Therapeutic in vivo delivery of gene editing agents

Abstract

In vivo gene editing therapies offer the potential to treat the root causes of many genetic diseases. Realizing the promise of therapeutic in vivo gene editing requires the ability to safely and efficiently deliver gene editing agents to relevant organs and tissues in vivo. Here, we review current delivery technologies that have been used to enable therapeutic in vivo gene editing, including viral vectors, lipid nanoparticles, and virus-like particles. Since no single delivery modality is likely to be appropriate for every possible application, we compare the benefits and drawbacks of each method and highlight opportunities for future improvements.

Figure 1.. Overview of therapeutic gene editing technologies

Nucleases create targeted double-strand DNA breaks (DSBs), which generally lead to uncontrolled mixtures of insertions and deletions (indels) that are useful for gene disruption. In certain types of dividing cells, DSBs in the presence of a DNA donor template can also lead to homology-directed repair (HDR) outcomes that can support gene correction, though indel byproducts typically accompany HDR outcomes. Base editors mediate targeted C•G-to-T•A, A•T-to-G•C, or C•G-to-G•C conversions with minimal indel byproducts. Prime editors enable targeted single-nucleotide conversions, insertions, deletions, and combinations thereof with minimal indel byproducts. See also Anzalone et al., 2020 for a more detailed description of gene editing mechanisms.

Figure 2.. Requirements for efficient in vivo delivery of gene editing agents

(A) An appropriate delivery vehicle (gray circles) for gene editing agents must efficiently encapsulate DNA or mRNA encoding gene editing agents, or gene editing proteins or ribonucleoproteins (RNPs). Delivery vehicles must protect their cargos from sequestration or degradation in vivo prior to encountering target cells. (B) Delivery vehicles must bind target cells, typically by engaging cell surface receptors with complementary molecules on the surface of the delivery vehicle. (C) Delivery vehicles must traverse the target cell membrane, typically through receptor-mediated endocytosis. (D) Following endocytosis, delivery vehicles must either escape endosomes and release their cargo, or fuse with endosomes to release their cargo into the target cell cytosol. The cargo must then be trafficked to the appropriate cellular compartment (typically the nucleus) for successful gene editing to occur.

Figure 3.. Overview and comparison of viral delivery methods

(A) Adeno-associated viruses are single-stranded DNA viruses with cargo capacity of 5 kb. (B) Lentiviral vectors are enveloped viruses with that package a single-stranded RNA genome of up to 10 kb. (C) Adenoviral vectors are double-stranded DNA viruses with a packaging capacity of 8 kb that can be expanded to 36 kb in “gutless” vectors devoid of all the viral protein-coding genes.

Figure 4.. Lipid nanoparticle (LNP) delivery

LNPs consist of four key components and can efficiently encapsulate various RNAs. Encapsulated mRNAs are typically modified by including alternative nucleotides during in vitro transcription, such as N1-methylpseudouridine, to increase cellular stability after delivery. Encapsulated guide RNAs are chemically modified at various positions, including with 2’-O-methylation and phosphorothioate linkages, which enhance the stability of the guide RNA.

Figure 5.. Virus-like particle (VLP) delivery

General schematic of the most important components of mRNA-packaging VLPs (left) and protein- or RNP-packaging VLPs (right). In both types of VLPs, the retroviral gag and gag-pro-pol polyproteins provide structural stability and the viral protease required for cleaving the polyproteins into distinct subunits during particle maturation. In mRNA-packaging VLPs, fusion of gag with an RNA-binding protein (RBP) enables encapsulation of mRNA cargo containing the RNA aptamer recognized by the RBP. If necessary, a guide RNA is typically encoded on an integration-deficient lentivirus (IDLV) genome. In RNP-packaging VLPs, fusion of gag with protein cargo via a viral protease-cleavable linker directs encapsulation of protein into particles as they form. Cleavage of the linker after particle maturation enables the release of free protein cargo into transduced cells. When packaging RNPs, guide RNAs can be co-packaged into particles due to the intrinsic affinity between the Cas9 protein and its guide RNA. In engineered VLPs (eVLPs), cargo packaging, release, and localization have been optimized through protein engineering (Banskota et al., 2022).