Project Summary CRISPR/Cas-based gene editing has ushered in a hopeful era that dreams of new therapies for currently untreatable genetic diseases. Because mutated proteins are produced in specific cells, there is a critical need to develop organ- and cell-specific delivery strategies to realize the full potential of genomic medicines. We recently overcame this challenge through development of the first class of non-viral nanoparticles for tissue-specific genome editing. Selective ORgan Targeting (SORT) lipid nanoparticles (LNPs) enable targeted intravenous delivery of nucleic acids and proteins to the lungs, liver, and spleen, plus local delivery to the muscle, brain, and skin. Tropism is driven by inclusion of SORT molecules, which create tissue-selective 5-component SORT LNPs that are compatible with multiple gene editing techniques, including mRNA, Cas9 mRNA / sgRNA, and Cas9 ribonucleoprotein (RNP) complexes. In this grant proposal, we Aim to (1) determine the mechanism of SORT, (2) improve the efficacy and tolerability of liver-, lung-, and spleen-targeting SORT LNPs, and (3) determine the cell-specific gene editing capabilities of SORT LNPs with the potential for expanded tropism. Results will determine the fundamental mechanisms and structure-activity relationships (SAR) for non-viral nanoparticle liver, lung, and spleen tropism. This will ultimately allow targeted and safer CRISPR/Cas gene editing in vivo. We will determine these factors by adapting a unique class of LNPs, called SORT LNPs, that we developed. We will employ human cells and genetically engineered mouse models that allow quantification of precise, cell specific gene editing events. Completion of the proposed studies will (1) Elucidate the fundamental mechanisms how and why SORT LNPs target extrahepatic tissues, (2) Determine how SORT molecules control efficacy and tolerability for improved gene editing outcomes, and (3) Determine and control cell-type gene editing specificity to expand targeted gene editing. Cumulatively, this will open new avenues for CRISPR/Cas-based correction of genetic diseases by developing efficacious, safe, and clinically translatable nanoparticle carriers.

Project Narrative Therapeutic use of CRISPR/Cas is currently limited by the lack of safe and effective delivery carriers that can mediate gene editing in vivo to targeted tissues and cells. In this grant, we study recently developed Selective ORgan Targeting (SORT) lipid nanoparticles (LNPs) that can mediate tissue-specific gene editing in the liver, lung, and spleen. Outcomes of this work will include elucidating the fundamental mechanisms how and why SORT LNPs target extrahepatic tissues, determining how SORT molecules control efficacy and tolerability for improved gene editing, quantifying cell-type gene editing specificity to expand targeted gene editing, and ultimately opening new avenues for CRISPR/Cas-based correction of genetic diseases by developing efficacious, safe, and translatable nanoparticle carriers.