Viral and nonviral nanocarriers for in vivo CRISPR-based gene editing

Abstract

The continued development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has the potential to greatly impact clinical medicine, particularly for disease diagnosis and treatment. Despite high demand for the in vivo delivery of CRISPR-based therapies, significant challenges persist. These include rapid degradation by enzymes, inefficient disease site targeting, and the risk of undesired off-target outcomes. Nanoparticulate platforms, with their tailorable properties, have been engineered to efficiently package CRISPR payloads in various formats, including as plasmid DNA, mRNA, and ribonucleoprotein complexes, for in vivo delivery. Among them, recombinant adeno-associated viruses, virus-like particles, and lipid nanoparticles have displayed exceptional promise. This review will discuss the development of these and other nanocarriers for in vivo CRISPR-based genome editing.

Keywords: clustered regularly interspaced short palindromic repeats (CRISPR); in vivo gene editing; lipid nanoparticle (LNP); nanocarriers; recombinant adeno-associated viruses (rAAVs); virus-like particle (VLP).

Figure 1

Overview of in vivo CRISPR delivery using nanocarriers. CRISPR payloads, including plasmid DNA, mRNA with sgRNA, and RNPs can be formulated into nanocarriers such as viral vectors, VLPs, and LNPs, thereby increasing their efficiency when delivered in vivo.

Figure 2

Genome editing using CRISPR/Cas. (a) Traditional CRISPR systems can knock-out or knock-in genes by two main strategies. Genomic DNA is cleaved to form a double-stranded break, which is subject to nonhomologous end joining. In the absence of donor DNA, this can create mutations that result in gene knock-out. Donor DNA with compatible overhangs can also be provided to facilitate gene knock-in. If the donor DNA is flanked by homology arms, the double-stranded break can go through the homology-directed repair pathway, which also results in gene knock-in. (b) CRISPR payloads come in three main formats: plasmid DNA, mRNA plus sgRNA, and RNPs.

Figure 3

AAV vectors for CRISPR gene editing. (a) Components and structure of an AAV. Reproduced with permission from Ref. [112], © Worner, T. P. et al. 2021. (b) Examples of all-in-one rAAV vectors for CRISPR delivery. Reproduced with permission from Ref. [132], © Ibraheim, R. et al. 2021.

Figure 4

Dual-rAAV platform for base editor delivery. (a) Design of a dual-rAAV system to deliver a base editor through protein trans-splicing. (b) Comparison of in vivo editing efficiency between systems using different recombination mechanisms. Reproduced with permission from Ref. [143], © Levy, J. M. et al., under exclusive licence to Springer Nature Limited 2020.

Figure 5

VLPs for the co-delivery of Cas9 mRNA and an sgRNA cassette. (a) Design and preparation of the all-in-one lentiviral particle CRISPR platform. (b) In a laser-induced wet age-related macular degeneration mouse model, the platform with sgRNA targeting Vegfa 2 was potent in inducing on-target indels and significantly reduced choroidal neovascularization (CNV) area. Reproduced with permission from Ref. [200], © Ling, S. et al., under exclusive licence to Springer Nature Limited 2021.

Figure 6

Optimization of VLPs for RNP delivery. (a) Design of a VLP for base editor delivery. Optimal placement of the cleavage site and nuclear export signal repeats is explored. (b) After systemic administration of the optimized VLP (v4 BE-eVLP) targeting PCSK9, high on-target editing efficiency and reduced serum Pcsk9 levels are observed. Reproduced with permission from Ref. [214], © Banskota, S. et al., published by Elsevier Inc. 2021.

Figure 7

Chemically engineered sgRNA to enhance LNP-mediated CRISPR editing in vivo. (a) The optimized structure of a chemically modified sgRNA. (b) An LNP formulated with Cas9 mRNA and an optimized sgRNA targeting PCSK9 considerably reduces serum Pcsk9 levels with high on-target editing efficiency. Reproduced with permission from Ref. [262], © Springer Nature America, Inc. 2017.

Figure 8

LNPs for organ-specific CRISPR delivery. (a) Addition of SORT lipids with different charges results in LNPs that can specifically deliver cargos to the lungs, spleen, or liver. (b) mRNA-based LNPs formulated with various SORT lipids demonstrate high gene editing efficiency in different organs. Reproduced with permission from Ref. [273], © Cheng, Q. et al., under exclusive licence to Springer Nature Limited 2020.

Figure 9

Antibody-modified LNPs for targeted CRISPR delivery. (a) LNPs loaded with CRISPR payloads (cLNPs) were modified with ASSET molecules, which enable subsequent antibody functionalization based on Fc binding. (b) Targeted cLNPs (T-cLNPs) loaded with sgRNA against PLK1 elicit potent gene editing efficiency in tumor cells and significantly improve the survival of tumor-bearing mice. Reproduced with permission from Ref. [279], © Rosenblum, D. et al., some rights reserved; exclusive licensee American Association for the Advancement of Science 2020.