ABCA4 Minigene rAAV Delivery for AAV Packaging Limits
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Solution Overview
Problem
The ABCA4 gene, which is associated with Stargardt disease, exceeds the packaging limit of conventional Adeno-associated Viral (AAV) vectors, making effective gene delivery challenging.
Innovation Solution
Development of gene therapy vectors, including rAAV vectors, that contain ABCA4 minigenes flanked by AAV inverted terminal repeats (ITRs), with tissue-specific promoters and capsids targeting ocular cells, for subretinal or intravitreal delivery to treat Stargardt disease.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional AAV vectors are used for gene delivery, then the vector packaging is simple and well-established, but the ABCA4 gene cannot be packaged because it exceeds the size limit
Solution Approach 1:
The ABCA4 gene is divided into multiple cDNA fragments that can be individually packaged into separate AAV vectors. Each fragment is then delivered to target cells where they assemble to form the complete functional protein, thereby overcoming the packaging size limitation of conventional AAV vectors.
Solution Approach 2:
Multiple cDNA fragments are designed to be co-packaged within a single AAV vector capacity by optimizing fragment size and arrangement. The fragments are nested within the vector genome along with necessary regulatory elements (promoters, polyA signals) to enable coordinated expression and assembly of the full-length protein.
2Reliability
If the complete ABCA4 gene is delivered, then full protein function is achieved, but the delivery system becomes incompatible with AAV packaging constraints
Solution Approach 1:
Non-essential portions of the ABCA4 gene sequence are extracted or optimized to create compact cDNA fragments that retain full protein-coding capability. Introns and regulatory sequences are removed, keeping only the essential coding regions that can be efficiently packaged and expressed in the target tissue.
Solution Approach 2:
The gene delivery approach changes from delivering a single complete gene to delivering multiple optimized cDNA fragments with modified parameters (size, sequence composition, regulatory elements). These parameter changes enable compatibility with AAV packaging while maintaining the ability to produce functional full-length protein through cellular assembly mechanisms.
3Adaptability or versatility
If tissue-specific promoters are used to target ocular cells, then delivery specificity is improved, but the promoter sequence adds to the overall vector size
Solution Approach 1:
Instead of using full-length traditional promoters, truncated or minimal versions of tissue-specific promoters are used that retain sufficient activity to drive expression in target ocular cells. This partial action approach reduces promoter sequence length while maintaining adequate tissue-specific expression capability.
Solution Approach 2:
Different cDNA fragments are equipped with different promoter elements or regulatory sequences optimized for their specific function and location within the protein. This allows each fragment to be independently regulated with minimal sequence requirements, reducing overall payload while maintaining tissue-specific expression of the complete protein.
Data Source
AI summary
Aspects of the disclosure relate to compositions and methods useful for delivering minigenes to a subject. Accordingly, the disclosure is based, in part, on isolated nucleic acids and gene therapy vectors, such as viral (e.g., rAAV) vectors, comprising one or more gene fragments encoding a therapeutic gene product, such as a protein or peptide (e.g., a minigene). In some embodiments, the disclosure relates to gene therapy vectors encoding a ABCA4 protein (e.g., the gene product of ABCA4 gene) or a portion thereof. In some embodiments, compositions described by the disclosure are useful for treating diseases associated with mutations in the ABCA4 gene, for example Stargardt disease.