A Business Engagement Fund project to test Lipid Nanoparticle (LNP)-mediated mRNA delivery and efficacy

The Medical Research Council provided £750K in funding for the National Mouse Genetics Network’s Business Engagement Fund with a call for applications in early 2023. The Business Engagement Fund supported 3–12-month projects, providing grants of £15–100K, with the expectation that matched funding would be provided by industry collaborators. Funded projects were designed to build and strengthen collaborations between the Network and businesses through feasibility, pilot, or initial studies. These activities aimed to explore ideas and generate initial data to support the development of competitive collaborative grant proposals.

We are now reporting on the first of these projects, highlighting how collaborative endeavours of this kind can help shape preclinical research and accelerate the development of therapeutic interventions.

The project was a partnership between Professor Nick Greene of University College London and OutFox Bio. Nick is a member of the Congenital Anomalies Cluster and a leading academic researcher studying a range of birth defects, with a long-standing interest in the role of folates in development and inherited metabolic disease. OutFox Bio is a delivery technology company focused on the development and optimisation of next-generation lipid nanoparticle (LNP) gene delivery technologies, designed to enable new gene therapy approaches and expand their potential applications.

A life-limiting incurable disease

Non-Ketotic Hyperglycinemia (NKH) is a life-limiting autosomal recessive neurometabolic disease that presents in neonates with lethargy, hypotonia, myoclonic jerks and apnoea. Affected children experience profound neurological impairment and complex epilepsy. Around one-third of infants with severe neonatal-onset NKH die within the first year, but age at death is highly variable, with some children surviving into their teenage years.

NKH is caused by mutations in genes that encode the glycine cleavage system (GCS). Most patients (80%) carry mutations in GLDC (glycine decarboxylase), with the remainder carrying mutations in AMT (aminomethyltransferase). The GCS decarboxylates glycine, with the concomitant transfer of a one-carbon (1C) group to tetrahydrofolate (THF), generating methylene-THF. Subsequent reactions in folate one-carbon metabolism (FOCM) provide 1C groups for multiple outputs, including nucleotide biosynthesis and methylation reactions. Hence, GCS dysfunction leads both to the accumulation of excess glycine in the body and to suppression of FOCM.

There is no cure for NKH; current treatments have limited efficacy. Prognosis remains very poor, highlighting an urgent unmet need for novel therapies. There is currently no established standard of care for NKH, although patients are typically treated with multiple anti-seizure medications. The most common treatment is sodium benzoate, which is administered to lower circulating glycine by stimulating glycine conjugation in the liver, generating hippurate (benzoylglycine) for excretion. Benzoate helps with seizure control but can be toxic and is associated with severe gastrointestinal side effects, necessitating long-term co-administration of proton-pump inhibitors which may carry additional risks. Replacement of benzoate has been highlighted as a priority during discussions with families of affected children.

To investigate NKH pathogenesis and develop novel treatments, Nick’s group developed a GLDC-deficient mouse model that recapitulates hallmark features of the disease, including elevated plasma and tissue glycine and neurological abnormalities. Loss of glycine cleavage system activity was confirmed by enzymatic assay and metabolic tracing using isotopically labelled glycine. Glycine is both a biomarker and a therapeutic target in NKH; both glycine and guanidinoacetate, a glycine–arginine conjugate, are epileptogenic.

In GLDC-deficient mice, the group observed that liver-specific reinstatement of GLDC expression or stimulation of hepatic glycine conjugation through benzoate administration led to normalisation of liver tissue glycine and glycine derivatives, correction of blood glycine concentrations, and reduction of glycine levels in the brain, the main site of NKH pathogenesis. These studies provide proof of principle for liver-directed therapy as a means of controlling systemic and brain glycine levels.

The causative genes are known, making NKH potentially amenable to therapies that restore gene expression. The aim is to develop RNA-based approaches to reinstate GLDC expression and normalise metabolism in NKH. Lipid nanoparticle (LNP)-mediated delivery of mRNA to the liver represents an attractive methodology for therapeutic gene expression. LNP systems have proven to be effective and safe for mRNA delivery and are already in clinical use for other conditions.

In this NMGN Business Engagement Fund project, undertaken in partnership with OutFox Bio, the team initially sought to address two key questions using a reporter-encoding mRNA.  First, they tested whether the liver in NKH remains amenable to LNP-mediated mRNA delivery despite abnormal metabolism. Second, they sought to identify the optimal LNP composition for mRNA delivery to the liver in the NKH GLDC-deficient mouse model. The team identified LNP compositions with improved efficacy compared with clinically approved benchmarks. They also confirmed that compromised glycine metabolism in the liver does not hinder uptake or expression of LNP-delivered mRNA in the NKH mouse model. For example, expression of LNP-mediated reporter expression was at least as high in GLDC-deficient mice as in wild-type mice following treatment with each LNP composition. These findings provided the proof of concept for extending the project to therapeutic mRNA, prioritising the lead LNPs.

The ongoing objective of the project is to develop an mRNA-based therapy that reinstates liver GLDC expression, normalises metabolism, and improves neurological outcomes in the GLDC-deficient NKH mouse model. Outputs from this project are expected to provide an evidence base for advancing this approach towards clinical trials in children with NKH.

Nick presented some of this work at the NKH Crusaders 11th Annual International Family Conference in Boston, where he gave a presentation and took part in several round-table discussions. The event was reported on social media, where Nick’s talk was also mentioned.

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