Dual mrna-lnp and gene targeting therapy for early-onset severe ornithine transcarbamylase deficiency

Overview

Early-onset severe ornithine transcarbamylase deficiency (OTCD) is a devastating urea cycle disorder with approximately 60% mortality at presentation and limited treatment options beyond liver transplantation. This project addresses a critical unmet medical need by proposing a dual therapeutic strategy that combines messenger RNA (mRNA) therapeutics with gene targeting technology.

The first component uses lipid nanoparticles (LNPs) to deliver codon-optimized human OTC mRNA directly to the liver, rapidly stabilizing the metabolic crisis caused by hyperammonemia. The second component uses CRISPR/Cas9 technology delivered via LNPs or AAV vectors to permanently integrate a codon-optimized human OTC cDNA into the albumin gene locus of hepatocytes, providing a lifelong cure.

This approach overcomes key limitations of current AAV-based gene therapy, including loss of episomal DNA during liver growth in young patients and the inability to re-administer AAV due to neutralizing antibodies. The platform is modular and can be adapted to treat other inherited metabolic diseases by replacing the therapeutic cDNA.

Technical specifications

Key components:

• OTC-mRNA-LNPs: Codon-optimized human OTC mRNA (3-4 times more efficient than wild-type cDNA) encapsulated in lipid nanoparticles for liver-targeted delivery
• SaCas9-LNPs: CRISPR/Cas9 nuclease delivered via LNPs to enable precise genome editing at the albumin locus
• AAV donor vector: Contains codon-optimized hOTC cDNA flanked by albumin homology arms for targeted integration
• Microfluidics-based LNP production: In-house Nanoassemblr platform for consistent LNP manufacturing

Validated capabilities:

• Liver-directed genome targeting demonstrated in neonatal mice for Crigler-Najjar syndrome, citrullinemia type I, Hemophilia B, and Fabry disease
• Successful rescue of neonatal lethality and symptom reversal in multiple metabolic disease models
• OTC knockout mouse model generated with complete Otc exon 2 deletion (hemizygous males die within 24 hours)
• Therapeutic efficacy demonstrated in Spf-Ash mice (late-onset OTCD model) using dual AAV-vector strategy

Safety assessment: Integration site analysis, liver transaminase monitoring, histology, immune response evaluation, inflammation markers, and on/off-target nuclease activity analysis.

Technology readiness level

The project is at an advanced preclinical stage with extensive proof-of-concept data already generated. The gene targeting approach has been validated across multiple disease models in neonatal mice, and the mRNA-LNP delivery system has been established using in-house microfluidics capabilities. The OTC knockout mouse model is available for efficacy testing, and codon-optimized hOTC cDNA has been developed and patented.

Immediate next steps involve preparing hOTC mRNA-LNPs and testing them in Spf-Ash and OTC knockout mice to determine optimal dosing and duration of therapeutic effect. The SaCas9-LNP delivery system will be optimized in wild-type mice, followed by combined approach testing in OTC knockout mice with survival, urinary orotic acid, and OTC enzymatic activity as efficacy endpoints.

The research team is seeking funding for LNP and mRNA preparation reagents and key personnel to advance this promising therapeutic platform toward clinical translation.