future. Integration of retroviruses and retroviral vectors within the target cell genome has been shown to be non-random. Human immunodeficiency virus-1 (HIV-1) and HIV-1 based vectors have a bias for integrating into genes, and more specifically genes that are active following infection with HIV-1. Murine leukemia virus (MLV) integration favors transcriptional start sites whilst Avian sarcoma-leukosis virus (ASLV) shows only a mild preference for transcriptional start sites. Previously, we have used Ligation mediated PCR (LMPCR) to map 458 integration sites for EIAV based lentiviral vectors and 162 control sites for HIV-1 based lentiviral vectors in human HEK293T cells. Like HIV-1, EIAV based vectors show a preference for active genes. In contrast to published analysis of MLV integration, EIAV and HIV-1 do not show a preference for the transcriptional start site or promoter region of genes. We have now determined the integration profile of EIAVbased vectors in rat primary astrocyte cultures, with 111 sites mapped. Integration favored transcriptionally active genes and gene dense regions. This profile is therefore similar to that previously described for EIAV integration within human HEK293T cells and has also been described for other retroviruses in a wide range of cell types and primary cells. doi:10.1016/j.bcmd.2007.10.046

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37 Successful gene therapy of canine leukocyte adhesion deficiency using foamy viral vectors Thomas R. Bauer, David Russell, Dennis D. Hickstein University of Washington School of Medicine, National Cancer Institute, USA The gammaretroviral vectors used in treating the genetic immunodeficiencies X-linked severe combined immunodeficiency (X-SCID) and chronic granulomatous disease have demonstrated the therapeutic potential of hematopoietic stem cell gene therapy; however, the vectors used in these trials have resulted in insertional activation of nearby oncogenes, oligoclonal hematopoiesis, and leukemia in several patients. These developments have prompted studies of modified or alternative vector systems. We treated five dogs with canine leukocyte adhesion deficiency or CLAD, a lethal genetic immunodeficiency disease caused by defects in the leukocyte integrin CD18, using a vector based on foamy virus (FV). All five CLAD dogs received non-myeloablative conditioning with 200 cGy TBI followed by infusion of FV transducedautologous CD34+ hematopoietic stem cells. One dog died within 8 days of transplant with intussception, a recognized complication of transplant in dogs. The four remaining dogs are now all 2 years or more from gene therapy with peripheral blood CD18+ neutrophil levels ranging from 3 to 6% with CD18+ lymphocyte levels ranging from 15 to 30%. In vitro testing of leukocytes has shown that the CD11/CD18 molecule present on FV vector transduced cells is appropriately regulated, with upregulation and activation of the CD18 molecule occurring only after stimulation, identical to results seen with normal cells. In vitro lymphocyte proliferation assays using Staphylococcal enterotoxin A and Concavalin A demonstrated that the increase in CD18+ T-lymphocyte levels compared to CD18+ neutrophil levels is due to a selective proliferation of the CD18+ Tlymphocytes in response to mitogens. Clinically, all four FV vector treated dogs have had complete and sustained reversal of the CLAD phenotype. In contrast, four CLAD control dogs were euthanized by 6 months of age due to complications from CLAD. To date, there have been no genotoxic complications in the four FV vector treated CLAD dogs, and integration site analysis has continued to demonstrate polyclonal marking of the transduced cells. The genotoxicity profile of the FV is considerably more favorable than conventional gammaretroviral vectors. These results support the use of FV vectors in the treatment of human hematopoietic stem cell diseases such as LAD. doi:10.1016/j.bcmd.2007.10.047

38 Ex vivo expansion of hematopoietic stem cells for use in nonmyeloablative transplantation S. Bakovic, H. Ohta1, S. Imren1, B. Cavilla1, C.J. Eaves1,2, R.K. Humphries1,3