Enhancing CRISPR/Cas gene editing through modulating cellular mechanical properties for cancer therapy

Abstract

Genome editing holds great potential for cancer treatment due to the ability to precisely inactivate or repair cancer-related genes. However, delivery of CRISPR/Cas to solid tumors for efficient cancer therapy remains challenging. Here, we targeted tumor tissue mechanics via a multiplexed dendrimer lipid nanoparticle (LNP) approach involving co-delivery of focal adhesion kinase (FAK) siRNA, Cas9 mRNA, and sgRNA (siFAK+CRISPR-LNPs) to enable tumor delivery and enhance gene editing efficacy. We show that gene editing was enhanced >10-fold in tumor spheroids due to increased cellular uptake and tumor penetration of nanoparticles mediated by FAK-knockdown. siFAK+CRISPR-PD-L1-LNPs reduced extracellular matrix stiffness and efficiently disrupted PD-L1 expression by CRISPR/Cas gene editing, which significantly inhibited tumor growth and metastasis in four mouse models of cancer. Overall, we provide evidence that modulating the stiffness of tumor tissue can enhance gene editing in tumors, which offers a new strategy for synergistic LNPs and other nanoparticle systems to treat cancer using gene editing.

Nonviral Delivery of CRISPR/Cas Systems in mRNA Format

“…[ 81 ] To enhance tissue penetration of CRISPR/Cas mRNA encapsulated in LNPs, Zhang et al exploited siRNA targeting a focal adhesion kinase (siFAK) as an assisted cargo for decreasing the tumor mechanics and extracellular matrix stiffness. [ 80 ] LNPs loaded with Cas9 mRNA, PD‐L1‐targeted sgRNA, and siFAK significantly inhibited tumor growth in the ID8‐Luc xenograft tumor model following i.t. injection and increased survival in the MYC‐driven mouse liver cancer model after i.v.…”

“…injection. [ 80 ] Besides the above tumor‐targeted delivery systems, a phenylboronic acid (PBA)‐contained LNPs was employed to selectively deliver CRISPR mRNA into sialic acid (SA) overexpressing cancer cell line by utilizing PBA/SA interaction. [ 89 ]…”

Lipid-mRNA nanoparticles landscape for cancer therapy

“…Zhang et al utilized dLNPs for the co-delivery of focal adhesion kinase (FAK) siRNA, Cas9 mRNA and sgRNA-PD-L1 (siFAK + CRISPR-LNPs) to enhance >10-fold gene-editing efficacy in tumor tissues. FAK-knockdown promoted cellular uptake and tumor EPR and CRISPR-LNPs significantly inhibited PD-L1 expression via CRISPR/Cas gene editing systems, which achieved a strong tumor growth inhibition and metastasis in four different mouse models of cancer ( Zhang et al, 2022 ). Peer’s laboratory synthesized a novel ionizable amino lipid involving co-delivery of Cas9 mRNA and sgRNAs-PLK1 (sgPLK1-cLNPs) to inhibit tumor growth and prolong survival by 80% ( Rosenblum et al, 2020 ).…”

Orthogonal Chemical Activation of Enzyme-Inducible CRISPR/Cas9 for Cell-Selective Genome Editing

“…It facilitates the elucidation of gene function in biology and the development of new treatments for genetic disorders. − The CRISPR/Cas9 system relies on the use of a single-guide RNA (sgRNA) to direct Cas9 nuclease to make a sequence-specific cleavage of target DNA . The convenience of designing a short sgRNA, usually around a hundred nucleotides, to target a gene of choice has significantly simplified the genome editing operation when compared to previously reported genome editing tools. − Therefore, it has greatly expanded the biomedical and therapeutic potential of genome editing technology, including gene therapy and disease diagnosis. − Despite the success of the CRISPR/Cas9 system for gene regulation and beyond, the less control over Cas9-mediated DNA cleavage to a specific time and location has compromised the precision of genome editing, leading to off-target editing and low disease cell selectivity. − In this regard, there is a great need for spatiotemporally controlled CRISPR/Cas9 systems by limiting the duration of Cas9 activity and constraining gene editing to specific types of cells . To this end, genetic engineering of inducible Cas9 protein has enabled controlled genome editing in response to chemicals and light regulation. − In alternative approaches, controlling sgRNA activity using exogenous signals has led to conditional genome editing with high efficiency. − These methods, however, usually show compromised Cas9 nuclease activity or require complicated chemical modification of sgRNA. , Moreover, their efficacy for disease cell-selective and in vivo genome editing remains limited, mostly due to the difficulty in designing an inducible CRISPR/Cas9 system that is selective to disease cells and the ineffective delivery of genome editing tools in vivo. − Therefore, it is highly desirable to control the activity of genome editing system that is both programmable and cell-selective for targeted gene regulation and therapy …”