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

This study lays the groundwork for future lentivirus-mediated gene therapy in patients with Diamond Blackfan anemia (DBA) caused by mutations in ribosomal protein S19 (RPS19), showing evidence of a new safe and effective therapy. The data show that, unlike patients with Fanconi anemia (FA), the hematopoietic stem cell (HSC) reservoir of patients with DBA was not significantly reduced, suggesting that collection of these cells should not constitute a remarkable restriction for DBA gene therapy. Subsequently, 2 clinically applicable lentiviral vectors were developed. In the former lentiviral 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 verified using a potentially novel method applicable to patients' blood cells, based on ribosomal RNA methylation analyses. Finally, in vivo safety studies and proviral insertion site analyses showed that lentivirus-mediated gene therapy was nontoxic.

Preclinical studies of efficacy thresholds and tolerability of a clinically ready lentiviral vector for pyruvate kinase deficiency treatment.

Pyruvate kinase deficiency (PKD) is a rare autosomal recessive disorder caused by mutations in the PKLR gene. PKD is characterized by non-spherocytic hemolytic anemia of variable severity and may be fatal in some cases during early childhood. Although not considered the standard of care, allogeneic stem cell transplantation has been shown as a potentially curative treatment, limited by donor availability, toxicity, and incomplete engraftment. Preclinical studies were conducted to define conditions to enable consistent therapeutic reversal, which were based on our previous data on lentiviral gene therapy for PKD. Improvement of erythroid parameters was identified by the presence of 20%-30% healthy donor cells. A minimum vector copy number (VCN) of 0.2-0.3 was required to correct PKD when corrected cells were transplanted in a mouse model for PKD. Biodistribution and pharmacokinetics studies, with the aim of conducting a global gene therapy clinical trial for PKD patients (RP-L301-0119), demonstrated that genetically corrected cells do not confer additional side effects. Moreover, a clinically compatible transduction protocol with mobilized peripheral blood CD34+ cells was optimized, thus facilitating the efficient transduction on human cells capable of repopulating the hematopoiesis of immunodeficient mice. We established conditions for a curative lentiviral vector gene therapy protocol for PKD.

TALEN mediated gene editing in a mouse model of Fanconi anemia.

The promising ability to genetically modify hematopoietic stem and progenitor cells by precise gene editing remains challenging due to their sensitivity to in vitro manipulations and poor efficiencies of homologous recombination. This study represents the first evidence of implementing a gene editing strategy in a murine safe harbor locus site that phenotypically corrects primary cells from a mouse model of Fanconi anemia A. By means of the co-delivery of transcription activator-like effector nucleases and a donor therapeutic FANCA template to the Mbs85 locus, we achieved efficient gene targeting (23%) in mFA-A fibroblasts. This resulted in the phenotypic correction of these cells, as revealed by the reduced sensitivity of these cells to mitomycin C. Moreover, robust evidence of targeted integration was observed in murine wild type and FA-A hematopoietic progenitor cells, reaching mean targeted integration values of 21% and 16% respectively, that were associated with the phenotypic correction of these cells. Overall, our results demonstrate the feasibility of implementing a therapeutic targeted integration strategy into the mMbs85 locus, ortholog to the well-validated hAAVS1, constituting the first study of gene editing in mHSC with TALEN, that sets the basis for the use of a new safe harbor locus in mice.