Engineering Targeted Delivery Vehicles for Genome Editing in Hematopoietic Stem Cells

About This Grant

PROJECT SUMMARY/ABSTRACT The advent of genome editing provides the opportunity to treat genetic diseases at their root cause. However, clinical translation remains limited by the challenge of delivering genome editors efficiently and specifically to target cells in vivo. Delivery vehicles are needed to protect genome editors, engage cell-surface receptors and release active enzymes into target cells. Inspired by viral tropism, we developed Enveloped Delivery Vehicles (EDVs), lentivirally derived lipid vesicles that encapsulate CRISPR–Cas9 ribonucleoproteins and display fusogens or engineered antibody fragments on their surface. EDVs enable receptor targeting, but how antibody fragment density and binding affinity control uptake, biodistribution and genome editing remain poorly defined. Building on our laboratory’s expertise with EDVs and CRISPR-Cas9 mechanistic biology, our goal is to define how antibody fragment presentation on EDVs influences their ability to target hematopoietic stem cells (HSCs), a clinically important cell population for treating blood and immune disorders. HSCs are a compelling target for blood cancers, because myeloid malignancies such as acute myeloid leukemia and myelodysplastic syndromes originate from malignant HSCs. Our central hypothesis is that an optimal range of ligand density and affinity (i.e., avidity) maximizes selective uptake and genome editing in HSCs while minimizing off-target uptake by bystander cells. We will test this hypothesis through two aims: 1) Quantify how EDV avidity affects uptake and genome editing in human HSCs ex vivo. 2) Quantify how EDV avidity affects biodistribution and genome editing specificity in humanized mouse models. Mouse models are indispensable for delivery vehicle development because invertebrate, in vitro, organoid, and computational systems cannot recapitulate the immune, spleen, liver, and bone marrow environments or the biophysics of blood flow and tissue perfusion that govern the biodistribution and elimination of delivery vehicles. Unlike prior approaches that focused solely on maximizing antibody affinity or surface presentation, this proposal systematically dissects how density and affinity interact to determine avidity, uptake, and editing efficiency. Completion of this project will produce new targeted delivery vehicles for HSC editing and establish quantitative rules for tuning delivery vehicle avidity to maximize potency and specificity. These results will be broadly applicable to other delivery platforms and accelerate the development of safe and effective in vivo genome editing therapies.