Hämatopoetische Stammzell-gerichtete Gentherapie für die Behandlung von Ataxia telangiectasia (A-T)
Zusammenfassung der Projektergebnisse
Ataxia Telangiectasia (A-T) is a monogenetic, autosomal recessive immunodeficiency caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The encoded ATM protein is a serine/threonine protein kinase with important roles in DNA repair and cell cycle control. Impaired function of ATM leads to a multisystemic disorder including immunodeficiency, progressive cerebellar degeneration, telangiectasia, radiation sensitivity, premature aging, and cancer predisposition. First symptoms are usually recognized early in childhood and life expectancy is severely compromised due to malignancy and chronic pulmonary disease (20-year survival rate 53.4%). Currently, there is no curative therapy for A-T patients. Symptomatic treatments are immunoglobulin replacement, antioxidants, and the administration of growth hormone or glucocorticoids as anti- inflammatory treatment. Allogenic stem cell transplantations proved to be beneficial for A-T patients correcting the immune deficiency, therefore, alternative autologous hematopoietic stem cell (HSC)- directed gene therapy is a promising concept. Gene therapy targeting the hematopoietic system is already clinical reality for several immunodeficiency disorders and a similar strategy could be implemented to correct the immunodeficiency in A-T patients. Ex vivo HSC gene therapy that corrects the ATM deficiency aims to cure the genetic cause of A-T with a “once-in–a lifetime” treatment. In our project, we developed several strategies for A-T gene therapy. First, we developed a lentiviral vector expressing the full length murine Atm coding sequence. Transduction of Atm-deficient cells with this lentiviral vector rescued the Atm kinase function tested in vitro by the detection of the phosphorylation of Atm target proteins, however, the transduction of HSPC was not efficient. This was probably because of the low titers of the lentiviral vectors as the long Atm coding sequence of 9.2 kb reaches the limit of packaging capacity of lentiviral vectors. We, therefore, developed split intein vectors that divide the cDNA onto two vectors and recombine the full length Atm by protein trans-splicing. In a third approach, we developed gene editing that inserts the full-length ATM cDNA after the first exon into the human ATM locus using CRISPR/Cas9 technology. To deliver the template DNA, ATM was split onto three AAV vectors that recombine during DNA second strand synthesis after transduction. Finally, we developed the HSPC transplantation in the Atm-deficient mouse as a preclinical model. Due to their DNA repair deficiency, Atm-deficient mice cannot be irradiated for conditioning prior transplantation. We evaluated different conditioning regimes using cyclophosphamide in combination with anti-CD4/CD8 antibodies, treatment with plerixafor and their combination. With the established in vivo model, our gene therapy strategies in the Atm-/- mouse model can be evaluated in further projects.
Projektbezogene Publikationen (Auswahl)
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Development of gene therapy vectors for Ataxia Telangiectasia (A‐T). Hum Gen Ther, Vol. 33, Issue 23-24, pp.1217- A235, P167
Hirch T., Brander N., Schenk F., Lindemann D., Schubert R. & Modlich U.
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Expression of a large coding sequence: Gene therapy vectors for Ataxia Telangiectasia. Scientific Reports, 13(1).
Hirch, Tanja; Brander, Nadine; Schenk, Franziska; Pöllmann, Simon J.; Reichenbach, Janine; Schubert, Ralf & Modlich, Ute
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Split intein‐based ATM delivery with lentiviral vectors. Hum Gen Ther, Vol 35, Issue 3.4, pp. A1-A336, P224
Hirch T., Fu Y., Schenk F., Schubert R. & Modlich U.
