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Gene therapy of DBA
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Figure 8. Impaired erythroid differentiation of RPS19-deficient CD34+ cord blood cells can be rescued by the EFS-RPS19 vector. (A) RPS19 mRNA expression in CD34+ cord blood cells transduced with shRNA. (B) Percentage of GFPhigh population in RPS19-deficient CD34+ cord blood cells treated or not with EFS-RPS19 during erythroid differentiation from stage I to stage III. (C) Fluorescnce activated cell sorting analysis of erythroid differentiation of RPS19-deficient cells treated or not with EFS-RPS19 on day 16. (D) Percentage of indicated cell outputs of GFPhigh populations on day 16. (E) Red blood cell pellets at the end of stage III initiated with equal numbers of CD34+ cord blood cells (data shown as mean ± standard deviation, ^P<0.05 compared to the shRNA1 group, #P<0.05 compared to the shRNA2 group, *P<0.05, **P<0.01, ***P<0.005 by a t-test, 3 independent experiments).
emergence of resident hematopoietic stem cells derived from the recipients, which contribute to the reconstitution of hematopoiesis as a protective mechanism against stress-induced exhaustion in the BM.41-43 In support of this concept, recipient-derived hematopoietic stem cells were observed in BM of the mock group at 16 weeks after trans- plantation, even though full lethal irradiation was per- formed prior to transplantation. Other unknown reasons may also contribute to this observation and the underlying mechanism is unknown.
As mentioned before, the majority of DBA patients have mutations in genes coding for ribosomal proteins, and 25% of them are RPS19-deficient (mostly because of point mutations or small deletions). Our findings indicate the possibility of developing SIN lentiviral vectors also tai- lored for other DBA mutations (e.g., RPL5) in the future.
In conclusion, our data show the safety and efficacy of a clinically applicable SIN lentiviral vector for the success- ful treatment of Rps19-deficient DBA in our mouse model and in human primary CD34+ cord blood cells. We did not observe any hematologic abnormalities in vivo due to enforced expression of RPS19. Our present study suggests
that the clinically applicable SIN lentiviral vector, EFS- RPS19, has the potential to be employed in a clinical gene therapy strategy for RPS19-deficient DBA patients.
Disclosures
No conflicts of interest to disclose.
Contributions
SK and YL conceived the project and directed the research; YL, MD, MR, ES, THMG, SW, and JC performed the exper- iments; YL, MR, AS, and SK analyzed the data; and YL and SK wrote the manuscript. Other co-authors provided feedback on the manuscript.
Acknowledgments
The authors thank Beata Lindqvist and Xiaojie Xian for lentivirus production, Zhi Ma for technical assistance, and Alexander Doyle for English language editing.
Funding
This work was supported by a Hemato-Linne grant from the Swedish Research Council Linnaeus, project grants from the
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