CEP290 Fragment Vector for AAV Packaging
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Solution Overview
Problem
Current therapeutic approaches for treating CEP290-related diseases, such as Leber congenital amaurosis, are hindered by the large size of the CEP290 gene, which prevents effective packaging in recombinant adeno-associated virus (AAV) vectors, limiting their delivery and treatment efficacy.
Innovation Solution
Development of a recombinant vector carrying a nucleic acid sequence encoding a fragment of the CEP290 gene lacking N-terminal and C-terminal inhibitory regions, under regulatory sequences for expression in mammalian cells, and a synthetic or recombinant protein comprising discontinuous CEP290 amino acid fragments spliced together in a single open reading frame, suitable for delivery using AAV vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the full-length CEP290 gene is used for therapeutic delivery, then the treatment addresses the complete gene function, but the large gene size prevents effective packaging in AAV vectors
Solution Approach 1:
The CEP290 gene is divided into functional segments, specifically retaining the N-terminal domain (amino acids 1-380) and C-terminal domain (amino acids 1695-1966) while removing the middle region. This segmentation creates a truncated gene version that fits within AAV packaging capacity while preserving essential microtubule-binding and ciliogenesis-promoting functions.
Solution Approach 2:
The patent extracts and removes the non-essential middle region of the CEP290 gene (amino acids 381-1694) that does not contribute to the core therapeutic function. This extraction reduces the overall gene size to enable AAV packaging while maintaining the functional integrity of the N-terminal and C-terminal domains required for treating ciliopathies.
2Adaptability or versatility
If the full-length CEP290 gene is used, then complete protein function is achieved, but the packaging capacity of AAV vectors is exceeded
Solution Approach 1:
The patent applies local quality by concentrating the essential functional properties of CEP290 into specific regions: the N-terminal domain (amino acids 1-380) provides microtubule-binding capability and the C-terminal domain (amino acids 1695-1966) provides ciliogenesis-promoting activity. By preserving these localized functional regions and removing non-essential sequences, the gene adapts to AAV packaging constraints while maintaining therapeutic versatility.
3Volume of moving object
If truncated CEP290 fragments are used, then the gene fits in AAV vectors, but there is concern about maintaining biological activity
Solution Approach 1:
The patent performs preliminary action by pre-identifying and preserving the critical functional domains of CEP290 (N-terminal and C-terminal regions) before truncation. Through prior structural and functional analysis, the inventors determined which regions are essential for microtubule-binding and ciliogenesis, allowing them to design truncated versions that maintain biological activity while reducing size for AAV packaging.
Solution Approach 2:
The patent applies parameter changes by modifying the gene length parameter from the full-length version to a truncated version (amino acids 1-380 and 1695-1966). This parameter change reduces the gene size to fit AAV packaging capacity while maintaining the functional parameters (microtubule-binding affinity and ciliogenesis-promoting activity) through preservation of key domains.
Data Source
AI summary
Compositions are provided that comprise a recombinant vector carrying a nucleic acid sequence encoding a fragment of CEP290 lacking all or part of its N-terminal and C-terminal inhibitory regions, under the control of regulatory sequences which express the product of said gene in a selected cell of a mammalian subject, and a pharmaceutically acceptable carrier. These and other compositions are disclosed with are useful in methods for treating a mammalian subject having a disease associated with a CEP290 mutation, such as Lebers Congenital Amaurosis.