Modified AAV6 Capsids for HDR Gene Editing in T Cells
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Solution Overview
Problem
Existing AAV vectors, particularly AAV6, are not optimally suited for targeted gene editing in T-cells, necessitating the development of novel AAV vectors for enhanced homology directed repair (HDR)-mediated gene editing and delivery of heterologous nucleic acids for immunotherapy.
Innovation Solution
Development of AAV capsid polypeptides with specific peptide modifications, such as insertions and substitutions in variable regions and amino acid positions, enhancing HDR efficiency in T-cells, and AAV vectors incorporating these polypeptides for targeted gene editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If natural AAV6 capsid is used, then the vector can infect T-cells, but HDR efficiency is not optimized for gene editing applications
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the AAV6 capsid protein sequence. Mutations at positions 492, 705, and 731 (among others) alter the physical-chemical properties of the capsid surface, thereby changing its interaction characteristics with T-cell receptors and nuclear import machinery to enhance HDR efficiency while maintaining T-cell infectivity
Solution Approach 2:
The invention creates composite capsid structures by combining wild-type AAV6 sequence regions with mutated regions. The capsid becomes a composite structure where specific domains retain original functions (e.g., cell attachment) while other domains acquire enhanced properties (e.g., nuclear import efficiency), achieving both T-cell targeting and optimized HDR
2Productivity
If AAV6 is used for T-cell targeting, then delivery to T-cells is achieved, but the vector is not optimally suited for targeted gene editing
Solution Approach 1:
The patent modifies capsid parameters including amino acid composition, surface charge distribution, and hydrophobicity at specific positions to enhance nuclear import efficiency and HDR-mediated gene editing productivity while preserving the T-cell delivery capability provided by the overall AAV6 capsid structure
3Reliability
If capsid mutations are introduced, then HDR efficiency increases, but capsid structure and stability may be affected
Solution Approach 1:
The patent applies local quality changes by introducing mutations only at specific, strategically chosen amino acid positions (492, 705, 731, etc.) rather than throughout the entire capsid. This localized approach allows enhancement of nuclear import and HDR efficiency at specific functional interfaces while preserving the overall capsid stability and self-assembly properties
Solution Approach 2:
The invention uses partial action by implementing a limited set of mutations (typically 3-10 specific amino acid changes) rather than comprehensive capsid redesign. This partial modification is sufficient to achieve enhanced HDR efficiency while minimizing disruption to capsid stability and assembly
Data Source
AI summary
Provided herein are AAV capsid polypeptides comprising peptide modifications relative to the wild-type AAV6 polypeptide that, when present in the capsid of an AAV vector, can facilitate homology directed repair (HDR)-mediated gene editing of human T cells. Also provided are AAV vectors comprising the capsid polypeptides, nucleic acid vectors comprising the encoding nucleic acid molecules, and host cells comprising the vectors, as well as methods of use of such AAV vectors, nucleic acid vectors and host cells.