Codon-Optimized RPGR Nucleic Acid for Stable AAV Vector Production
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Solution Overview
Problem
The instability of nucleic acid sequences encoding the human retinitis pigmentosa GTPase regulator (RPGR) protein during large-scale production of AAV vectors leads to mutations and deletions, limiting their use in gene therapy applications for treating X-linked retinitis pigmentosa.
Innovation Solution
A codon-optimized nucleic acid sequence (SEQ ID NO: 1) for the human RPGR protein is developed, which is stable during large-scale production, used in combination with an expression cassette and vectors like rAAV and rHSV, and an IRBP promoter for targeted expression in photoreceptor cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the wild type RPGR cDNA is used in large-scale production of AAV vectors, then the production process can be initiated, but the nucleic acid sequence becomes unstable leading to mutations and deletions
Solution Approach 1:
The patent applies codon optimization to change the nucleotide sequence parameters of RPGR cDNA while maintaining the same amino acid sequence. This involves replacing codons with synonymous alternatives that are more stable and less prone to mutations during large-scale AAV vector production, thereby resolving the contradiction between productivity and reliability
Solution Approach 2:
The patent creates a codon-optimized copy of the wild type RPGR cDNA that serves as a stable template for AAV vector production. This optimized copy (SEQ ID NO: 1) replicates the functional properties of the original gene while eliminating the instability issues that prevent large-scale production
2Reliability
If the RPGR gene contains the ORF15 repetitive region, then the complete protein function is maintained, but the region becomes a mutation hot spot during cloning and vector preparation
Solution Approach 1:
The patent modifies the nucleotide sequence parameters within the ORF15 region by applying codon optimization, replacing the unstable repetitive sequence with a codon-optimized version that maintains the same glutamate and glycine amino acid repeats but exhibits significantly reduced mutation rates during cloning and vector preparation
Solution Approach 2:
The patent converts the harmful repetitive sequence into a beneficial codon-optimized sequence by maintaining the functional amino acid repeats while eliminating the mutation-prone nucleotide patterns, thereby transforming a liability into an asset for manufacturing precision
Data Source
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AI summary
This invention relates generally to a codon optimized nucleic acid encoding a retinitis pigmentosa GTPase regulator (RPGR) protein. The nucleic acid has enhanced stability during plasmid production relative to a wildtype cDNA encoding the RPGR protein. The invention also relates to expression cassettes, vectors, and host cells comprising the codon optimized nucleic acid. Methods for preparing a recombinant adeno-associated (rAAV) expression vector comprising the codon optimized nucleic acid sequence are also provided. The nucleic acids, expression cassettes, vectors, and host cells provided may be useful in the large scale production of rAAV expression vectors for gene therapy applications.