P1358: mining the genome for erythroid specific enhancers to optimize gene therapy vectors for beta-hemoglobinopathies

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: The evolution of gene therapy has led to the development of innovative therapeutic strategies for beta-hemoglobinopathies, including the gene addition of beta- or gamma-globin gene or the induction of HbF reactivation. Notwithstanding the promising results emerged from the lentiviral vector gene therapy using autologous hematopoietic stem cells(HSCs), some limitations still exist. Incorporation of the long in size β-LCR(~3 kb), to enhance transgene expression results in low viral titers and reduced transducibility of HSCs, thus negatively affecting efficiency and the overall cost. Aims: The main scope of the present study was to explore our previously published high-resolution temporal atlas of developmentally responsive DNase I hypersensitive sites during human adult erythropoiesis, to identify novel strong, compact, erythroid specific enhancers able to challenge the canonical enhancer of beta-hemoglobinopathy-vectors, β-LCR. Methods: We performed a FACS-based MPRA assay to interrogate the enhancer ability of a library of 15,000 candidate genomic sequences. Among these sequences ~6000 were shown to bare enhancer activity. A subset of these elements was then tested individually and the new enhancers displayed in vitro and in vivo erythroid specificity coupled with a broad range of temporal activity. Two of these enhancers were selected and cloned into a BCL11A-shRNA lentiviral vector to be compared with the micro β-LCR. The new vectors’ efficiency was evaluated in an erythroid cell line, in healthy, beta-thalassemia major- and sickle cell disease- derived CD34+ cells in vitro and in vivo. Results: The novel enhancers yielded a 2-5 fold increase in viral titers during production compared to the uLCR respective vector and exhibited elevated transduction levels in an immortalized erythroid progenitor cell line. Within the transduced populations γ-globin expression was equally increased in all groups compared to untransduced cells(p<0.004 in all vs untransduced). Following transduction with NE-shRNA or uLCR-shRNA vector, CD34+ cells from 5 thalassemic patients were cultured under erythroid differentiation conditions. At the end of the culture we observed a significantly increase of HbF+ cells (%) within the enucleated cells population, with the difference being higher in the case of the NE transduced cells (p=0.009 NE-shRNA and p=0.02 μLCR-shRNA vs untransduced cells). Additionally, we observed increased gamma globin production (α/γ-globin:p=0.024 and p=0.36, μLCR and NE vs untransduced) that resulted in an almost complete restoration of the imbalance between alpha and beta-like globins (beta-like/a-globin:p=0.036 NE-shRNA vs p=0.024 μLCR-shRNA). The elevated HbF levels reduced the oxidative stress in both transduced groups and improved both cell expansion and erythroid differentiation and maturation. Similar results were observed after transduction and erythroid differentiation of non mobilized SCD-derived HSPCs. Specifically, increased gamma globin chain production and reduction of βς -globin to a-globin ratio was observed only in the case of NE-shRNA transduced cells. Summary/Conclusion: Collectively, our data provide evidence that the replacement of μLCR by a novel, compact erythroid enhancer, is feasible and successful, leading to at least equally efficient transgene expression and HbF induction in both thalassemic and SCD erythroid cells. Overall, the new viral vectors are able to address the current issues of low viral titers, low HSC transducibility, in vivo performance and effect in cells from patients still seeking a robust and radical therapy. Keywords: Hematopoietic stem cell, Gene therapy, beta thalassemia