AAV Capsid Screening via Human Neurons for Motor Neuron Targeting
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Solution Overview
Problem
Current gene therapy approaches for neuromuscular and neuromotor disorders, such as spasticity, face challenges in specificity and efficacy due to the inability to target specific neurons effectively, leading to off-target effects and limited transferability from animal models to humans.
Innovation Solution
A method involving the use of induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) to derive human neurons for screening adeno-associated virus (AAV) capsid libraries, allowing for the identification of capsids that efficiently infect specific neuronal types, including motor neurons, and enabling personalized gene therapy vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If gene therapy is directed to specific neuronal types using promoter genes and viral vectors, then treatment specificity is improved, but device complexity and manufacturing complexity increase due to the need for precise targeting mechanisms
Solution Approach 1:
The patent modifies the capsid protein parameters (amino acid sequences) of AAV vectors to change their tropism and specificity for neuronal types. By altering capsid parameters through directed evolution and screening, the therapy achieves high targeting specificity without requiring complex promoter-gene combinations or delivery systems
Solution Approach 2:
The patent uses screening methods to identify and copy successful capsid sequences from AAV libraries that demonstrate high neuronal targeting efficiency. These copied capsid designs are then applied across different therapeutic contexts, simplifying the overall system by reusing proven targeting mechanisms
2Productivity
If animal models are used to screen AAV capsid libraries, then screening efficiency is improved, but transferability to humans deteriorates due to species-specific differences in viral tropism
Solution Approach 1:
The patent introduces human neuronal cell lines as an intermediary screening platform that bridges animal model efficiency and human applicability. These cell lines allow high-throughput screening while maintaining human-specific viral tropism characteristics, serving as a mediator between preclinical models and clinical applications
Solution Approach 2:
The patent changes the screening substrate from animal tissues to human-derived neuronal cells, altering the biological parameter of species origin. This enables screening in a human-relevant context while maintaining the efficiency of in vitro cell-based assays
3Ease of manufacture
If wild-type AAV serotypes are used for gene delivery, then ease of manufacture is improved, but targeting specificity deteriorates because wild serotypes infect multiple tissue and cell types
Solution Approach 1:
The patent applies local quality modification by introducing specific mutations into the capsid protein sequence to create localized changes in tropism. These focused modifications at specific capsid regions enable neuronal-type-specific targeting while maintaining overall capsid structure and manufacturability
Solution Approach 2:
The patent creates dynamic capsid variants through directed evolution processes that generate diverse capsid sequences. These dynamic, evolving capsid designs allow optimization of both specificity and manufacturability through iterative screening and selection
4Reliability
If multiple rounds of mutagenesis and screening are performed to increase evolutionary pressure, then capsid targeting efficiency is improved, but loss of time increases due to iterative screening processes
Solution Approach 1:
The patent performs preliminary screening of large AAV capsid libraries before committing to iterative mutagenesis cycles. By pre-identifying promising capsid variants through initial high-throughput screening, the need for multiple time-consuming evolutionary rounds is reduced
Solution Approach 2:
The patent implements feedback mechanisms where screening results from each round directly inform the design of subsequent mutagenesis experiments. This feedback-driven approach optimizes the evolutionary process by focusing mutations on capsid regions that show promise, reducing the number of iterations needed
Data Source
AI summary
The invention provides methods of screening for capsid-encoding nucleotide sequences of adeno-associated virus (“AAV”) particles capable of infecting a subject's neurons, or sub-compartments thereof, involving neurons derived from induced pluripotent stem cells or embryonic stem cells. The invention also provides AAV capsids, capsid-encoding nucleotide sequences, expression vectors, viral particles, cells and kits for use in methods of treating neuromuscular or neuromotor disorders, such as spasticity. Sequences generated from the methods may lead to new gene therapies targeted to select populations of neurons, such as motor neurons innervating muscle cells, thus providing high specificity that may be personalised to the subject.