Delivery of Gene Writers In Vivo to Hematopoietic Stem Cells and T Cells Using Targeted and Untargeted Lipid Nanoparticles (LNP)
Authors: Monte Michael T. , Tozzi Lorenzo , Palchaudhuri Rahul , Wangweerawong Apiwat , Heshmati Yaser , Schiroli Giulia , Gu Han , Cao Yu , Salomon William E. , Bothmer Anne , Rodriguez Jason , Gupta Mohit , Rottman James B. , Querbes William , Cotta-Ramusino Cecilia , Holmes Michael C , Pujar Hari , Wang Z. Jane 
Published: December 2024

Genomic medicines have the therapeutic potential to cure rare genetic diseases and cancers, but most treatments rely on ex vivo cell editing due to the challenge of delivering genome editing cargos in vivo to the cell types of interest. In vivo delivery of genomic medicines could significantly lower cost, decrease manufacturing hurdles and provide broader patient access, thus potentially unlocking new therapeutic frontiers. In recent years, lipid nanoparticles (LNPs) have emerged as versatile delivery solutions for RNA therapeutics but have seldom been used for in vivo delivery beyond liver and intramuscular vaccines. Challenges of extra-hepatic delivery of LNPs include the biodistribution to organs and cell types of interest and sufficiently high delivery efficacy of cargos to enable therapeutic levels of genome editing. To address these challenges, we have developed novel LNPs designed to deliver RNA Gene Writer payloads to either T cells or hematopoietic stem cells (HSCs) in vivo.
To develop an LNP carrier for gene editing of hemoglobin subunit beta gene (HBB) to treat sickle cell disease, we designed novel ionizable lipids to improve HSC delivery, optimized LNP formulations that de-target the liver, and utilize active targeting moieties to improve specificity. We have screened a library of proprietary ionizable lipids and identified several candidates across multiple lipid families that can deliver reporter payloads to humanized NBSGW mouse bone marrow HSCs. We then optimized our LNP formulations to achieve 11-fold lower liver delivery of GFP reporter mRNA in liver while maintaining high levels of HSC delivery and longer circulation times. Lastly, by tuning our LNP assembly process, we achieved further improvements in GFP delivery efficiency, enabling delivery to 95% of long-term HSCs in humanized NBSGW mice at 1 mpk (LT-HSCs defined as Lin-CD34+CD38-CD90+CD45RA- cells).
While delivery of GFP and other reporter mRNA cargos is useful for delivery optimization, these cargos are not fully representative of true genome editing cargos which are complex, including both large mRNAs and smaller guide RNAs, and require a higher threshold of delivery for activity. To address this gap, we established representative gene writing reagents (delivered as Gene Writer mRNA and template guide RNA) that leverage target-primed reverse transcription (TPRT) as a mechanism to knock out the beta-2 microglobulin (B2M) gene. In this system, our HSC-tropic LNPs achieved an average of 76% B2M rewriting efficiency in LT-HSCs in humanized NBSGW mice in a single dose, as determined by amplicon sequencing. A similar LNP carrier in non-human primates (NHP) also enabled 76% rewriting of the B2M gene in LT-HSCs 30 days after dose. The ability to deliver both reporter and gene editing cargos suggests our LNP carrier is robust and versatile for in vivo HSC delivery.
Utilizing our learnings from LNP optimization for HSC delivery, we also developed T cell-tropic LNPs that can deliver to T cells in vitro and in vivo. We have previously identified LNP formulations that can deliver GFP reporter mRNA to T cells. Here, we disclose additional optimizations that have enabled us to deliver Gene Writers and achieve integration of GFP in the genome of both activated and resting T cells in vitro. Furthermore, our optimized T cell LNP formulation enables in vivo delivery of editing cargos to peripheral blood T cells in an NSG mouse model (implanted with human T cells 14 days prior to study). This combination of in vivo delivery to HSCs and T cells and TPRT-based gene writing has the potential to dramatically impact the significant unmet needs in sickle cell disease and oncology.