In utero delivery of targeted ionizable lipid nanoparticles facilitates in vivo gene editing of hematopoietic stem cells.

Monogenic blood diseases are among the most common genetic disorders worldwide. These diseases result in significant pediatric and adult morbidity, and some can result in death prior to birth. Novel ex vivo hematopoietic stem cell (HSC) gene editing therapies hold tremendous promise to alter the therapeutic landscape but are not without potential limitations. In vivo gene editing therapies offer a potentially safer and more accessible treatment for these diseases but are hindered by a lack of delivery vectors targeting HSCs, which reside in the difficult-to-access bone marrow niche. Here, we propose that this biological barrier can be overcome by taking advantage of HSC residence in the easily accessible liver during fetal development. To facilitate the delivery of gene editing cargo to fetal HSCs, we developed an ionizable lipid nanoparticle (LNP) platform targeting the CD45 receptor on the surface of HSCs. After validating that targeted LNPs improved messenger ribonucleic acid (mRNA) delivery to hematopoietic lineage cells via a CD45-specific mechanism in vitro, we demonstrated that this platform mediated safe, potent, and long-term gene modulation of HSCs in vivo in multiple mouse models. We further optimized this LNP platform in vitro to encapsulate and deliver CRISPR-based nucleic acid cargos. Finally, we showed that optimized and targeted LNPs enhanced gene editing at a proof-of-concept locus in fetal HSCs after a single in utero intravenous injection. By targeting HSCs in vivo during fetal development, our Systematically optimized Targeted Editing Machinery (STEM) LNPs may provide a translatable strategy to treat monogenic blood diseases before birth.

Predictive High-Throughput Platform for Dual Screening of mRNA Lipid Nanoparticle Blood-Brain Barrier Transfection and Crossing.

Lipid nanoparticle (LNP)-mediated nucleic acid therapies, including mRNA protein replacement and gene editing therapies, hold great potential in treating neurological disorders including neurodegeneration, brain cancer, and stroke. However, delivering LNPs across the blood-brain barrier (BBB) after systemic administration remains underexplored. In this work, we engineered a high-throughput screening transwell platform for the BBB (HTS-BBB), specifically optimized for screening mRNA LNPs. Unlike most transwell assays, which only assess transport across an endothelial monolayer, HTS-BBB simultaneously measures LNP transport and mRNA transfection of the endothelial cells themselves. We then use HTS-BBB to screen a library of 14 LNPs made with structurally diverse ionizable lipids and demonstrate it is predictive of in vivo performance by validating lead candidates for mRNA delivery to the mouse brain after intravenous injection. Going forward, this platform could be used to screen large libraries of brain-targeted LNPs for a range of protein replacement and gene editing applications.

Ionizable Lipid Nanoparticles for In Vivo mRNA Delivery to the Placenta during Pregnancy.

Ionizable lipid nanoparticles (LNPs) are the most clinically advanced nonviral platform for mRNA delivery. While they have been explored for applications including vaccines and gene editing, LNPs have not been investigated for placental insufficiency during pregnancy. Placental insufficiency is caused by inadequate blood flow in the placenta, which results in increased maternal blood pressure and restricted fetal growth. Therefore, improving vasodilation in the placenta can benefit both maternal and fetal health. Here, we engineered ionizable LNPs for mRNA delivery to the placenta with applications in mediating placental vasodilation. We designed a library of ionizable lipids to formulate LNPs for mRNA delivery to placental cells and identified a lead LNP that enables in vivo mRNA delivery to trophoblasts, endothelial cells, and immune cells in the placenta. Delivery of this top LNP formulation encapsulated with VEGF-A mRNA engendered placental vasodilation, demonstrating the potential of mRNA LNPs for protein replacement therapy during pregnancy to treat placental disorders.

Influence of ionizable lipid tail length on lipid nanoparticle delivery of mRNA of varying length.

RNA-based therapeutics have gained traction for the prevention and treatment of a variety of diseases. However, their fragility and immunogenicity necessitate a drug carrier. Lipid nanoparticles (LNPs) have emerged as the predominant delivery vehicle for RNA therapeutics. An important component of LNPs is the ionizable lipid (IL), which is protonated in the acidic environment of the endosome, prompting cargo release into the cytosol. Currently, there is growing evidence that the structure of IL lipid tails significantly impacts the efficacy of LNP-mediated mRNA translation. Here, we optimized IL tail length for LNP-mediated delivery of three different mRNA cargos. Using C12-200, a gold standard IL, as a model, we designed a library of ILs with varying tail lengths and evaluated their potency in vivo. We demonstrated that small changes in lipophilicity can drastically increase or decrease mRNA translation. We identified that LNPs formulated with firefly luciferase mRNA (1929 base pairs) and C10-200, an IL with shorter tail lengths than C12-200, enhance liver transfection by over 10-fold. Furthermore, different IL tail lengths were found to be ideal for transfection of LNPs encapsulating mRNA cargos of varying sizes. LNPs formulated with erythropoietin (EPO), responsible for stimulating red blood cell production, mRNA (858 base pairs), and the C13-200 IL led to EPO translation at levels similar to the C12-200 LNP. The LNPs formulated with Cas9 mRNA (4521 base pairs) and the C9-200 IL induced over three times the quantity of indels compared with the C12-200 LNP. Our findings suggest that shorter IL tails may lead to higher transfection of LNPs encapsulating larger mRNAs, and that longer IL tails may be more efficacious for delivering smaller mRNA cargos. We envision that the results of this project can be utilized as future design criteria for the next generation of LNP delivery systems for RNA therapeutics.

Optimized microfluidic formulation and organic excipients for improved lipid nanoparticle mediated genome editing.

mRNA-based gene editing platforms have tremendous promise in the treatment of genetic diseases. However, for this potential to be realized in vivo, these nucleic acid cargos must be delivered safely and effectively to cells of interest. Ionizable lipid nanoparticles (LNPs), the most clinically advanced non-viral RNA delivery system, have been well-studied for the delivery of mRNA but have not been systematically optimized for the delivery of mRNA-based CRISPR-Cas9 platforms. In this study, we investigated the effect of microfluidic and lipid excipient parameters on LNP gene editing efficacy. Through in vitro screening in liver cells, we discovered distinct trends in delivery based on phospholipid, cholesterol, and lipid-PEG structure in LNP formulations. Combination of top-performing lipid excipients produced an LNP formulation that resulted in 3-fold greater gene editing in vitro and facilitated 3-fold greater reduction of a therapeutically-relevant protein in vivo relative to the unoptimized LNP formulation. Thus, systematic optimization of LNP formulation parameters revealed a novel LNP formulation that has strong potential for delivery of gene editors to the liver to treat metabolic disease.