Dual AAV-MYO7A Vector Split Design to Eliminate Truncated Protein
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Solution Overview
Problem
Existing AAV vector systems are unsuitable for delivering large genes, such as those encoding MYO7A protein, due to their limited DNA packaging capacity, leading to truncated protein production and cytotoxicity, which is inadequate for treating conditions like Usher syndrome.
Innovation Solution
Development of modified dual AAV vector systems that shift the coding sequence for the MYO7A tail domain and alter overlapping coding sequences to eliminate truncated protein production, increasing packaging efficiency and transduction efficiency in the retina.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If standard AAV vector systems are used to deliver large genes like MYO7A, then the vector can be packaged and delivered, but the gene is truncated and produces cytotoxic truncated protein
Solution Approach 1:
The patent divides the large MYO7A gene into two separate AAV vectors (first and second vectors), each carrying a portion of the coding sequence. The first vector contains the 5' portion and the second vector contains the 3' portion, with an overlapping region that allows proper reconstitution of the full-length gene through homologous recombination in the target cell, avoiding production of truncated proteins
Solution Approach 2:
The patent introduces an overlapping coding sequence region between the two AAV vectors that serves as an intermediary element. This overlapping region contains homology sequences that facilitate homologous recombination in the target cell, acting as a mediator to properly join the two separate gene portions into a complete, functional MYO7A gene without generating cytotoxic truncated proteins
2Reliability
If the coding sequence is shifted to eliminate truncated protein, then full-length MYO7A is expressed, but the vector design becomes more complex
Solution Approach 1:
The patent segments the MYO7A coding sequence into two distinct AAV vectors with clearly defined boundaries and an overlapping region. The first vector encompasses the 5' portion up to a specific breakpoint, while the second vector contains the 3' portion starting from the overlapping region. This systematic segmentation ensures proper gene reconstitution while maintaining manageable vector design
Solution Approach 2:
The patent strategically changes the parameters of the vector design by introducing an overlapping coding sequence region with specific homology. This parameter change (adding overlap) enables reliable full-length gene expression through homologous recombination while providing a systematic approach that, although more complex than single-vector systems, follows predictable design rules for split-gene delivery
3Productivity
If dual vector systems are used to package large genes, then packaging efficiency increases, but transduction efficiency in retina was previously limited
Solution Approach 1:
The patent employs AAV serotype 2 (AAV2) capsids for both vectors in the dual-vector system, providing universality in the delivery mechanism. This consistent use of AAV2 capsids ensures that both the first and second vectors share the same tropism and transduction efficiency characteristics in retinal cells, overcoming previous limitations of dual-vector systems with different serotypes
Solution Approach 2:
The patent segments the therapeutic payload (MYO7A gene) into two manageable portions that can be efficiently packaged into separate AAV vectors, while maintaining the same viral capsid type. This segmentation approach allows each vector to be optimally packaged within AAV's ~4.7 kb capacity limit while ensuring both vectors exhibit identical transduction properties in retinal photoreceptors and RPE cells
Data Source
AI summary
Disclosed are compositions and methods for treating diseases of the mammalian eye, and in particular, complications of the retina associated with Usher syndrome 1B (USH1B). Further disclosed are compositions and methods for treating diseases of the mammalian inner ear, and in particular, complications of ear hair cells associated with Usher syndrome 1B (USH1B). The disclosure provides improved AAV-based, dual vector systems that facilitate the expression of full-length proteins whose coding sequences exceed that of the polynucleotide packaging capacity of an individual AAV vector. Described herein are modified hybrid dual vector systems that shift the coding sequence for the MYO7A tail domain from the front-half vector to the back-half vector by altering the split point (e.g., from between exons 23 and 24, to between exons 21 and 22), in order to eliminate the production of truncated MYO7A protein. Further described herein are improved, codon-modified hybrid and overlap vector systems in which putative stop codons and residual sequences in non-coding sequences are removed.