Development of Third-Generation Gold Nanoparticles (CRISPR-AuNP) for Enhanced CRISPR Delivery to Hematopoietic Stem and Progenitor Cells

Abstract

Efficient CRISPR ribonucleoprotein (RNP) delivery into hematopoietic stem and progenitor cells (HSPC, CD34+) enables stable genome editing with potential for lifelong therapeutic benefits from a single intervention. However, current methods like electroporation require specialized equipment and procedures, whereas viral vectors are costly and require living cells to assemble, limiting access in research and clinical translation. Synthetic nanoparticles offer a promising alternative. We previously reported a gold-based CRISPR-AuNP nanoformulation that delivered gene editing in HSPCs, favoring Cas12a over Cas9. Here, we demonstrate that Cas9 loads poorly into this formulation. By elucidating Cas9's physicochemical interactions with AuNP surfaces, we optimized the nanoformulation by pre-forming RNP complexes in a 2nd generation CRISPR-AuNP. This formulation demonstrated increased active Cas9 and Cas12a loading in tube but failed to deliver gene editing in primary cells in vitro. Further analysis of the CRISPR-AuNP physiochemistry with HPSC endosomes guided further optimization. Preformed RNP polyplexes were formed with thiolated poly(ethyleneimine)-poly(ethylene glycol) (PEI-PEG-SH) polymers at a 2 N/P ratio and conjugated to AuNP. This 3rd generation Cas9 CRISPR-AuNP achieved 13.23 ± 0.12% indels at the β-2-microglobulin (B2M) gene in HSPCs at a 100 pmol dose, with no observed loss in cell viability and cell entry within 6 hours. The platform's modularity extended to two additional CRISPR systems: Cas12a (15.07 ± 1.9% indels) and MG29-1 (13.39 ± 1.5% indels), highlighting its versatility. Most importantly, this nanoformulation can be assembled in a few hours at benchtop for <$70 per 1 million HSPC treated. We also demonstrate early data that this formulation can be modified with surface antibodies to facilitate active cellular targeting, with a future goal of testing these formulations in vivo for possible clinical translation. Very preliminary in vivo studies using untargeted 3rd generation Cas9 CRISPR-AuNP targeting the murine B2m gene in wild-type C57Bl6 mice showed detectable gene editing in multiple tissues with no elevated inflammatory responses or increased liver enzymes. These advancements position gold-polymeric nanoparticle hybrids as a simple, low-cost non-viral delivery system for CRISPR into HSPCs. The ability to rapidly assemble these particles with different CRISPR systems and polymers will greatly increase research access for multiple cell types.