AAV Hollow Particle Encapsulation Without Capsid Denaturation
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Solution Overview
Problem
Existing methods for encapsulating nucleic acids in empty AAV particles are complex and inefficient, leading to reduced viral infectious activity and safety concerns.
Innovation Solution
A method involving the preparation of a linear nucleic acid fragment containing an AD sequence and a gene of interest, introduced into a cell that produces empty AAV particles, followed by culturing, to efficiently encapsulate the nucleic acid while maintaining initial infectious activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If denaturing agents such as urea are used to incorporate drugs into empty particles, then the drug incorporation efficiency is improved, but the initial infectious activity of the capsid is lowered and the function as a delivery carrier is impaired
Solution Approach 1:
The invention extracts and removes the denaturing step from the traditional protocol. Instead of using urea or other denaturing agents to open the capsid for drug incorporation, the method directly introduces the drug-nucleic acid complex into intact empty particles through a simplified transfection process, thereby preserving capsid integrity and infectious activity while achieving efficient drug incorporation
Solution Approach 2:
The invention uses a drug-nucleic acid complex as an intermediary that can be directly packaged into empty particles without requiring capsid denaturation. This complex serves as the mediator that bridges the drug and the capsid, enabling efficient incorporation while maintaining capsid functionality
2Adaptability or versatility
If conventional viral vectors including AAV are used for gene introduction, then the transmission capability and immunogenicity characteristics are achieved, but the acquisition of self-renewal ability and contamination with wild-type virus occur
Solution Approach 1:
The invention extracts and removes the viral genome from the AAV particle, creating empty particles that lack self-renewal ability. By using only the capsid without the viral genome, the method eliminates the risk of wild-type virus contamination and acquisition of self-renewal ability while preserving the capsid's ability to specifically recognize and deliver cargo to target cells
Solution Approach 2:
The invention creates a simplified version of the viral vector by using only the capsid structure without the complete viral genome. This 'copy' of the viral delivery mechanism retains the beneficial characteristics of cell specificity and low immunogenicity while eliminating the harmful aspects of self-renewal and wild-type contamination risks
3Productivity
If complex encapsulation methods are used to encapsulate nucleic acids in empty AAV particles, then the encapsulation efficiency is improved, but the process complexity and time consumption increase
Solution Approach 1:
The invention removes complex steps from the traditional encapsulation protocol, including denaturation, multiple purification steps, and extensive optimization requirements. The simplified method directly introduces the drug-nucleic acid complex into empty particles through a single transfection step, dramatically reducing process complexity while maintaining high encapsulation efficiency
Solution Approach 2:
Instead of following the traditional approach of creating empty particles first and then attempting to incorporate nucleic acids through complex denaturation and reconstitution steps, the invention inverts the process by directly introducing the drug-nucleic acid complex into empty particles in their native state, simplifying the overall workflow
Data Source
AI summary
A method for producing a nucleic acid-encapsulated adeno-associated virus (AAV) hollow particle, comprising the following steps: (1) preparing a linear nucleic acid fragment comprising a sequence for A region and a sequence for D′ region in an AAV inverted terminal repeat (ITR) (i.e., an AD sequence) or a sequence complementary to the AD sequence and a target gene sequence; (2) introducing the nucleic acid fragment prepared in step (1) into a cell capable of producing an AAV hollow particle; and (3) culturing the cell obtained in step (2).