PROJECT SUMMARY Despite advances in the medical care of sickle cell disease (SCD), most patients continue to experience severe pain, poor quality of life, progressive organ deterioration and premature death. Allogeneic hematopoietic stem cell transplantation (HSCT) can cure SCD but is associated with numerous toxicities and only 20% of patients have Human Leukocyte Antigen (HLA)-matched donors. Therefore, improved and more widely accessible curative therapies are needed. Genetic modification of autologous HSCs is a promising experimental approach for treating SCD that circumvents some of the problems associated with allogeneic HSCT, although the optimal technical strategies are not yet established. This proposal explores the use of adenosine base editors (ABEs) and prime editors (PEs) for genetic correction of SCD. In contrast to conventional genome editing, these novel approaches create precise nucleotide alterations independent of double-stranded DNA breaks (DSBs), which can cause structural DNA abnormalities, cell death or malignant transformation. Adenosine base editors convert targeted A·T base pairs to G·C pairs. Prime editors copy edited sequence information from a guide RNA template into a targeted DNA locus. We will test these potentially transformative tools in 3 different strategies for SCD therapy. Aim 1 employs ABEs to create HSC alterations that recapitulate hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition that alleviates the pathophysiology of co-inherited SCD by inducing the expression of red blood cell (RBC) fetal hemoglobin (HbF), a potent anti-sickling agent. We have used protein evolution strategies to create new high-efficiency ABEs that generate HPFH mutations at frequencies of up to 60% in CD34+ hematopoietic stem and progenitor cells (HSPCs), with HbF being induced to levels that inhibit hypoxic sickling of erythroid progeny. Aim 2 uses ABEs to convert the mutant SCD codon from valine to alanine, thereby generating “Hemoglobin Makassar (HbG)”, a naturally occurring benign non- sickling variant. We have developed an altered PAM-specific ABE that converts HbS alleles to HbG in SCD donor HSPCs at frequencies of up to 80%, with inhibition of RBC sickling. Aim 3 employs prime editing to revert the mutant SCD codon to normal (Val→Glu), which we have shown to occur efficiently in the HEK293T cell line and now aim to optimize in HSPCs from affected individuals. Overall, our preliminary studies have shown proof of principle for three novel, independent editing approaches to treating SCD without the need to enrich for edited cells or to create DSBs. Through the proposed research, we seek to optimize the efficiency of these approaches in primary HSPCs and to further determine their safety and efficacy by using mouse models, in vitro culture methods and biochemical assays. Developing three approaches simultaneously will enable us to compare their outcomes directly and to determine the best therapeutic strategy to pursue in future clinical studies. More generally, our planned studies have the potential to generate new paradigms for using base editors and PEs to treat numerous genetic blood disorders via precise genetic manipulation of HSCs.

NARRATIVE This application investigates new strategies to treat sickle cell disease using two potentially transformative technologies for precision genetic manipulation, termed base editing and prime editing. Specifically, we will use these new tools to modify the genome of blood forming cells from affected individuals to either directly repair the sickle cell disease mutation or circumvent its damaging effects. If successful, our strategies will lead to a safe and effective cure for a devastating disorder that affects 100,000 Americans and millions worldwide.