Orphan Motif Transgene Expression via BANP Binding
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Solution Overview
Problem
Current gene therapy methods face challenges in achieving high and specific expression of heterologous transgenes in target tissues due to limitations in vector packaging capacity and regulatory element size, particularly with AAV vectors, which restrict the delivery of larger genetic payloads.
Innovation Solution
Incorporating at least two copies of a specific orphan regulatory motif bound by protein BANP, which acts as a strong transcriptional activator, to enhance the expression of heterologous transgenes, allowing for increased expression of therapeutic proteins and nucleic acids like shRNA or gRNA, and enabling larger genetic payloads by reducing the size requirements of regulatory elements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If AAV vectors are used for gene delivery, then transduction efficiency and safety are improved, but packaging capacity is limited to approximately 4.7 kb for ssAAV and 2.4 kb for scAAV
Solution Approach 1:
The invention extracts and removes the restrictive packaging capacity limitation by using a hybrid vector system that separates the therapeutic payload (which can be larger than 4.7 kb) from the AAV vector backbone. The hybrid particle contains AAV capsid proteins but incorporates larger genetic payloads that exceed traditional AAV packaging limits, effectively extracting the payload size constraint while maintaining AAV safety and transduction properties.
Solution Approach 2:
The invention creates a composite vector system combining AAV capsid proteins with larger genetic payloads that would normally exceed AAV packaging capacity. This hybrid structure integrates the safety and transduction efficiency of AAV with the ability to carry larger therapeutic genes, effectively creating a composite material that exhibits properties of both components while overcoming the packaging limitation.
2Productivity
If regulatory elements are included in AAV vectors, then transgene expression is enabled, but the size of heterologous nucleic acid that can be packaged is further restricted
Solution Approach 1:
The invention extracts the regulatory elements from the constrained AAV packaging system and places them in a separate plasmid or delivery mechanism. This allows the AAV vector to focus on delivering the therapeutic payload while the regulatory elements are provided separately, thereby removing the constraint that regulatory elements impose on the size of heterologous nucleic acid that can be packaged.
Solution Approach 2:
The invention introduces a second plasmid or delivery system as an intermediary that carries the regulatory elements (promoters, enhancers, etc.) separately from the AAV vector. This intermediary provides the necessary transcriptional control elements without consuming valuable packaging capacity within the AAV capsid, allowing larger therapeutic payloads to be delivered while maintaining robust transgene expression.
3Adaptability or versatility
If larger genetic payloads are delivered, then therapeutic protein size and complexity are increased, but AAV vector packaging capacity is exceeded
Solution Approach 1:
The invention segments the genetic delivery system into multiple components: the AAV vector delivers the therapeutic coding sequence (which can be larger than traditional limits), while regulatory elements and other necessary genetic components are delivered separately via additional plasmids or vectors. This segmentation allows each component to be optimized independently and overcomes the packaging capacity limitation by distributing the total genetic payload across multiple delivery vehicles.
Solution Approach 2:
The invention creates a composite delivery system where AAV particles are combined with other nucleic acid delivery vehicles (such as plasmids or other viral vectors). This composite approach allows the therapeutic protein coding sequence to be delivered within the safe and efficient AAV platform while supplementary genetic elements are provided by the companion delivery system, effectively enabling delivery of larger and more complex therapeutic proteins without exceeding AAV packaging capacity.
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 approach significantly increases the expression of heterologous transgenes by a factor greater than two compared to single motif copies, facilitating more effective gene therapy by enhancing the delivery and expression of therapeutic proteins and nucleic acids in specific tissues.
Implementation Method 1
a thus far orphaned regulatory motif in mammalian, when bound by protein BANP, acts as a strong transcriptional activator
Data Source
AI summary
The present invention provides an isolated nucleic acid comprising, operably linked to an heterologous transgene, at least two copies of a sequence selected from the group of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, said at least two copies being selected independently from one another.