Recombinant AAV Capsid Protein Segmentation for Targeted Delivery
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Solution Overview
Problem
Current AAV vectors face challenges in targeting specific cell populations due to their natural tropism for heparin sulfate proteoglycan, limiting their ability to infect non-dividing cells and requiring modifications to insert peptide ligands within the capsid open reading frame, which can disrupt the capsid structure and reduce the size of insertions.
Innovation Solution
The development of recombinant AAV vectors that mutate the capsid proteins' start codons to express individual capsid proteins independently, allowing for the production of infectious virions without VP2 or VP3, enabling larger peptide insertions and improved control over targeting by using helper vectors to provide exogenous proteins, and modifying the capsid to reduce heparin sulfate binding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If peptide ligands are inserted within the capsid open reading frame to target specific cell populations, then targeting specificity is improved, but capsid structure stability deteriorates and insertion size is limited
Solution Approach 1:
The patent divides the capsid protein expression into separate components by mutating start codons, allowing independent expression of VP1, VP2, and VP3 proteins from separate plasmids. This segmentation enables peptide ligands to be inserted into specific capsid proteins without disrupting the overall capsid structure, as each protein can be independently optimized for targeting functionality while maintaining structural integrity through separate expression and assembly.
2Productivity
If the natural tropism for heparin sulfate proteoglycan is maintained, then infection efficiency is improved, but specificity for non-dividing cells deteriorates
Solution Approach 1:
The patent applies local quality by maintaining heparin sulfate proteoglycan binding capability in specific regions of the capsid while introducing peptide ligands at other locations. This allows the vector to simultaneously exhibit natural tropism for efficient infection and enhanced specificity for non-dividing cells through localized peptide insertion that does not compromise the heparin binding function.
3Adaptability or versatility
If larger peptide insertions are made to improve targeting, then targeting capability is improved, but capsid structure integrity deteriorates
Solution Approach 1:
By segmenting capsid protein expression and allowing independent optimization of each protein, the patent enables larger peptide insertions into specific capsid proteins without compromising overall capsid integrity. The separate plasmid expression system allows for tailored peptide sequences that can be inserted at optimal locations while maintaining structural stability through independent protein folding and assembly.
Data Source
AI summary
Disclosed are improved recombinant adeno-associated viral (rAAV) vectors having mutations in one or more capsid proteins. Exemplary vectors are provided that have altered affinity for heparin or heparin sulfate, as well as vectors, expression systems, and rAAV virions that lack functional VP2 protein expression, but are nevertheless, fully virulent. Also provided by the invention are rAAV vector-based compositions, virus particles, host cells, and pharmaceutical formulations that comprise them useful in the expression of selected therapeutic proteins, polypeptides, peptides, antisense oligonucleotides and/or ribozymes in selected mammals, including organs, tissues, and human host cells.