Codon-optimized REP1 nucleic acids for choroideremia gene therapy
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Solution Overview
Problem
Current gene therapy approaches for choroideremia face challenges due to suboptimal expression levels of wild-type cDNA in human photoreceptors and retinal pigment epithelium, limiting their effectiveness in treating the disease.
Innovation Solution
Development of codon-optimized nucleic acid molecules encoding human Rab escort protein-1 (REP1) with enhanced expression levels, utilizing a recombinant adeno-associated virus (rAAV) vector and optimized promoters to achieve higher and more specific expression in target cells, including the use of a CAG promoter and Woodchuck hepatitis virus posttranscriptional regulatory element for enhanced transgene expression in the retina.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild type cDNA of CHM is used for gene therapy, then the disease phenotype can be rescued, but the expression level in human photoreceptors and RPE is suboptimal
Solution Approach 1:
The patent applies codon optimization to change the nucleotide sequence parameters of the CHM cDNA while maintaining the amino acid sequence. This involves replacing codons with synonymous codons that are more frequently used in human photoreceptors and RPE, thereby optimizing translation efficiency and increasing protein expression levels without altering the functional properties of the REP1 protein
Solution Approach 2:
The patent uses tissue-specific promoters (such as the RPE65 promoter) to drive expression of the codon-optimized CHM cDNA specifically in retinal pigment epithelium cells. This local quality approach ensures high expression levels precisely where needed in the retina, rather than uniform expression throughout all cell types
2Quantity of substance
If codon optimized nucleic acid molecules are used, then expression levels are significantly higher, but the nucleic acid sequence is modified from wild type
Solution Approach 1:
The patent modifies the nucleotide sequence parameters through codon optimization, changing the DNA composition while preserving the encoded amino acid sequence. This allows achieving higher expression levels through optimized codon usage patterns that match host cell tRNA availability, without altering the functional protein product
Solution Approach 2:
The patent creates a codon-optimized copy of the wild type CHM cDNA that replicates the amino acid sequence information but uses an optimized nucleotide sequence. This copied version maintains functional equivalence while improving expression characteristics through rational nucleotide sequence design
3Adaptability or versatility
If rAAV vector with optimized promoters is used, then specific expression in target cells is enhanced, but the vector complexity increases
Solution Approach 1:
The patent employs tissue-specific promoters (such as RPE65 promoter or CAG promoter) within the rAAV vector to drive expression specifically in retinal pigment epithelium or photoreceptor cells. This local quality approach ensures that the therapeutic gene is expressed precisely in the target cell types within the retina, enhancing therapeutic relevance while using well-characterized promoter elements
Data Source
AI summary
The present disclosure provides codon optimized nucleotide sequences encoding human REP1, vectors, and host cells comprising codon optimized REP1 sequences, and methods of treating retinal disorders such as choroideremia comprising administering to the subject a codon optimized sequence encoding human REP1.