Mutated AAV Capsid Cysteine Sites for Targeted Thioether Coupling
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Solution Overview
Problem
Current AAV vectors face challenges with broad tropism, leading to reduced transduction efficacy and safety concerns due to transduction of non-target tissues, and low efficiency for certain cell types, necessitating improvements in cell-specific targeting and transduction.
Innovation Solution
Introduce cysteine residues on the surface of AAV capsid proteins through mutation, enabling covalent modification via thioether binding for attaching ligands, nanoparticles, or drugs, allowing targeted cell entry and increased coding capacity through dual vector systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If AAV vectors are used for gene delivery, then transduction of both dividing and non-dividing cells is achieved, but broad tropism leads to transduction of non-target tissues reducing safety and efficacy
Solution Approach 1:
The patent applies local quality by introducing cysteine residues at specific locations on the AAV capsid surface through site-directed mutagenesis. These localized cysteine modifications enable site-specific chemical conjugation of targeting ligands to particular regions of the capsid, allowing precise control over vector targeting without altering the overall capsid structure or function.
Solution Approach 2:
The patent uses chemical linkers as intermediaries to connect cysteine residues on the AAV capsid to targeting ligands. This intermediary approach allows modular attachment of various ligands (antibodies, peptides, aptamers) to the viral vector, enabling targeted delivery to specific cell types while maintaining the natural transduction capabilities of AAV.
2Duration of action of stationary object
If AAV vectors are used for gene delivery, then long term gene expression is achieved, but coding capacity is limited
Solution Approach 1:
The patent employs merging by combining multiple AAV vectors into a single transduction event. Through chemical conjugation of targeting ligands, the system can deliver multiple vector genomes simultaneously to target cells, enabling co-expression of multiple genes or delivery of larger genetic payloads that exceed the capacity of a single AAV vector.
Solution Approach 2:
The patent applies the nested doll principle by packaging multiple genetic elements within the AAV vector system. Chemical modification of the capsid allows incorporation of additional genetic material, such as multiple transgenes or regulatory elements, within the constrained packaging capacity of the viral vector, effectively nesting multiple functional units within a single particle.
3Reliability
If AAV vectors are used for gene delivery, then apathogenicity and low immunogenicity are achieved, but transduction efficiency for certain cell types remains low
Solution Approach 1:
The patent applies preliminary action by pre-modifying the AAV capsid with cysteine residues and attaching targeting ligands before transduction. This pre-functionalization of the vector surface with cell-type-specific ligands enhances recognition and binding to target cells, improving transduction efficiency for difficult-to-transduce cell types while preserving the safe profile of AAV.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enhances cell-specific targeting and transduction efficiency, overcoming broad tropism issues and expanding the coding capacity of AAV vectors, while maintaining infectivity and safety.
Implementation Method 1
enabling covalent modification via thioether binding for attaching ligands, nanoparticles, or drugs
Data Source
AI summary
In a first aspect, the present invention relates to a mutated adeno-associated virus (AAV) capsid protein or fragment thereof wherein a substitution of a wild type non-cysteine amino acid into a cysteine is present whereby the wild type non-cysteine amino acid is exposed on the outer surface of the capsid of an AAV particle. In a further aspect, a mutated AAV particle comprising the AAV capsid protein or fragment thereof according to the present invention is provided. In addition, a nucleic acid encoding the AAV capsid protein according to the present invention is identified together with a corresponding nucleic acid vector, in particular, a plasmid or a gene string. In addition, a host cell containing the nucleic acid vector or the nucleic acid according to the present invention as well as a composition comprising at least an infectious (transducing) AAV particle containing a mutated AAV capsid protein as defined herein together with a non-infectious AAV particle containing a mutated AAV capsid protein as e.g. defined herein is disclosed. Further, a method for the modification of a mutated AAV particle is disclosed allowing a specific modification of the same including an embodiment using a reagent addressing the cysteine residues for binding reaction such as a thioether binding.