Engineered rAAV Capsids With Peptide Inserts for Tissue-Selective Delivery
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Solution Overview
Problem
Current methods for identifying recombinant adeno-associated viruses (rAAVs) with enhanced tropism to specific tissues and improved transduction efficiency are limited, particularly for treating muscle disorders like dystrophinopathies, and there is a need for improved methods to target and deliver therapies effectively using lower dosing.
Innovation Solution
Engineered capsid proteins with peptide insertions in variable regions IV or VIII of the AAV capsid, such as AAV9, to enhance tissue targeting and transduction, promoting delivery to muscle, CNS, and retinal tissues, while reducing transduction in undesirable tissues like liver.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If natural diversity of AAV sequences is mined to identify capsids with desirable properties, then tissue targeting and transduction efficiency may be improved, but the complexity of identifying and selecting optimal capsids increases
Solution Approach 1:
The capsid protein is segmented into functional regions, with focus on variable regions (VR-IV and VR-VIII) that are responsible for tissue tropism. By inserting peptides at these specific segmented locations, the invention achieves targeted tissue tropism without needing to redesign the entire capsid structure, thus reducing overall selection complexity.
Solution Approach 2:
Instead of randomly mutating the entire capsid sequence, the invention applies local modifications by inserting specific peptides at defined variable regions. This localized approach allows optimization of tissue targeting properties while maintaining the functional integrity of the rest of the capsid structure.
2Productivity
If conventional selection methods are used to identify rAAV capsids with enhanced properties, then transduction efficiency in target tissues may be improved, but the dosing requirement remains high
Solution Approach 1:
The invention changes the structural parameters of the capsid by inserting peptides at specific variable regions, which alters the capsid's interaction with target cells. This parameter modification results in enhanced transduction efficiency at lower dosing levels compared to conventional capsids.
3Productivity
If peptide insertions are made in variable regions of the capsid to enhance tissue targeting, then transduction efficiency in target tissues increases, but transduction in non-target tissues may also increase
Solution Approach 1:
The invention uses local peptide insertions at specific variable regions (VR-IV and VR-VIII) to confer tissue-specific tropism. The chosen peptide sequences are designed to interact specifically with receptors or markers present in target tissues while avoiding non-target tissues, thus achieving selective transduction.
Solution Approach 2:
The inserted peptides act as intermediary molecules that mediate the interaction between the AAV capsid and target cell surface receptors. These peptide intermediaries facilitate specific binding to target tissue markers, directing the vector to the intended destination while preventing off-target effects.
Data Source
AI summary
The present invention relates to recombinant adeno-associated viruses (rAAVs) having capsid proteins engineered to include amino acid sequences that confer and/or enhance desired properties. In particular, the invention provides engineered capsid proteins comprising peptide insertions from heterologous proteins inserted within or near variable region IV (VR-IV) of the virus capsid, such that the insertion is surface exposed on the AAV particle. The invention also provides capsid proteins that direct rAAVs to target tissues, in particular, capsid proteins comprising peptides that are inserted into surface-exposed variable regions using a method for replacing a stop codon in the capsid gene with such peptide insert. The invention provides such methods for making the rAAV vectors into vector libraries and selecting the rAAV vectors having peptide inserts that target the rAAV to particular tissues of interest, and thus deliver therapeutics for treating disorders.