Expression of a large coding sequence: Gene therapy vectors for Ataxia Telangiectasia

Ataxia telangiectasia is a monogenetic disorder caused by mutations in the ATM gene. Its encoded protein kinase ATM plays a fundamental role in DNA repair of double strand breaks (DSBs). Impaired function of this kinase leads to a multisystemic disorder including immunodeficiency, progressive cerebellar degeneration, radiation sensitivity, dilated blood vessels, premature aging and a predisposition to cancer. Since allogenic hematopoietic stem cell (HSC) transplantation improved disease outcome, gene therapy based on autologous HSCs is an alternative promising concept. However, due to the large cDNA of ATM (9.2 kb), efficient packaging of retroviral particles and sufficient transduction of HSCs remains challenging.We generated lentiviral, gammaretroviral and foamy viral vectors with a GFP.F2A.Atm fusion or a GFP transgene and systematically compared transduction efficiencies. Vector titers dropped with increasing transgene size, but despite their described limited packaging capacity, we were able to produce lentiviral and gammaretroviral particles. The reduction in titers could not be explained by impaired packaging of the viral genomes, but the main differences occurred after transduction. Finally, after transduction of Atm-deficient (ATM-KO) murine fibroblasts with the lentiviral vector expressing Atm, we could show the expression of ATM protein which phosphorylated its downstream substrates (pKap1 and p-p53)

Hematopoietic stem cell transplantation restores naïve t-cell populations in atm-deficient mice and in preemptively treated patients with ataxia-telangiectasia

Background: Ataxia-telangiectasia (A-T) is a multisystem disorder with progressive cerebellar ataxia, immunodeficiency, chromosomal instability, and increased cancer susceptibility. Cellular immunodeficiency is based on naïve CD4+ and CD8+ T-cell lymphopenia. Hematopoietic stem cell transplantation (HSCT) offers a potential to cure immunodeficiency and cancer due to restoration of the lymphopoietic system. The aim of this investigation was to analyze the effect of HSCT on naïve CD4+ as well as CD8+ T-cell numbers in A-T. Methods: We analyzed total numbers of peripheral naïve (CD45RA+CD62L+) and memory (CD45RO+CD62L−) CD4+ and CD8+ T-cells of 32 A-T patients. Naïve (CD62LhighCD44low) and memory (CD62LlowCD44high) T-cells were also measured in Atm-deficient mice before and after HSCT with GFP-expressing bone marrow derived hematopoietic stem cells. In addition, we analyzed T-cells in the peripheral blood of two A-T patients after HLA-identic allogeneic HSCT. Results: Like in humans, naïve CD4+ as well as naïve CD8+ lymphocytes were decreased in Atm-deficient mice. HSCT significantly inhibited thymic lymphomas and increased survival time in these animals. Donor cell chimerism increased up to more than 50% 6 months after HSCT accompanied by a significant increase of naïve CD4 and CD8 T-cell subpopulations, but not of memory T-cells. This finding was also identified in the blood of the A-T patients after HSCT. Conclusion: HSCT seems to be a feasible strategy to overcome immunodeficiency and might be a conceivable strategy to avoid T-cell driven cancer in A-T at higher risk for malignancy. Naïve CD4 and CD8 T-cells counts are suitable markers for monitoring immune reconstitution post-HSCT. However, risks and benefits of HSCT in A-T have to be properly weighted