Peptide-Tagged AAV Conjugates for Cas9 Protein Delivery
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Solution Overview
Problem
Current AAV-based gene delivery systems face limitations in packaging size, restricting the delivery of large or multiple genes, and permanent protein expression leads to off-target effects, particularly with Cas9, necessitating improved vector systems for transient protein delivery.
Innovation Solution
A modular AAV-based gene and protein delivery system using peptide tags that form stable bonds, allowing AAV capsids to link together and tether proteins, expanding carrying capacity and enabling transient expression of biologically active proteins like Cas9.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If AAV vectors are used for gene delivery, then delivery efficiency is improved, but packaging capacity is limited to 4.7 kB
Solution Approach 1:
The invention divides the gene delivery function into separate components: one AAV vector delivers the Cas9 protein (as a transduced protein rather than encoded gene), while another AAV vector delivers the large gene of interest. This segmentation allows each vector to specialize - one carries protein for immediate function, the other carries large DNA for sustained expression, overcoming the 4.7 kB packaging limit.
Solution Approach 2:
The invention uses an intermediary approach by delivering Cas9 as a pre-formed protein complex rather than as a gene. This intermediary form (protein instead of DNA) bypasses the packaging size constraint, allowing the AAV to deliver functional Cas9 without needing to accommodate the large gene sequence within its capsid.
2Reliability
If Cas9 is permanently expressed via viral vectors, then gene editing capability is maintained, but off-target effects increase
Solution Approach 1:
The invention implements periodic (transient) expression of Cas9 by delivering it as a protein that degrades naturally over time, rather than permanent expression from integrated viral DNA. The Cas9 protein is expressed temporarily to perform gene editing, then disappears through natural protein turnover, providing a time-limited window of activity that reduces off-target effects while maintaining editing capability during the active period.
3Quantity of substance
If AAV vector size is increased to deliver large genes, then gene delivery capacity is improved, but vector stability decreases
Solution Approach 1:
The invention segments the delivery payload into two separate stable AAV vectors: one optimized for delivering the large gene (maintaining AAV stability and tropism), and another delivering the Cas9 protein. This segmentation allows each vector to remain within the stable 4.7 kB packaging limit while collectively delivering the full functionality of large gene + Cas9 system, preserving vector stability.
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
This system doubles the carrying capacity of AAV vectors, ensuring efficient delivery of large genes and multiple genes while minimizing off-target effects by providing transient protein expression, enhancing the utility of AAV-mediated gene delivery and editing.
Implementation Method 1
peptide tags that form a bond (a non-limiting example of which can be a covalent bond) with a binding-partner
Implementation Method 2
the inventive AAV vector can link (such as, for example, covalently link) capsids to each other and/or to proteins, creating a larger infectious unit
Data Source
AI summary
In one embodiment, the invention provides an Adeno-Associated Virus (AAV) comprising an exterior surface, which surface comprises one or more peptide tags that form a bond with a binding-partner, wherein the AAV is a live virus. In another embodiment, the invention provides a conjugate comprising at least one such AAV and at least one polypeptide comprising a first domain which is the binding-partner for the tag and a second domain, which is a bioactive polypeptide. In another embodiment, the invention provides a conjugate comprising at least one such AAV (first AAV) and at least one second AAV, which second AAV comprises a second exterior surface, which second exterior surface comprises at least one binding-partner for the tag or for a third linker molecule, wherein the at least one first AAV and the at least one second AAV are bound.