Modulating Nucleotide Metabolism Enhances Prime Editing in Hematopoietic Stem and Progenitor Cells

Therapeutic prime editing (PE) of hematopoietic stem cells (HSCs) holds great potential to correct pathogenic mutations causative of severe blood disorders. While recent advances have improved the technology, efficient prime editing remains challenging in primary hematopoietic cells. Since SAMHD1 has been identified as an antiviral factor restricting the nucleotide pool available for reverse transcription and virus replication, we hypothesized that modulating this metabolic pathway could enhance reverse transcription and thus prime editing in HSCs. We devised strategies to modulate nucleotide metabolism by supplementing deoxynucleosides (dNs) and delivering the viral accessory protein Vpx using virus-like particles (VLPs). Exogenous dNs supplementation and Vpx-mediated proteasomal degradation of SAMHD1 markedly increased PE efficiency in HSPCs from different healthy donors from an average of 14% to 32% editing with the PE2max system, and from 51% to 70% editing with the PE3max system. Supplementing individual dNs or all dNs minus one also boosted prime editing, but all four dNs were needed for maximal enhancement of PE from an average of 41% to 64% editing at ATP1A1. Using a prime editing guide RNA designed to correct the HBB-E6V mutation causative of sickle cell disease, we achieved an average of 58% and 44% editing with the PE3max and PE3bmax systems, respectively. Modulation of nucleotide metabolism also ameliorated prime editing in both resting and activated CD3 ⁺ T cells, although to a lesser extent than in HSPCs. Of relevance, our strategies had little to no impact on PE in K562 and Jurkat cells, suggesting that nucleotide metabolism does not restrict PE in cancer cell lines. Altogether, modulation of nucleotide metabolism should enhance prime editing to facilitate the clinical translation of gene therapies for severe blood disorders. This work highlights a key metabolic restriction for therapeutic prime editing of primary cells and could help design future approaches to engineer quiescent HSCs in vivo.