2023 Nov 8;13(1):19386.
 doi: 10.1038/s41598-023-46332-4.

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

Affiliations 

Affiliations

  • 1Division of Veterinary Medicine, RG Gene Modification in Stem Cells, Paul-Ehrlich-Institute, Langen, Germany.
  • 2Department of Gene and Cell Therapy, Institute for Regenerative Medicine - IREM, University of Zurich, Schlieren, Switzerland.
  • 3Deptartment of Somatic Gene Therapy, University Children's Hospital Zurich, Zurich, Switzerland.
  • 4Competence Center for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Zurich, Switzerland.
  • 5Department for Children and Adolescents, University Hospital Frankfurt, Goethe University, Frankfurt, Germany.
  • 6Division of Veterinary Medicine, RG Gene Modification in Stem Cells, Paul-Ehrlich-Institute, Langen, Germany. ute.modlich@irem.uzh.ch.
  • 7Department of Gene and Cell Therapy, Institute for Regenerative Medicine - IREM, University of Zurich, Schlieren, Switzerland. ute.modlich@irem.uzh.ch.
    • PMID: 37938627
    Free PMC article

Abstract

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).