Lentivirus-mediated gene therapy corrects ribosomal biogenesis and shows promise for Diamond Blackfan anemia

Abstract

This study lays the groundwork for future lentiviral-mediated gene therapy in Diamond Blackfan anemia patients caused by mutations in RPS19, showing evidence that should constitute a new safe and effective therapy.. The data show that, unlike patients with Fanconi anaemia (FA), the hematopoietic stem cell (HSC) reservoir of DBA patients is not significantly reduced; suggesting that collection of these cells should not constitute a remarkable restriction for DBA gene therapySubsequentially, two clinically applicable lentiviral vectors were developed. In the former vector; PGK.CoRPS19 LV, a codon-optimized version of RPS19 was driven by the phosphoglycerate kinase promoter (PGK), already used in different gene therapy trials including FA gene therapy. In the latter one; EF1α.CoRPS19 LV, RPS19 expression was driven by the elongation factor alpha short promoter, EF1α(s). Preclinical experiments showed that transduction of DBA-patient CD34+ cells with the PGK.CoRPS19 LV restored erythroid differentiation, and demonstrated the long-term repopulating properties of corrected DBA CD34+ cells providing evidence of improved erythroid maturation. Concomitantly, long-term restoration of ribosomal biogenesis was confirmed using a novel method applicable to patients' blood cells, based on ribosomal RNA methylation analyses. Finally, in vivo safety studies and proviral insertion sites analyses showed that lentivirus-mediated gene therapy was non-toxic.