Kassandra Bisson
A recent study describes the novel development of engineered virus-like particles for delivery of base editors in vivo for therapeutic applications with increased efficiency and decreased off target affects compared to other viral and nonviral delivery strategies.
As genomic technologies rapidly improve, the prospect of using gene therapies to cure genetic diseases is becoming brighter. Base editors (BEs) have recently been developed that allow for single base substitutions, insertions, or deletions without requiring double stranded DNA breaks1. They thereby avoid unwanted consequences and widen the breadth of potential applications1. Both nonviral and viral strategies such as lipid nanoparticle (LNP) and adeno-associated viruses (AAVs) respectively, have been previously established to deliver DNA encoding BEs to desired tissues for correcting pathogenic point mutations1. There are, however, respective disadvantages to the established methods including off-target editing due to prolonged expression in transduced cells and potential for oncogenesis due to viral vector integration into the genome2.
To combat those major disadvantages, a novel approach developed by Banskota et al., uses rational design to engineer virus-like particles (eVLPs) that can mediate therapeutic levels of postnatal in vivo gene editing for many possible applications2. DNA-free VLPs are assemblies of viral proteins allowing infection of cells yet lack viral genetic material. They can therefore be used as vehicles for delivering gene editing proteins like Cas9 ribonucleoproteins (RNPs) or BEs to target tissues allowing correction of pathogenic point mutations. This approach utilizes the viral delivery strategy without risking viral genome integration, prolonged expression of BEs and reduces off target editing2. Prior VLP-mediated approaches had limited validation of in vivo therapeutic efficacy2. This rational design method of eVLPs by Banskota et al.as depicted in Figure 1A, yields greater efficiency in both delivery and packaging of base editors than any of the previous viral and nonviral approaches.
The benefit of eVLP rational design was demonstrated in this study through use of mouse models, wherein single injections of eVLPs achieved therapeutic levels of base editing in multiple targeted tissues inclusive of the liver and eyes. In this study, in vivo liver base editing was investigated through targeting the proprotein convertase subtilisin/kexin type 9(PCSK9) gene which is known to be involved in cholesterol homeostasis3. Loss of function (LOF) mutations of PCSK9 can result in lower blood levels of low-density lipoprotein (LDL) leading to a reduced risk of atherosclerotic cardiovascular disease. Banksokta et al.’s eVLP design targeted and disrupted the splice donor at the boundary of PCSK9 exon 1 and intron 1 creating an LOF mutation which was a previously established BE strategy for knockdown of PCSK9 in the mouse liver3. Adult C57BL/6J mice were injected with the eVLPs retro-orbitally and base editing in the liver was monitored one week after the injection as seen in Figure 1B. From this test, 63% editing in bulk liver with the highest dose of 7 x 10^11 eVLPs was observed, which was comparable to the editing efficiency of the AAV-mediated and the LNP delivery systems. There was however, no detectable off-target editing above background levels observed in the eVLP method unlike in the AAV and LNP methods using the same BE and single guide RNA (sgRNA)4,5. These observations demonstrate the comparable efficiency alongside the reduced off target editing in vivo compared to existing strategies.

To demonstrate the applications of rationally designing eVLPs even further, Banskota et al. sought to use eVLPs to correct a disease-causing point mutation. Leber congenital amaurosis (LCA) in adult mice is an eye disorder primarily affecting the retina6. The mutation studied was in the retinoid isomerohydrolase(RPE65) gene (c.120C>T, p.R44X)which resulted in almost complete loss of visual function6. This murine pathogenic variant has a homologous mutation identified in humans that also causes LCA thereby further demonstrating the relevance of eVLPs for human applications6. The designed eVLPs encapsulated the adenine base editor ABE7.10-NG RNPs which converts A•T-to-G•C and sought to target the pathogenic point mutation causing LCA for base editing correction7. Adult rd12 mice were injected subretinally (Figure 1C) with the ABE7.10-NG-eVLPs resulting in a 12% correction of the R44X mutation in the RPE genomic DNA. This eVLP performance was compared to a previous lentiviral (LV) delivery method wherein a lentivirus encoding the same sgRNA and the ABE7.10-NG constructs generated an 11.5% correction2. In comparison, the use of eVLPs resulted in a 1.4-fold improvement in bystander-free correction relative to the LV treatment. This demonstrates that the eVLPs have comparable and slightly higher correction efficacy relative to the alternative LV method8. In addition, the rationally designed eVLPs were able to efficiently correct the pathogenic mutations in the mouse model resulting in improvements in visual function.
As with many of the new gene therapy approaches, this strategy offers a promising outlook for gene therapy however, there are still many more obstacles to overcome before it can be readily used in clinical practice. The main outcome of this study is that rationally designed eVLP treatments have been demonstrated to be more efficient for in vivo base editing in multiple organs than other BE delivery strategies. Studies investigating other tissues can help further the therapeutic potential of eVLPs. While eVLPs were investigated in mice models alongside preliminary primary human cell lines, further pharmacokinetic studies should be undertaken in other models. In particular, nonhuman primate models can help determine dosing requirements, residence time and the cargoes of eVLPs for other applications.
References:
1. Newby GA & Liu DR. In vivo somatic cell base editing and prime editing. Mol Ther. 29(11):3107-3124. (2021). PMID: 34509669.
2. Banskota S, et al. Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins. Cell. 185(2):250-265.e16 (2022). PMID: 35021064
3. Fitzgerald K, et al. Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled, phase 1 trial. Lancet. 383(9911):60-68. (2014). PMID: 24094767.
4. Musunuru K, et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature. 593(7859):429-434. (2021). PMID: 34012082.
5. Rothgangl, T, et al. In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels. Nat Biotechnol 39, 949–957 (2021).
6. Pang JJ, et al. Retinal degeneration 12 (rd12): a new, spontaneously arising mouse model for human Leber congenital amaurosis (LCA). Mol Vis. 11:152-62. (2005). PMID: 15765048.
7. Richter, M.F., et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol 38, 883–891 (2020).
8. Suh S, et al. Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing. Nat Biomed Eng. 5(2):169-178. (2021). PMID: 33077938