Circular ssDNA Vector with Nuclease Cleavage Site
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Solution Overview
Problem
Single-stranded nucleic acids are quickly degraded within cells due to enzymatic activity and can trigger immune responses when present in cytoplasm or endosomes, limiting their therapeutic efficacy and requiring effective delivery methods that avoid immune recognition.
Innovation Solution
A delivery vector comprising a circular single-stranded polynucleotide with a duplex section and a loop section, where the duplex includes a recognition sequence for a targeted nuclease, allowing controlled release of the linear single-stranded nucleic acid within the cell, thereby protecting it from degradation and immune activation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If single-stranded nucleic acid is delivered directly to cells, then it is immediately available for transcription and translation, but it is quickly degraded by enzymatic activity
Solution Approach 1:
The single-stranded nucleic acid is nested within a circular single-stranded DNA vector structure, where the therapeutic ssDNA is protected inside the vector's duplex and loop sections. This nesting provides physical protection against enzymatic degradation while maintaining the ability to deliver the active ssDNA to the cell nucleus.
Solution Approach 2:
The circular single-stranded DNA vector acts as an intermediary carrier that protects the therapeutic single-stranded nucleic acid during delivery. The vector's structure with duplex and loop sections serves as a mediator that shields the vulnerable ssDNA from nucleolytic degradation by cellular exonucleases while still allowing the therapeutic payload to reach its target.
2Ease of operation
If single-stranded nucleic acid is present in cytoplasm or endosomes, then delivery to cell is achieved, but immune responses are triggered
Solution Approach 1:
The design extracts the vulnerable single-stranded region into a protected loop structure within the circular vector, while the duplex sections provide immunity from immune recognition. This separation allows the ssDNA to be delivered without triggering immune responses in the cytoplasm and endosomes.
Solution Approach 2:
Different sections of the vector have different properties: the duplex sections provide stability and immune evasion, while the loop section contains the single-stranded therapeutic payload. This local differentiation allows the molecule to simultaneously achieve delivery efficiency and avoid immune detection.
3Reliability
If chemical modifications are added to improve stability, then degradation resistance increases, but ability to bind to proteins is affected
Solution Approach 1:
The vector is segmented into distinct functional regions: duplex sections that provide stability and protection from degradation, and a loop section that contains the single-stranded therapeutic sequence. This segmentation allows different parts to optimize for their specific functions without compromising the other.
Solution Approach 2:
The vector structure changes the physical parameters of the nucleic acid by forming a circular configuration with duplex and loop sections. This structural parameter change provides stability against exonuclease degradation without requiring chemical modifications that would affect protein binding, as the stability is achieved through structure rather than chemistry.
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
The delivery vector enables the controlled and covert delivery of single-stranded nucleic acids, enhancing their stability and therapeutic efficacy by preventing premature degradation and immune activation, while ensuring targeted release within the cell for applications like gene editing and antisense therapy.
Implementation Method 1
a duplex formed from a first section and a third section of said polynucleotide, said sections including sequences which are complementary
Implementation Method 2
said vector comprising: (a) a duplex formed from a first section and a third section of said polynucleotide, said sections including sequences which are complementary; (b) a loop formed from a second section, said section separating the first and third sections; wherein said duplex includes a recognition sequence for a targeted nuclease
Data Source
AI summary
The invention relates to a delivery vector for the delivery of a single-stranded nucleic acid. Said vector is a closed circular polynucleotide comprised of at least three sections, two of which have sufficient complementarity to form a duplex, and an intervening sequence containing the single-stranded nucleic acid to be delivered. Said duplex includes a recognition sequence for a targeted nuclease such that under appropriate conditions the single-stranded nucleic acid is released.