Journal
NATURE BIOTECHNOLOGY
Volume 24, Issue 1, Pages 89-94Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt1176
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Funding
- NATIONAL CANCER INSTITUTE [P30CA008748, P01CA059350] Funding Source: NIH RePORTER
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R01HL057612] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [Z01DK027011] Funding Source: NIH RePORTER
- NCI NIH HHS [CA08748, CA59350] Funding Source: Medline
- NHLBI NIH HHS [HL57612] Funding Source: Medline
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The application of RNA interference (RNAi) to stem cell-based gene therapies will require highly specific and lineage-restricted gene silencing. Here we show the feasibility and therapeutic potential of coregulating transgene expression and RNAi in hematopoietic stem cells. We encoded promoterless small-hairpin RNA (shRNA) within the intron of a recombinant c-globin gene. Expression of both c-globin and the lariat-embedded small interfering RNA (siRNA) was induced upon erythroid differentiation, specifically downregulating the targeted gene in tissue- and differentiation stage-specific fashion. The position of the shRNA within the intron was critical to concurrently achieve high-level transgene expression, effective siRNA generation and minimal interferon induction. Lentiviral transduction of CD34(+) cells from patients with sickle cell anemia led to erythroid-specific expression of the gamma-globin transgene and concomitant reduction of endogenous beta(S) transcripts, thus providing proof of principle for therapeutic strategies that require synergistic gene addition and gene silencing in stem cell progeny.
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