Genetic Correction of Sickle Cell Disease by Co-Regulated γ-Globin Transgene Expression and ßS Interference.
Authors: Samakoglu Selda , Usachenko Yelena , Budak-Alpdogan Tulin , Acuto Santina , DiMarzo Rosalba , Maggio Aurelio , Tisdale John , Riviere Isabelle , Sadelain Michel 
Published: April 2020

Abstract
RNA interference (RNAi) is a promising therapeutic strategy, but its application to stem cell-based gene therapy for the treatment of congenital or acquired disorders will require highly specific gene silencing. To ensure co-expression of a therapeutic transgene and a small interfering RNA (siRNA), we hypothesized that a promoter-less small hairpin RNA (shRNA) embedded within an intron could yield siRNA in tissue-specific fashion and thus achieve regulated RNAi. We demonstrate here that γ-globin expression and erythroid-specific siRNA generation can be achieved in mammalian cells, including human CD34+ cells. The shRNA was encoded under the transcriptional control of the human β-globin promoter, a prototypic tissue-specific Pol II promoter, and positioned at two different sites in the second intron or in the 5′-UTR of a recombinant human γ-globin gene. Three different genes were targeted in mouse erythroleukemia (MEL) cells, green fluorescent protein (EGFP), human sickle β-globin (β S) and endogenous mouse β-gobin. When cloned immediately upstream of the branch point, the siRNA was efficiently generated without altering γ-globin mRNA expression and processing, suggesting that hairpin positioning near the branch point is not detrimental to RNA splicing. When cloned near the 5′-end of the intron, the siRNA was structurally impaired, and the γ-globin mRNA levels greatly diminished. This strong effect of shRNA positioning is consistent with a quality control pathway of gene transcription, whereby introns harboring dsRNA stem loops are degraded if splicing is altered. The strong induction of interferon type I genes associated with the latter position but not the former correlated with the formation of small shRNA degradation products. Positioning of the shRNA in the 5′-UTR did not induce major interferon responses but severely compromised γ-globin expression. To further validate these findings in a clinically relevant model, we engineered an RNAi lentiviral vector in which the human sickle β-globin specific (β S) siRNA is embedded the second intron of a recombinant γ-globin gene. Following transduction of CD34+ cells from patients with sickle cell disease, γ-globin transgene expression was induced upon erythroid differentiation concomitant with a dramatic decrease of the β S transcripts. These findings fully support the principle of synergistic gene delivery and lariat-encoded RNAi in human CD34+ cells, demonstrating the feasibility of using lariat-embedded siRNA to potentiate globin gene transfer by reducing competition from endogenous β S globin chains. Importantly, a moderate decrease in β S expression may substantially improve SCD and abrogate the need for high level expression of the vector-encoded globin gene. This approach to regulate RNAi may find broad applicability in a wide range of disorders where the concomitant expression of a transgene and RNAi will enhance treatment safety and/or efficacy.