Capsid-Free ceDNA Vectors for PAH Expression Beyond AAV Limits
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Solution Overview
Problem
Current gene therapy methods using adeno-associated virus (AAV) vectors are limited by their small viral packaging capacity, patient immune response, and slow gene expression, making them ineffective for treating conditions like Phenylketonuria (PKU), which requires sustained expression of the phenylalanine hydroxylase (PAH) enzyme.
Innovation Solution
The use of capsid-free, covalently-closed DNA (ceDNA) vectors that include PAH nucleic acid sequences, allowing for efficient and repeatable delivery of PAH protein to cells, enabling sustained expression and correction of the genetic defect in PKU, without the limitations of viral capsids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If AAV vectors are used for gene delivery, then the vectors can infect a wide variety of cell types and persist as episomes, but the viral packaging capacity is limited to about 4.5 kb of heterologous DNA
Solution Approach 1:
The patent extracts and removes the viral capsid structure from the DNA vector system, transitioning from encapsidated AAV vectors to capsid-free ceDNA vectors. This extraction eliminates the packaging capacity limitation imposed by viral capsid size constraints while preserving the ability to deliver genetic material to target cells through non-viral mechanisms such as lipofection or electroporation.
2Object-affected harmful factors
If AAV vectors are used for gene delivery, then the vectors are relatively poor immunogens, but pre-existing neutralizing antibodies can eliminate the vector from the patient
Solution Approach 1:
The patent removes the viral capsid component that serves as the primary immunogen, thereby eliminating the problem of pre-existing neutralizing antibodies against AAV capsids. The capsid-free ceDNA vectors lack the structural proteins that trigger immune responses, enabling repeated administrations without immunological interference while maintaining vector persistence and therapeutic efficacy.
3Productivity
If AAV vectors are used for gene delivery, then the vectors can be administered repeatably, but the onset of gene expression is relatively slow
Solution Approach 1:
The patent replaces the biological mechanism of viral capsid-mediated transduction with a physical delivery mechanism such as lipofection or electroporation. This substitution enables direct introduction of ceDNA into cells, bypassing the slow process of viral replication and gene expression initiation, thereby achieving rapid onset of therapeutic protein production.
Data Source
AI summary
The application describes ceDNA vectors having linear and continuous structure for delivery and expression of a transgene. ceDNA vectors comprise an expression cassette flanked by two ITR sequences, where the expression cassette encodes a transgene encoding PAH protein. Some ceDNA vectors further comprise cis-regulatory elements, including regulatory switches. Further provided herein are methods and cell lines for reliable gene expression of PAH protein in vitro, ex vivo and in vivo using the ceDNA vectors. Provided herein are method and compositions comprising ceDNA vectors useful for the expression of PAH protein in a cell, tissue or subject, and methods of treatment of diseases with said ceDNA vectors expressing PAH protein. Such PAH protein can be expressed for treating disease, e.g., Phenylketonuria (PKU).