Mini-PCDH15 AAV Packaging Within 4.7 kb Capacity
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Solution Overview
Problem
Current AAV vectors are limited by their capacity to accommodate only ~4.7 kb of transgene, making it difficult to deliver the ~5.8 kb PCDH15 coding sequence for treating Usher 1F, which affects hearing and vision, and they lack efficient expression in inner and outer hair cells and eye cells.
Innovation Solution
Development of a mini-PCDH15 nucleic acid sequence, truncated to fit within a single AAV genome, comprising extracellular, transmembrane, and cytoplasmic domains, packaged with AAV capsids like AAV5, AAV7, or AAV9, for targeted delivery to inner ear and eye cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the full-length PCDH15 coding sequence (~5.8 kb) is used for gene therapy, then complete functional expression is achieved, but it exceeds the AAV capsid capacity limit (~4.7 kb)
Solution Approach 1:
The patent extracts and removes specific non-essential extracellular EC domains (EC4, EC5, EC6, EC7, EC8, EC9, EC10) from the full-length PCDH15 protein, retaining only the critical EC1-EC3 and EC11 domains along with the transmembrane and cytoplasmic domains. This extraction reduces the coding sequence from ~5.8 kb to fit within the AAV capsid while preserving essential tip-link function for hearing and balance.
Solution Approach 2:
The patent segments the PCDH15 protein into essential and non-essential domains, creating a mini-PCDH15 construct that contains only the critical functional regions (EC1-EC3, EC11, transmembrane, and cytoplasmic domains). This segmentation allows the gene to be divided into a deliverable size for AAV while maintaining the core functional units needed for stereocilia tip-link formation.
2Reliability
If conventional AAV vectors are used for delivery, then safety is maintained, but efficient expression in inner ear and eye cells is not achieved
Solution Approach 1:
The patent applies local quality by using tissue-specific promoters (such as Atoh1 promoter for hair cells and CRX promoter for photoreceptors) to drive mini-PCDH15 expression specifically in target cells within the inner ear and eye. This ensures high expression efficiency in the relevant cell types while maintaining the safety profile of conventional AAV vectors.
Data Source
Figure 1A~1B
Figure 1C~1D
Figure 1E~1F
AI summary
Aspects of the disclosure relate to compositions, nucleic acids, vectors, viruses, and methods useful for treating hearing loss and/or blindness, for example, Usher Syndrome type IF. The present disclosure provides isolated nucleic acids, vectors, and rAAV.9.PHP.B comprising a transgene encoding a mini-PCDH15, and methods of treating hearing loss using the same. The present disclosure also provides a gRNA associated with a base editor to correct one or more mutations in PCDH15 for treating hearing loss and/or vision loss.