Correction of ATM-deficiency by lentiviral vector gene transfer
Authors:   , Hirch Tanja 
Published: October 2024

Ataxia Telangiectasia (A-T) is a rare monogenetic, autosomal recessive disorder with an incidence of 1 in 40,000-100,000 live births caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The encoded serine/threonine protein kinase (ATM) plays a major role in DNA damage response as well as apoptosis, cell cycle regulation, cell survival, oxidative stress response and genomic stability. Biallelic mutations result in partial or complete loss of ATM expression and/or ATM protein activity. A-T is a disease characterized by progressive cerebellar degeneration, telangiectasia, immunodeficiency (impaired B- and T-cell development), recurrent sinopulmonary infections, radiation sensitivity, premature aging, and a predisposition to cancer. Life expectancy of these patients is highly compromised, with only around 50% expected to reach 20 years of age. Malignancies and pulmonary diseases are the two main causes of death. There is currently no therapy available for A-T patients. There are symptomatic treatments available (e.g. immunoglobulin replacement therapy, therapy with antioxidants, and the administration of growth hormone or glucocorticoids as anti- inflammatory hormones) and in some patients, allogeneic hematopoietic stem cell transplantations from matched donors were performed with improved disease outcome. Unfortunately, suitable donors are not available for most patients. An autologous hematopoietic stem cell (HSC)-directed gene therapy approach is a promising alternative, since no matching donor is needed. The patient’s own cells are used, modified ex-vivo (e.g. delivering a healthy copy of the gene with viral vectors or directly correcting the mutation with gene editing). Afterwards, modified HSCs are given back to the patient thereby repopulating the bone marrow and re-establishing the whole blood system. The aim of this project was to develop a gene transfer tool for Atm. In the first part of this project, retroviral vectors containing the full-length murine Atm cDNA were generated. Gene transfer of Atm with retroviral vectors is challenging, as the Atm cDNA is 9.1 kb in size reaching the packaging capacity of retroviral vectors. Although the foamy viral vector is described to have superior abilities to transfer large sequences, produced titers of the foamy viral Atm vectors were low and transductions of Atm-deficient fibroblasts were inefficient. In contrast, gene transfer of Atm with gammaretroviral and lentiviral vectors was possible, and because lentiviral vectors harboring the full-length Atm coding sequence were produced with the highest viral titers, this vector was used to transduce Atm-deficient fibroblasts. Following transduction, ATM protein levels were restored (40 - 50% of wild-type level). In addition, transduced cells showed increased levels of phosphorylated ATM downstream substrates (γH2AX, pKap1 and p-p53) after irradiation, demonstrating functional reconstitution. However, efficient transduction of murine lineage marker negative cells, the target cells for an Atm gene therapy approach, was not possible and viability of these cells was highly compromised after transduction. Therefore, a dual vector system was developed in the second part of the project to circumvent the packaging limit of retroviral vectors. Protein halves were fused with split inteins which catalyze their self-excision followed by the formation of a full-length protein in a process called protein trans-splicing. The split Atm cDNA was delivered with lentiviral vectors and sufficient viral titers were achieved for efficient double transduction of Atm-deficient fibroblasts. Whereas the reconstitution of full-length ATM protein was low in cells transduced with vectors containing Npu split inteins, the use of Rma split inteins showed superior reconstitution. When comparing reconstitution levels with two different split sites within the ATM protein, no major differences were observed. Because a proof of ATM functionality could not be shown with these vector pairs, the F2A site used to co-deliver a marker gene was replaced by an IRES element. After transduction with split intein Atm vectors containing IRES elements, the level of ATM protein reached only 10% of the wild-type level. Nevertheless, an increased amount of pKap1 and p-p53 was detected demonstrating a functional kinase activity of reconstituted ATM protein. Furthermore, a partial repair of cell cycle defects in Atm-deficient fibroblasts was demonstrated. In parallel to the development of a gene transfer tool for Atm, preliminary experiments were performed in Atm-deficient mice to create optimal transplantation conditions for gene-corrected HSCs that could be performed in the future. Because Atm-deficient mice are highly sensitive to irradiation, conventional conditioning regimes (e.g. total body irradiation or myeloablative conditioning with chemotherapeutics) cannot be used prior to HSC transplantation. Therefore, Atm-deficient mice were pretreated with different conditioning regimens and subsequently received a bone marrow transplantation. Mice that did not receive preconditioning prior to transplantation showed no chimerism in peripheral blood, bone marrow or spleen samples, indicating that preconditioning of mice is required for donor cell engraftment. A non-myeloablative conditioning regimen with cyclophosphamide and immunosuppressive CD4 and CD8 antibodies and the application of a mobilizing agent (Plerixafor) one hour before transplantation showed the highest chimerism in recipient mice. None of the mice developed a thymic tumor, and lymphoid-biased differentiation of the donor cells was observed, as chimerism was highest in T cells in the blood, bone marrow and spleen. In addition, chimerism was higher in lymphoid progenitor cells than in myeloid progenitors. Blood counts (white blood cell and lymphocyte counts) were normal 20 weeks after transplantation (comparable to wild-type mice), making this preconditioning regime suitable for Atm-deficient mice. Taken together, this data paves the way for using split intein-based lentiviral vectors for Atm delivery in preclinical models and opens new possibilities for developing gene therapy for A-T patients.