AAV Capsid Tropism Redirection via TRACER Platform
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Solution Overview
Problem
Current methods for improving the tropism of adeno-associated virus (AAV) capsids for gene delivery to the adult central nervous system (CNS) are limited, with low transduction efficiency and neutralization by pre-existing antibodies, and previous approaches are not tractable in large animals or clinically translatable.
Innovation Solution
A broadly-applicable TRACER platform for cell type-specific biopanning that uses RNA-driven screening to increase selective pressure for capsid variants, allowing specific recovery and subcloning of capsid mRNA without transgenic animals or helper virus co-infection, using cell-type specific promoters like synapsin-1 for neurons or GFAP for astrocytes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional AAV capsids are used for gene delivery to the adult CNS, then the method is simple and broadly applicable, but the transduction efficiency is too low for clinical applications
Solution Approach 1:
The patent applies parameter changes by systematically modifying capsid protein sequences through multiple approaches: randomization of specific residues, substitution of amino acids at defined positions, and shuffling of capsid gene segments. These parameter changes in the capsid structure enable enhanced transduction efficiency for CNS delivery while maintaining the fundamental AAV vector system
Solution Approach 2:
The patent employs preliminary action through in vitro selection and directed evolution of capsid variants before clinical application. Capsid libraries are generated and screened in cell culture models to pre-identify variants with improved CNS tropism, reducing the risk of low transduction efficiency in actual clinical use
2Adaptability or versatility
If capsid sequence randomization is used to improve tropism, then novel capsid variants with enhanced properties are obtained, but the transgenic CRE system is not tractable in other animal species and large animals
Solution Approach 1:
The patent extracts the cell-type specificity requirement from the complex transgenic CRE system and implements it through simpler, species-independent methods. Instead of requiring transgenic animals with CRE recombinase, the patent uses in vitro selection systems that can be applied across different species including non-human primates, thereby taking out the limiting complexity of the transgenic system
Solution Approach 2:
The patent creates universal capsid variants that can be applied across multiple species through directed evolution in cell culture. The selected capsid variants exhibit enhanced tropism for human CNS cells and are presumed to function similarly in other species, providing a universal solution that doesn't require species-specific transgenic systems
3Productivity
If adenovirus co-infection is used to trigger exponential replication of infectious AAV DNA, then transduction of target cell populations is improved, but the specific Ad tropism biases the library distribution and many CNS tissues are not readily accessible
Solution Approach 1:
The patent uses cell culture systems as an intermediary to perform directed evolution of capsids, replacing the need for in vivo adenovirus co-infection. This intermediary approach allows high-throughput selection of capsid variants with improved CNS tropism without the limitations of tissue accessibility and adenovirus tropism bias associated with in vivo methods
4Reliability
If pre-existing neutralizing antibodies are present, then treatment of a large proportion of patients is prevented, but no alternative delivery method is provided
Solution Approach 1:
The patent applies parameter changes to the capsid protein sequences through systematic randomization and substitution at specific positions. These changes alter the antigenic properties of the capsid while maintaining or improving its ability to deliver therapeutic genes, thereby providing variants that can evade pre-existing neutralizing antibodies
Solution Approach 2:
The patent generates composite capsid variants by shuffling gene segments from different AAV serotypes and combining them with randomized regions. This creates hybrid capsids with novel surface properties that can overcome antibody neutralization while retaining functional domains from parent serotypes
Data Source
AI summary
The disclosure relates to compositions, methods, and processes for the preparation, use, and/or formulation of adeno-associated virus capsid proteins, wherein the capsid proteins comprise targeting peptide inserts for enhanced tropism to a target tissue.