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A genetic strategy to treat sickle cell anemia by coregulating globin transgene expression and RNA interference


The application of RNA interference (RNAi) to stem cell–based 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 γ-globin gene. Expression of both γ-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 γ-globin transgene and concomitant reduction of endogenous βS 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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Figure 1: Erythroid-specific RNA interference mediated by G9 RNAi vectors.
Figure 2: Structural analysis of G9-encoded globin transcripts and siRNA.
Figure 3: Specificity and efficacy of sickle β-globin-specific siRNA.
Figure 4: γ-globin transgene expression and concomitant βS interference in sickle cell patient erythroid cells.


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The authors thank Valentina Motta and Hao Ho for excellent technical assistance. This work was supported by the US National Institutes of Health (grants HL57612, CA08748 and CA59350) and the Leonardo Giambrone Foundation and the Associacione per la ricerca Piera Cutino.

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Corresponding author

Correspondence to Michel Sadelain.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Inducible accumulation of transgene expression (PDF 744 kb)

Supplementary Table 1

Sequences of oligonucleotides and PCR primers (PDF 24 kb)

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Samakoglu, S., Lisowski, L., Budak-Alpdogan, T. et al. A genetic strategy to treat sickle cell anemia by coregulating globin transgene expression and RNA interference. Nat Biotechnol 24, 89–94 (2006).

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