Tuesday, May 13, 2025
5/13/2025
Self-Assembling Peptide Nanoparticles for in vivo Genome Editor Delivery to Hematopoietic Stem Cells - Project Summary Sickle cell disease and transfusion-dependent ?-thalassemia are the most common monogenic diseases worldwide, affecting ~400,000 newborns per year globally and shortening life expectancy by decades. Ongoing clinical trials have established the potential of genome editing as a curative strategy for these disorders. While promising, genome editing of hematopoietic stem and progenitor cells (HSPCs) currently requires resource-intensive ex vivo processes, limiting equitable access to a potentially curative intervention. Vectors for in vivo delivery of genome editors to HSPCs could circumvent this issue, but reported HSPC delivery systems are limited by vector immunogenicity, low editing efficiencies, and a lack of cell targeting specificity. As a self-assembling, human-derived protein polymer amenable to facile incorporation of targeting domains, elastin-like polypeptides (ELPs) have been established for nanoparticle-mediated drug delivery; however, an approach for ELP-mediated genome editor delivery has not been defined. The objective of the proposed work is to generate ELP nanoparticles for the delivery of mRNA-encoded genome editors to hematopoietic stem cells in vivo. In Aim 1, ELPs domain will be optimized for delivery of Cas9 mRNA and single guide RNA (sgRNA) targeting housekeeping gene HPRT1. A library of ELPs will be screened to identify an optimal domain design for cargo complexation, release, and intracellular delivery in cell lines. In Aim 2, HSPC specific monoclonal antibodies (mAbs) that enable in vitro genome editor delivery to HSPCs will be identified. Antibodies against markers enriched on murine HSPCs will be conjugated to ELP nanoparticles with varying valency to promote HSPC specific uptake. Cell-specific delivery of Cas9 will be evaluated in HSPCs isolated from Ai9-SauSpyCas9 mice, which enable fluorescent detection of editing activity. In Aim 3, the utility of mAb-labeled ELP nanoparticles for genome editor delivery to HSPCs in vivo will be determined. The biodistribution and HSPC tropism of mAb-labeled ELP nanoparticles in wild type mice will be evaluated by conducting pooled screens of nanoparticles loaded with unique DNA barcodes. The lead nanoparticle formulation identified from biodistribution studies will be assessed for Cas9 delivery efficiency in Ai9-SauSpyCas9 mice. The effects of mobilizing HSPCs to the peripheral blood on both nanoparticle biodistribution and HSPC editing efficiency will be defined. These studies will establish a non-viral delivery vector for in vivo genome editing of HSPCs, enabling the treatment of a wide variety of inherited hematologic disorders. By defining critical physicochemical principles that govern nucleic acid complexation and delivery by ELP nanoparticles, this work will also form a foundation for establishing ELPs as a platform for nucleic acid delivery to other cells and tissues.
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