Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases.

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.

Rationally designed AAV2 and AAVrh8R capsids provide improved transduction in the retina and brain.

The successful application of adeno-associated virus (AAV) gene delivery vectors as a therapeutic paradigm will require efficient gene delivery to the appropriate cells in affected organs. In this study, we utilized a rational design approach to introduce modifications to the AAV2 and AAVrh8R capsids and the resulting variants were evaluated for transduction activity in the retina and brain. The modifications disrupted either capsid/receptor binding or altered capsid surface charge. Specifically, we mutated AAV2 amino acids R585A and R588A, which are required for binding to its receptor, heparan sulfate proteoglycans, to generate a variant referred to as AAV2-HBKO. In contrast to parental AAV2, the AAV2-HBKO vector displayed low-transduction activity following intravitreal delivery to the mouse eye; however, following its subretinal delivery, AAV2-HBKO resulted in significantly greater photoreceptor transduction. Intrastriatal delivery of AAV2-HBKO to mice facilitated widespread striatal and cortical expression, in contrast to the restricted transduction pattern of the parental AAV2 vector. Furthermore, we found that altering the surface charge on the AAVrh8R capsid by modifying the number of arginine residues on the capsid surface had a profound impact on subretinal transduction. The data further validate the potential of capsid engineering to improve AAV gene therapy vectors for clinical applications.

Preexisting immunity and low expression in primates highlight translational challenges for liver-directed AAV8-mediated gene therapy.

Liver-directed gene therapy with adeno-associated virus (AAV) vectors effectively treats mouse models of lysosomal storage diseases (LSDs). We asked whether these results were likely to translate to patients. To understand to what extent preexisting anti-AAV8 antibodies could impede AAV8-mediated liver transduction in primates, commonly preexposed to AAV, we quantified the effects of preexisting antibodies on liver transduction and subsequent transgene expression in mouse and nonhuman primate (NHP) models. Using the highest viral dose previously reported in a clinical trial, passive transfer of NHP sera containing relatively low anti-AAV8 titers into mice blocked liver transduction, which could be partially overcome by increasing vector dose tenfold. Based on this and a survey of anti-AAV8 titers in 112 humans, we predict that high-dose systemic gene therapy would successfully transduce liver in >50% of human patients. However, although high-dose AAV8 administration to mice and monkeys with equivalent anti-AAV8 titers led to comparable liver vector copy numbers, the resulting transgene expression in primates was ~1.5-logs lower than mice. This suggests vector fate differs in these species and that strategies focused solely on overcoming preexisting vector-specific antibodies may be insufficient to achieve clinically meaningful expression levels of LSD genes using a liver-directed gene therapy approach in patients.