A Duplex CRISPR-Cas9 Ribonucleoprotein Nanomedicine for Colorectal Cancer Gene Therapy

Abstract

Based on the high frequency of concurrent adenomatous polyposis coli (APC) and KRAS mutations and their strong cooperative interaction in human colorectal cancer (CRC) promotion, we herein develop a CRISPR-Cas9-based genome-editing nanomedicine to target both APC and KRAS mutations for the treatment of CRC. To this end, a hyaluronic acid (HA)-decorated phenylboronic dendrimer (HAPD) was designed for the targeted delivery of Cas9 ribonucleoprotein (RNP), by which both APC and KRAS genetic mutations harboring in CRC cells can be synergistically disrupted. Systemic administration of Cas9 RNP targeting APC and KRAS enabled by HAPD significantly inhibits tumor growth on xenografted and orthotopic CRC mouse models and also greatly prevents CRC-induced liver metastasis and lung metastasis. Thus, this duplex genome-editing system provides a promising gene therapy strategy for the treatment of human CRC and can be extended to other types of cancers with activated Wnt/β-catenin and RAS/extracellular signal-regulated kinase (ERK) pathways.

Tumor-microenvironment activated duplex genome-editing nanoprodrug for sensitized near-infrared titania phototherapy

“…Since carcinogenesis, insertional mutagenesis, and immunogenicity of viral vectors limits its clinical application, nonviral vectors are growing an alternative strategy 22 , 23 , 24 , 25 , 26 , 27 , 28 . Several nanomaterials-based platforms have been developed to encapsulate Cas9/single-guide RNA (sgRNA) complexes into nanoparticles for delivery of CRISPR, including polymeric nanoparticles, metal–organic frameworks (MOFs), cationic liposomes, gold nanoparticles, and other nanomaterials 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 . Nevertheless, these vectors still face limitations including moderate transfection efficiencies or low targeting effect, which greatly prompts the need for improved CRISPR delivery systems 38 .…”

Toward Gene Transfer Nanoparticles as Therapeutics

“…[ 50 ] An alternative approach using a phenylboronic dendrimer conjugated with hyaluronic acid demonstrated the ability to deliver Cas9 ribonucleoprotein following systemic delivery, leading to antitumor activity. [ 22 ] In the case of gene editing, a particularly innovative approach utilized biodegradable ionizable dendrimer‐based lipid nanoparticles (dLNPs) to enable in vivo homology‐directed repair (HDR) of ≈20% following a direct injection of the dLNPs formulated with DOPE, cholesterol, and PEG–DMG and carrying Cas9 mRNA, gRNA, and a donor ssDNA template. [ 15 ] The convergence of biomaterial technologies (including polymer/lipid hybrid materials) is showing great promise in the design of next‐generation nanomaterials for nucleic acid delivery and gene editing.…”

Nanocarriers for intracellular co-delivery of proteins and small-molecule drugs for cancer therapy

“…Multiple co-delivery systems loaded with antineoplastic drug combinations have been developed to treat tumors, such as two chemotherapeutics, chemotherapeutic and gene, chemotherapeutic and protein, etc. ( Yin et al, 2018 ; Zhou et al, 2018 ; Gao et al, 2019 ; Wan et al, 2021 ) Among them, it is relatively difficult to load small-molecule drugs and macromolecular proteins together in the nanocarriers ( Fumoto and Nishida 2020 ). Because small-molecule drugs and proteins have unique physical and chemical properties, such as size, hydrophilic/hydrophobic properties, charge properties, specificity, stability and cell membrane permeability ( Peng et al, 2019 ).…”