Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo

Abstract

Prime editing enables precise installation of genomic substitutions, insertions and deletions in living systems. Efficient in vitro and in vivo delivery of prime editing components, however, remains a challenge. Here we report prime editor engineered virus-like particles (PE-eVLPs) that deliver prime editor proteins, prime editing guide RNAs and nicking single guide RNAs as transient ribonucleoprotein complexes. We systematically engineered v3 and v3b PE-eVLPs with 65- to 170-fold higher editing efficiency in human cells compared to a PE-eVLP construct based on our previously reported base editor eVLP architecture. In two mouse models of genetic blindness, single injections of v3 PE-eVLPs resulted in therapeutically relevant levels of prime editing in the retina, protein expression restoration and partial visual function rescue. Optimized PE-eVLPs support transient in vivo delivery of prime editor ribonucleoproteins, enhancing the potential safety of prime editing by reducing off-target editing and obviating the possibility of oncogenic transgene integration.

Rapid modeling of an ultra-rare epilepsy variant in wild-type mice by in utero prime editing

“…While technically challenging, the potential for direct somatic cell prime editing in vivo would, in principle, allow for the generation of animal models without the labor-intensive and timeconsuming process of germline transmission, which requires multiple rounds of breeding. Such prime editing directly in vivo was recently been demonstrated to be feasible in hepatocytes, retinal cells, lung epithelia, astrocytes, and cardiomyocytes [33][34][35][36][37][38][39][40][41] . Despite this potential, a disease model producing a clinical phenotype using direct in vivo prime editing in wild-type animals has not yet emerged.…”

“…Prime editing has been successfully applied to a variety of genomic loci in vitro and more recently in somatic cell editing in vivo [33][34][35][36][37][38][39][40][41] , suggesting this approach is likely applicable to a wide range of genetic conditions, including diverse genetic epilepsies. We propose that this pipeline may be a valuable tool for assessing personalized pharmacotherapy options for individual patients, and for in vivo preclinical screens for drugs against ultrarare and neglected genetic disease.…”

Engineering PEG10 assembled endogenous virus-like particles with genetically encoded neoantigen peptides for cancer vaccination

“…Specifically, the POI can be inserted into the C-terminal of matrix protein (MA), core capsid (CA), nucleocapsid (NC), or even integrase (IN) with a protease site, allowing for independent functions upon cleavage by viral protease (Voelkel et al, 2010). For example, in the delivery of CRISPR-Cas9 ribonucleoproteins (RNP), the lentiviral gag domain, including MA and NC, were fused with Cas9 and mixed with the full-length gag-pol domain for virus-like particle (VLP) packaging; upon transfection, Cas9 was cleaved by the protease from the viral pol domain in the host cell, leading to excellent gene editing efficiency (Mangeot et al, 2019; Hamilton et al, 2021; An et al, 2024; Hamilton et al, 2024). To enhance the delivery efficacy of VLP, the ratio between gag-POI and gag-pol should be optimized.…”

Revival of Bioengineered Proteins as Carriers for Nucleic Acids

“…Enveloped VLPs protect their nucleic acid cargo within a lipid bilayer and efficiently escape the endosome via incorporation of fusogenic membrane proteins . VLPs have been developed by David Liu et al to codeliver effector proteins such as prime editors in vivo . However, the required assembly inside cells makes them more akin to viral vectors than other protein-based carriers.…”