In Vivo HSC Gene Editing for Correction of the Sickle Cell Mutation Using RNA Gene Writers
Authors: Tozzi Lorenzo , Schiroli Giulia , Heshmati Yaser , Cao Yu , Monte Michael T. , Wangweerawong Apiwat , Rottman James B. , Palchaudhuri Rahul , Ribeil Jean-Antoine , Manis John , Pujar Hari , Querbes William , Bothmer Anne , Cotta-Ramusino Cecilia , Wang Z. Jane , Salomon William E. , Holmes Michael C 
Published: December 2024

Tessera Therapeutics is pioneering a new approach to genome engineering through the development of our Gene Writing and delivery platforms. Our RNA Gene Writers, which leverage target-primed reverse transcription, are designed to introduce a broad range of edits to the genome, from writing in a transgene, inserting an exon, to introducing single nucleotide changes. We believe the mechanism of the RNA Gene Writer to precisely write DNA at the single nucleotide level makes it an ideal modality to correct the sickle cell disease (SCD) causing mutation, HBB E6V, in hematopoietic stem cells (HSCs). We previously demonstrated that RNA Gene Writers can introduce any type of single nucleotide change and revert the SCD causing mutation to the wild-type sequence in patient derived CD34+ cells. In addition, we observed our RNA Gene Writers did not trigger a DNA damage response when compared to classic nuclease treatment. Lastly, we demonstrated that primary HSCs edited at the HBB locus did not show functional impairment and achieved long-term engraftment in a secondary transplantation mouse model.

RNA Gene Writers are designed to be delivered as an all-RNA composition, which enables in vivo, non-viral delivery by lipid nanoparticles (LNPs). Our proprietary HSC-targeting LNPs delivered a GFP reporter to over 90% of long-term HSCs (LT-HSCs defined as Lin-CD34+CD38-CD90+CD45RA- cells) in humanized NBSGW mice (hNBSGW) and non-human primates (NHP), through a single intravenous administration. Through systematic optimization of our RNA Gene Writers and LNP delivery carriers, we achieved correct installation of the HBB Makassar variant (E6A) in more than 30% of LT-HSCs with a single dose in NBSGW mice engrafted with healthy donor CD34+ cells. Furthermore, we also demonstrated that, with a second administration of our LNPs, we achieved a near linear increase in the percentage of HBB editing in LT-HSC. To compare rewriting efficiency in hNBSGW mice and NHP, we employed a Gene Writer to knockout expression of the B2M gene, which is highly homologous in human and NHP, with expression easily detected by flow cytometry. In vivo delivery of LNPs containing the B2M template RNA in hNBSGW mice resulted in more than 70% B2M rewriting in hematopoietic stem and progenitor cells, as well as LT-HSCs. In cynomolgus macaque, we systemically administered our LNPs containing the B2M template and collected a bone marrow aspirate to assess activity by amplicon sequencing and flow cytometry. On day 30 after LNPs administration, we observed 76% B2M rewriting in LT-HSCs by amplicon sequencing, and flow cytometry analysis further confirmed that over 60% of LT-HSCs had complete B2M knock out. Altogether these data demonstrate our RNA Gene Writers, in combination with our proprietary LNP delivery platform, have the potential to correct the SCD causing mutation by systemic delivery to HSCs in vivo.