Segmented RNA Molecules for Full-Length Protein Reconstitution
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Solution Overview
Problem
Existing gene therapy vectors like AAV face challenges in packaging large disease-linked genes due to limited capacity and inadequate regulatory sequences, hindering efficient expression of target proteins.
Innovation Solution
A system comprising synthetic nucleic acid molecules with promoters, dimerization domains, and splice junctions allows for the reconstitution of full-length proteins by linking N-terminal, middle, and C-terminal portions of target proteins through RNA recombination, using vectors like AAV or lentiviral vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If AAV vectors are used for gene replacement therapy, then delivery efficiency is improved, but packaging capacity is limited to about 5000 nucleotides
Solution Approach 1:
The patent divides the large gene into multiple smaller cDNAs (e.g., dystrophin cDNA split into 5 separate cDNAs), each fitting within AAV packaging capacity. These segmented cDNAs are then reconstituted in host cells to produce the full-length functional protein, thereby overcoming the 5000 nucleotide packaging limit while maintaining efficient AAV delivery.
2Quantity of substance
If the replacement gene size is reduced to fit AAV capacity, then packaging is enabled, but space for regulatory sequences is insufficient
Solution Approach 1:
The gene is segmented into multiple cDNAs, each with its own minimal regulatory sequences (promoters, polyadenylation signals). This segmentation allows each cDNA to be independently packaged in AAV with sufficient regulatory elements, while the collective reconstitution achieves full gene function.
Solution Approach 2:
Host cell machinery serves as an intermediary to reconstitute the full-length functional protein from multiple separate cDNA transcripts. The host's splicing and translation mechanisms combine the segmented cDNAs into the complete functional protein, eliminating the need to package the entire gene with all regulatory sequences in a single AAV vector.
3Reliability
If traditional gene therapy approaches are used, then treatment of small genes is effective, but treatment of large disease-linked genes remains challenging
Solution Approach 1:
The patent applies segmentation to make gene therapy applicable to large genes by dividing them into multiple AAV-compatible cDNAs. This approach maintains treatment effectiveness for small genes while extending versatility to treat large disease-linked genes that previously could not be packaged in AAV vectors.
Solution Approach 2:
The segmented cDNA approach creates a universal platform that can treat both small and large genes. The same AAV delivery system and host cell reconstitution mechanism work for genes of various sizes, making the therapy broadly applicable across different disease contexts.
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 enables efficient expression of large proteins by overcoming packaging constraints, achieving high reconstitution efficiency and effective protein production in target tissues.
Implementation Method 1
a second dimerization domain operably linked to the second promoter, and having reverse complementarity to the first dimerization domain
Data Source
AI summary
Provided herein are synthetic RNA molecules for reconstitution of RNA molecules, including compositions and methods of using these molecules. For example, such molecules can be used to deliver a protein coding sequence over two or more viral vectors (such as AAVs), resulting in reconstitution of the full-length protein in a cell. Such methods can be used to deliver a therapeutic protein, for example to treat a genetic disease or cancer.