Improved specificity and efficacy of base-editing therapies with hybrid guide RNAs.

Phenylketonuria (PKU), hereditary tyrosinemia type 1 (HT1), and mucopolysaccharidosis type 1 (MPSI) are autosomal recessive disorders linked to the phenylalanine hydroxylase (PAH) gene, fumarylacetoacetate hydrolase (FAH) gene, and alpha-L-iduronidase (IDUA) gene, respectively. Potential therapeutic strategies to ameliorate disease include corrective editing of pathogenic variants in the PAH and IDUA genes and, as a variant-agnostic approach, inactivation of the 4-hydroxyphenylpyruvate dioxygenase (HPD) gene, a modifier of HT1, via adenine base editing. Here we evaluated the off-target editing profiles of therapeutic lead guide RNAs (gRNAs) that, when combined with adenine base editors correct the recurrent PAH P281L variant, PAH R408W variant, or IDUA W402X variant or disrupt the HPD gene in human hepatocytes. To mitigate off-target mutagenesis, we systematically screened hybrid gRNAs with DNA nucleotide substitutions. Comprehensive and variant-aware specificity profiling of these hybrid gRNAs reveal dramatically reduced off-target editing and reduced bystander editing. Lastly, in a humanized PAH P281L mouse model, we showed that when formulated in lipid nanoparticles (LNPs) with adenine base editor mRNA, selected hybrid gRNAs revert the PKU phenotype, substantially enhance on-target editing, and reduce bystander editing in vivo. These studies highlight the utility of hybrid gRNAs to improve the safety and efficacy of base-editing therapies.

A base editing strategy using mRNA-LNPs for in vivo correction of the most frequent phenylketonuria variant.

The c.1222C>T (p.Arg408Trp) phenylalanine hydroxylase (PAH) variant is the most frequent cause of phenylketonuria (PKU), an autosomal recessive disorder characterized by accumulation of blood phenylalanine (Phe) to neurotoxic levels. Here we devised a therapeutic base editing strategy to correct the variant, using prime-edited hepatocyte cell lines engineered with the c.1222C>T variant to screen a variety of adenine base editors and guide RNAs in vitro, followed by assessment in c.1222C>T humanized mice in vivo. We found that upon delivery of a selected adenine base editor mRNA/guide RNA combination into mice via lipid nanoparticles (LNPs), there was sufficient PAH editing in the liver to fully normalize blood Phe levels within 48 h. This work establishes the viability of a base editing strategy to correct the most common pathogenic variant found in individuals with the most common inborn error of metabolism, albeit with potential limitations compared with other genome editing approaches.

Orexin A, an amphipathic α-helical neuropeptide involved in pleiotropic functions in the nervous and immune systems: Synthetic approach and biophysical studies of the membrane-bound state.

This research reports on the membrane interactions of orexin A (OXA), an α-helical and amphipathic neuropeptide that contains 33 residues and two disulfide bonds in the N-terminal region. OXA, which activates the orexins 1 and 2 receptors in neural and immune cell membranes, has essential pleiotropic physiological effects, including at the levels of arousal, sleep/wakefulness, energy balance, neuroprotection, lipid signaling, the inflammatory response, and pain. As a result, the orexin system has become a prominent target to treat diseases such as sleep disorders, drug addiction, and inflammation. While the high-resolution structure of OXA has been investigated in water and bound to micelles, there is a lack of information about its conformation bound to phospholipid membranes and its receptors. NMR is a powerful method to investigate peptide structures in a membrane environment. To facilitate the NMR structural studies of OXA exposed to membranes, we present a novel synthetic scheme, leading to the production of isotopically-labeled material at high purity. A receptor activation assay shows that the 15N-labeled peptide is biologically active. Biophysical studies are performed using surface plasmon resonance, circular dichroism, and NMR to investigate the interactions of OXA with phospholipid bilayers. The results demonstrate a strong interaction between the peptide and phospholipids, an increase in α-helical content upon membrane binding, and an in-plane orientation of the C-terminal region critical to function. This new knowledge about structure-activity relationships in OXA could inspire the design of novel therapeutics that leverage the anti-inflammatory and neuro-protective functions of OXA, and therefore could help address neuroinflammation, a major issue associated with neurological disorders such as Alzheimer's disease.

Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration.

Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP containing an ionizable lipid. Herein, we report a method that can predict the LNP pKa from the structure of the ionizable lipid. We used theoretical, NMR, fluorescent-dye binding, and electrophoretic mobility methods to comprehensively measure protonation of both the ionizable lipid and the formulated LNP. The pKa of the ionizable lipid was 2-3 units higher than the pKa of the LNP primarily due to proton solvation energy differences between the LNP and aqueous medium. We exploited these results to explain a wide range of delivery efficiencies in vitro and in vivo for intramuscular (IM) and intravascular (IV) administration of different ionizable lipids at escalating ionizable lipid-to-mRNA ratios in the LNP. In addition, we determined that more negatively charged LNPs exhibit higher off-target systemic expression of mRNA in the liver following IM administration. This undesirable systemic off-target expression of mRNA-LNP vaccines could be minimized through appropriate design of the ionizable lipid and LNP.