Lentivirus-mediated gene therapy corrects ribosomal biogenesis and shows promise for Diamond Blackfan anemia
Yari Giménez, Manuel Palacios, Rebeca Sánchez-Domínguez, Christiane Zorbas, Jorge Peral, Alexander Puzik, Laura Ugalde, Omaira Alberquilla, Mariela Villanueva, Paula Río, Eva Gálvez, Lydie Da Costa, Marion Strullu, Albert Catala, Anna Ruiz-Llobet, Jose Carlos Segovia, Julián Sevilla, Brigitte Strahm, Charlotte M. Niemeyer, Cristina Beléndez, Thierry Leblanc, Denis L.J. Lafontaine, Juan Bueren, Susana Navarro
Yari Giménez, Manuel Palacios, Rebeca Sánchez-Domínguez, Christiane Zorbas, Jorge Peral, Alexander Puzik, Laura Ugalde, Omaira Alberquilla, Mariela Villanueva, Paula Río, Eva Gálvez, Lydie Da Costa, Marion Strullu, Albert Catala, Anna Ruiz-Llobet, Jose Carlos Segovia, Julián Sevilla, Brigitte Strahm, Charlotte M. Niemeyer, Cristina Beléndez, Thierry Leblanc, Denis L.J. Lafontaine, Juan Bueren, Susana Navarro
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Research Article Hematology Therapeutics

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

Abstract

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.

Authors

Yari Giménez, Manuel Palacios, Rebeca Sánchez-Domínguez, Christiane Zorbas, Jorge Peral, Alexander Puzik, Laura Ugalde, Omaira Alberquilla, Mariela Villanueva, Paula Río, Eva Gálvez, Lydie Da Costa, Marion Strullu, Albert Catala, Anna Ruiz-Llobet, Jose Carlos Segovia, Julián Sevilla, Brigitte Strahm, Charlotte M. Niemeyer, Cristina Beléndez, Thierry Leblanc, Denis L.J. Lafontaine, Juan Bueren, Susana Navarro

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Figure 5

Safety studies mediated by the overexpression of RPS19 in cord blood CD34+ cells from HDs transduced with the therapeutic PGK.CoRPS19 LV.

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Safety studies mediated by the overexpression of RPS19 in cord blood CD3...
Graphs refer to HD cord blood (CB) CD34+ cells transduced with therapeutic vector PGK.CoRPS19 LV or the control vector PGK.EGFP LV. (A) Growth curves of HD HSPC progenitors maintained in HSC expansion medium, after transduction. The graph shows the mean and standard deviation of 5 experiments. Mann-Whitney test. x axis, days. (B and C) Healthy donor umbilical cord CD34+ cells transduced with PGK.CoRPS19 LV or PGK.EGFP LV (control). (B) Number of granulocyte-macrophage progenitor colony-forming units (CFU-GMs) per 105 mononuclear cells seeded. (C) The number of BFU-Es per 105 mononuclear cells seeded. The degree of significance was determined with the Mann-Whitney test. (D) Body weight of NSG mice transplanted with HD CD34+ cells transduced with the therapeutic vector PGK.CoRPS19 LV or the control vector PGK.EGFP LV. (E) The repopulation potential was determined by quantifying the percentage of hCD45+ cells in NSG recipient mice up to day 120. (F) VCN in human HSPC engrafted into NSG recipient mice. The graph shows the mean and standard error of the mean of 2 different experiments after transducing healthy donor CD34+ cells from CB and transplanting them into NSG mice. Statistical significance was determined with the Mann-Whitney test (P value; * ≤ 0.05). (G) Distribution of myeloid cells (CD33+), lymphoid cells (CD19+), and HSCs (CD34+) in NSG recipients. (H) Determination of hematological parameters in NSG mice transplanted: erythrocytes, leukocytes, platelets, and neutrophils. The graph shows the mean and standard error of the mean of 2 different experiments conducted to analyze safety and in vivo toxicity effects of the therapeutic vectors after transfecting HD CD34+ cells from CB and transplanting them into NSG mice. Statistical significance was determined with the 2-tailed Mann-Whitney test. x axis, days.