Targeted homology-directed repair in blood stem and progenitor cells with CRISPR nanoformulations

Abstract

Ex vivo CRISPR gene editing in hematopoietic stem and progenitor cells has opened potential treatment modalities for numerous diseases. The current process uses electroporation, sometimes followed by virus transduction. While this complex manipulation has resulted in high levels of gene editing at some genetic loci, cellular toxicity was observed. We have developed a CRISPR nanoformulation based on colloidal gold nanoparticles with a unique loading design capable of cellular entry without the need for electroporation or viruses. This highly monodispersed nanoformulation avoids lysosomal entrapment and localizes to the nucleus in primary human blood progenitors without toxicity. Nanoformulation-mediated gene editing is efficient and sustained with different CRISPR nucleases at multiple loci of therapeutic interest. Engraftment kinetics of nanoformulation-treated primary cells in humanized mice are better relative to nontreated cells, with no differences in differentiation. Here we demonstrate nontoxic delivery of the entire CRISPR payload into primary human blood progenitors. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Multifunctional Gold Nanoparticles: A Novel Nanomaterial for Various Medical Applications and Biological Activities

“…This shows promise application in its application for gene delivery systems. For example, Shahbazi et al (2019) synthesized AuNPs core using the citrate reduction method, and developed a CRISPR nanoformulation, using colloidal AuNPs (AuNPs/CRISPR), with guide RNA and nuclease on the surface of AuNPs, with or without a single-strand DNA (ss DNA) template to support homology-directed repair. The outcome was an efficient gene editing.…”

Surface chemistry of gold nanoparticles for health-related applications

“…CRISPR-Cas9 systems have been developed to manipulate mammalian genomes and have shown great potential in treating generelated diseases and cancer. 133,134 However, the delivery of the plasmid that encodes the CRISPR-Cas9 system into cells and nuclei is very challenging due to its strongly negative charges and large size (>10 000 kp). Viral carriers show high-efficiency gene transfection.…”

In vivo gene delivery mediated by non-viral vectors for cancer therapy

“…The production of the protein in the bacterial hosts might be expensive and the endotoxin contamination could be considered as an additional obstacle for their large-scale production. Various non-viral carriers have been used to transfer such platforms into the target cells including gold NPs, graphene oxide, carboxylated branched poly (β-amino ester) NPs, β-cyclodextrin-conjugated low-molecular-weight PEI, microbubble-nanoliposomal particles, pH-responsive silica-metal-organic framework (SMOF) hybrid NPs consisting of both silica and zeolitic imidazole framework (ZIF) as well as cell-penetrating peptides and DNA nanoclews [319][320][321][322][323][324][325][326][327][328][329][330][331].…”