Nearly 50% of mouse and human genomes are composed of repetitive sequences. Transcription of these sequences is tightly controlled during development to prevent genomic instability, inappropriate gene activation and other maladaptive processes. Here, we demonstrate an integral role for H1 linker histones in silencing repetitive elements in mouse embryonic stem cells. Strong H1 depletion causes a profound de-repression of several classes of repetitive sequences, including major satellite, LINE-1, and ERV. Activation of repetitive sequence transcription is accompanied by decreased H3K9 trimethylation of repetitive sequence chromatin. H1 linker histones interact directly with Suv39h1, Suv39h2, and SETDB1, the histone methyltransferases responsible for H3K9 trimethylation of chromatin within these regions, and stimulate their activity toward chromatin in vitro. However, we also implicate chromatin compaction mediated by H1 as an additional, dominant repressive mechanism for silencing of repetitive major satellite sequences. Our findings elucidate two distinct, H1-mediated pathways for silencing heterochromatin.

Through its ability to bind the ends of poly(ADP-ribose) (PAR) chains, the function of the histone variant macroH2A1.1, including its ability to regulate transcription, is coupled to PAR polymerases (PARPs). PARP1 also has a major role in DNA damage response (DDR) signaling, and our results show that macroH2A1 alters the kinetics of PAR accumulation following acute DNA damage by both suppressing PARP activity and simultaneously protecting PAR chains from degradation.

Using adenine base editors, we aim to treat sickle cell disease by generating single nucleotide polymorphisms in human CD34+ hematopoietic stem and progenitor cells (HSPCs) at specific target sites by mediating A-T to G-C base conversions. While ex vivo gene editing approaches show great therapeutic promise, access is limited due to the requirement of an autologous hematopoietic stem cell (HSC) transplant to deliver the ex vivo edited cells. To further increase the number of patients eligible for base editing therapy, we are developing an alternative approach to directly deliver base editors to HSCs in vivo through non-viral delivery methods. Lipid Nanoparticles (LNPs) are a clinically validated, non-viral approach that enables the delivery of nucleic acid payloads, which may avoid the challenges associated with ex vivo approaches including the transplantation of edited CD34+ HSPCs. Here we describe the development and characterization of LNPs for the delivery of messenger RNA (mRNA) to HSPCs in vivo in both mice and cynomolgus macaques. By screening >1,000 chemically distinct LNPs in vivo utilizing a DNA barcoding technology, we identified several hit LNPs capable of biodistribution to HSPCs. Upon individual validation of these hit LNPs by delivery of Cre recombinase mRNA in a Cre-reporter mouse model (Ai14), which expresses the fluorescent protein tdTomato under a constitutive CAG promoter following Cre-meditate gene editing, we confirmed that several LNPs efficiently delivered Cre recombinase mRNA to mouse Lin-Sca-1+c-Kit+ (LSK) HSPCs. We next confirmed the most potent hit LNP (LNP-HSC1) identified from the in vivo screen to transfect LSK HSPCs in a dose-dependent manner between 0.1 and 1.0 mg/kg Cre recombinase mRNA, transfecting over 40% of LSK HSPCs in Ai14 mice at 1.0mg/kg. In a transfection durability study using Ai14 mice, we observed maintenance of tdTomato+ LSK HSPCs levels in the bone marrow at 10 weeks post-LNP delivery. As LNP-HSC1 had been identified and validated in mice of a C57BL6/j background, we next confirmed its ability to transfect a reporter mRNA into HSPCs in Balb/c mice and in 5 cynomolgus macaques. LNP-HSC1 efficiently transfected LSK HSPCs in Balb/c mice at doses ranging from 0.3 to 1.0 mg/kg. In 5 cynomolgus macaques (n=5 across two experiments), we observed a dose-dependent increase in reporter mRNA delivery with an average of 19% of bone marrow-derived CD34+ HSPCs (n=3) expressing the reporter protein at the highest dose tested. Taken together, these data demonstrate the value of our in vivo high-throughput LNP screening approach to identify novel LNPs capable of delivering to HSPCs, providing a promising delivery platform for an in vivo HSC gene editing approach for the treatment of hemoglobinopathies. Disclosures Sago: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Campbell: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lutz: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Patwardhan: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hamilton: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wong: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Keating: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Murray: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Singh: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ciaramella: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

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