Modified OTC Protein and Codon-Optimized mRNA for OTC Deficiency
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Solution Overview
Problem
Current treatments for ornithine transcarbamylase (OTC) deficiency, such as dietary restrictions, medications, and liver transplantation, are cumbersome, risky, and do not effectively stabilize OTC protein for efficient delivery to mitochondria, leading to challenges in managing ammonia levels.
Innovation Solution
A modified human OTC protein (SEQ ID NO: 4) with optimized ubiquitination sites and a codon-optimized mRNA construct is developed to enhance stability and efficiency of OTC protein delivery to mitochondria, using a 5'UTR derived from Arabidopsis thaliana for improved expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type OTC protein is used, then the protein can be synthesized, but it is rapidly degraded by ubiquitin ligases due to ubiquitination sites, resulting in insufficient stable OTC protein for effective treatment
Solution Approach 1:
The patent modifies specific amino acid residues at predicted ubiquitination sites (lysine, arginine, or asparagine) to non-ubiquitinated amino acids (alanine, glycine, serine, threonine, valine, or proline). This parameter change at the molecular level eliminates ubiquitination sites while preserving the protein's catalytic function, thereby increasing protein stability and half-life without altering the overall protein structure or function
Solution Approach 2:
The patent extracts and removes the problematic ubiquitination sites from the OTC protein sequence by site-directed mutagenesis. By specifically targeting and eliminating the lysine, arginine, or asparagine residues that serve as ubiquitination substrates, the invention separates the harmful degradation pathway from the essential catalytic function of the protein
2Productivity
If mRNA therapy is used to deliver OTC protein, then protein expression can be achieved, but poor stability and inefficient delivery to mitochondria limit therapeutic effectiveness
Solution Approach 1:
The patent performs preliminary optimization of the mRNA construct before delivery, including codon optimization for enhanced translation efficiency and selection of stable mRNA sequences. The modified OTC protein sequence is pre-engineered with eliminated ubiquitination sites, ensuring that once the mRNA is translated, the resulting protein is inherently stable and resistant to degradation, thus preparing the therapeutic agent in advance for maximum effectiveness
Data Source
AI summary
The present disclosure provides a modified human OTC protein having improved properties for the treatment of OTC deficiency in a patient. Preferably, the protein of the disclosure is produced from a codon optimized mRNA suitable for administration to a patient suffering from OTC deficiency wherein upon administration of the mRNA to the patient, the protein of the disclosure is expressed in the patient in therapeutically effective amounts to treat OTC deficiency. The present disclosure also provides codon optimized mRNA sequences encoding wild type human OTC comprising a 5′ UTR derived from a gene expressed by Arabidopsis thaliana for use in treating OTC deficiency in a patient.