Benjamin W. Burgstone

Lipid nanoparticle (LNP) delivery of clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity and scalable in vivo genome editing if efficacious RNP-LNP complexes can be reliably produced. Here we engineer a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100× more genome editing of cells and organs compared with the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP-LNP complexes edit a variety of cell types and induce homology-directed repair in cells receiving codelivered single-stranded DNA templates. Using tissue-selective LNP formulations, we observe genome-editing levels of 16‒37% in the liver and lungs of reporter mice that receive single intravenous injections of iGeoCas9 RNP-LNPs. In addition, iGeoCas9 RNPs complexed to biodegradable LNPs edit the disease-causing SFTPC gene in lung tissue with 19% average efficiency, representing a major improvement over genome-editing levels observed previously using viral or nonviral delivery strategies. These results show that thermostable Cas9 RNP-LNP complexes can expand the therapeutic potential of genome editing.

Abstract Lipid nanoparticle (LNP) delivery of CRISPR ribonucleoproteins (RNPs) has the potential to enable high-efficiency in vivo genome editing with low toxicity and an easily manufactured technology, if RNP efficacy can be maintained during LNP production. In this study, we engineered a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) using directed evolution to generate iGeoCas9 evolved variants capable of robust genome editing of cells and organs. iGeoCas9s were significantly better at editing cells than wild-type GeoCas9, with genome editing levels >100X greater than those induced by the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP:LNP complexes edited a variety of cell lines and induced homology-directed repair (HDR) in cells receiving co-delivered single-stranded DNA (ssDNA) templates. Using tissue-selective LNP formulations, we observed genome editing of 35‒56% efficiency in the liver or lungs of mice that received intravenous injections of iGeoCas9 RNP:LNPs. In particular, iGeoCas9 complexed to acid-degradable LNPs edited lung tissue in vivo with an average of 35% efficiency, a significant improvement over editing efficiencies observed previously using viral or non-viral delivery strategies. These results show that thermostable Cas9 RNP:LNP complexes are a powerful alternative to mRNA:LNP delivery vehicles, expanding the therapeutic potential of genome editing.

In utero gene editing with mRNA-based therapeutics has the potential to revolutionize the treatment of neurodevelopmental disorders. However, a critical bottleneck in clinical application has been the lack of mRNA delivery vehicles that can efficiently transfect cells in the brain. In this report, we demonstrate that in utero intracerebroventricular (ICV) injection of densely PEGylated lipid nanoparticles (ADP-LNPs) containing an acid-degradable PEG-lipid can safely and effectively deliver mRNA for gene editing enzymes to the fetal mouse brain, resulting in successful transfection and editing of brain cells. ADP-LNPs containing Cre mRNA transfected 30% of the fetal brain cells in Ai9 mice and had no detectable adverse effects on fetal development and postnatal growth. In addition, ADP-LNPs efficiently transfected neural stem and progenitor cells in Ai9 mice with Cre mRNA, which subsequently proliferated and caused over 40% of the cortical neurons and 60% of the hippocampal neurons to be edited in treated mice 10 weeks after birth. Furthermore, using Angelman syndrome, a paradigmatic neurodevelopmental disorder, as a disease model, we demonstrate that ADP-LNPs carrying Cas9 mRNA and gRNA induced indels in 21% of brain cells within 7 days postpartum, underscoring the precision and potential of this approach. These findings demonstrate that LNP/mRNA complexes have the potential to be a transformative tool for in utero treatment of neurodevelopmental disorders and set the stage for a frontier in treating neurodevelopmental disorders that focuses on curing genetic diseases before birth.