New Advances in Using Virus-like Particles and Related Technologies for Eukaryotic Genome Editing Delivery

Abstract

The designer nucleases, including Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas), have been widely used for mechanistic studies, animal model generation, and gene therapy development. Clinical trials using designer nucleases to treat genetic diseases or cancers are showing promising results. Despite rapid progress, potential off-targets and host immune responses are challenges to be addressed for in vivo uses, especially in clinical applications. Short-term expression of the designer nucleases is necessary to reduce both risks. Currently, delivery methods enabling transient expression of designer nucleases are being pursued. Among these, virus-like particles as delivery vehicles for short-term designer nuclease expression have received much attention. This review will summarize recent developments in using virus-like particles (VLPs) for safe delivery of gene editing effectors to complement our last review on the same topic. First, we introduce some background information on how VLPs can be used for safe and efficient CRISPR/Cas9 delivery. Then, we summarize recently developed virus-like particles as genome editing vehicles. Finally, we discuss applications and future directions.

Keywords: CRISPR/Cas9; RNA; TALEN; ZFN; aptamer; aptamer-binding protein; delivery; designer nuclease; genome editing; ribonucleoprotein; viral capsid; virus-like particle (VLP).

Figure 1

Structure of four RNA aptamers.

Figure 2

Strategies for modifying the Gag protein for designer nuclease RNP delivery by VLPs. (A) Diagram illustrating protein- or RNP-delivering VLPs. The RNA, if present, does not contain a long terminal repeat, so reverse transcription cannot happen. (B) Strategy for packaging proteins and RNPs into extracellular vesicles. A dashed line indicates non-covalent interactions. The VLPs and exosomes were reported in references [5], [10] and [12] respectively.

Figure 3

Using VLPs to deliver mRNA for genome editing. (A) Viral capsid-derived RNA-delivering VLPs. Aptamers are added in Cas9 mRNA and sgRNA so that they can be packaged via aptamer/ABP interactions. The RNAs do not contain a long terminal repeat, so reverse transcription cannot happen. (B) Mammalian transposon protein-derived VLPs for RNA delivery. The VLPs were reported in references [11], [15] and [16] respectively.

Figure 4

Strategies for modifying the Gag protein for mRNA and sgRNA delivery by VLP/LV hybrid particles. (A) Diagram illustrating mRNA- and sgRNA-co-delivery of VLP/LV hybrid particles. The mRNAs can only serve as the templates for translation since they do not contain viral sequences necessary for reverse transcription, so reverse transcription cannot happen. (B) Different approaches to Gag modification for Cas9-mRNA and sgRNA-expression cassette co-delivery. The VLPs were reported in references [6], [7] and [13] respectively.