In Vivo correction of the sickle cell disease mutation in hematopoietic stem cells using RNA gene writers

Abstract Tessera Therapeutics is pioneering a novel approach to in vivo genome editing that uses RNA Gene Writers and delivery of all RNA-based components to introduce precise edits into the genome of cells in vivo by target-primed reverse transcription. Our RNA Gene Writers have been engineered to enable a wide range of genomic modifications, from transgene insertion to single nucleotide changes, offering a highly versatile platform for the treatment of inherited and acquired diseases. In this study, we applied RNA Gene Writers to correct the HBB E6V mutation, the genetic cause of sickle cell disease (SCD), in hematopoietic stem cells (HSCs), with the aim of achieving therapeutically relevant levels of gene correction in vivo. We report that intravenous administration of the RNA-based Gene Writer, delivered via targeted lipid nanoparticles (LNPs), achieved therapeutic levels of HBB editing in HSCs in both SCD patient-derived mouse models and non-human primates (NHPs). Our approach would eliminate the need for HSC mobilization, intensive myeloablative conditioning, and ex vivo HSC manipulation, offering a potentially curative treatment that is safer, simpler, and more scalable for patients with SCD. We first assessed the efficiency and impact of our RNA Gene Writers to edit the HBB locus in HSCs by introducing the HBB Makassar variant (E6A) into ex vivo cultured wild-type human CD34+ cells and we achieved an average editing efficiency of 74%. Edited cells transplanted into immunodeficient mice demonstrated robust engraftment and stable maintenance of approximately 70% HBB editing in long-term HSCs (LT-HSCs) and multi-lineage progeny at 16 weeks post-transplantation. Similar editing levels were sustained following secondary transplantation, confirming the successful and durable editing of HSCs. We then evaluated RNA Gene Writers designed to make the wild-type correction in cultured HSCs derived from SCD donors and observed ~70% correction. Upon in vitro erythroid differentiation, these cells exhibited a 98% restoration of adult hemoglobin (HbA) levels, as measured by Liquid Chromatography-Mass Spectrometry (LC-MS) and reduced sickling under hypoxic conditions. To edit HSCs in vivo, we developed HSC-targeted LNPs that we have previously shown could deliver GFP mRNA to ~95% of LT-HSCs (Lin-CD34+CD38-CD90+CD45RA-) in both humanized mice and non-human primates (NHPs) following intravenous administration. Using humanized NBSGW mice engrafted with human CD34+ cells, we first demonstrated in vivo installation of the HBB Makassar variant in 62% of LT-HSCs. We repeated the experiment with HSCs from multiple donors and consistently observed editing efficiencies of > 50% across all samples. Comparable editing levels were maintained in LT-HSCs and their multi-lineage progeny after transplantation of in vivo edited CD34+ cells into secondary recipients, providing additional evidence of successful in vivo targeting of LT-HSCs and preservation of stem cell function. Importantly, using this same approach in cynomolgus NHPs, we achieved an average of 24% in vivo HBB Makassar editing in LT-HSCs by day 14 post-treatment. Editing levels remained stable in LT-HSCs for up to nine months, with edited cells detected across multiple hematopoietic lineages above 20%. Longitudinal tracking of edited cells revealed early and sustained multi-lineage output, with stable contributions observed as early as two months post-treatment, providing key insights into the kinetics of hematopoietic reconstitution following in vivo editing. Collectively, these results demonstrate the robustness of our Gene Writer and HSC delivery platforms to target and durably edit multiple loci in LT-HSCs in vivo in both murine and NHP models, supporting their therapeutic potential in the treatment of SCD and other hematologic disorders.