Modified U7 snRNA for TDP-43 Cryptic Exon Repression
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Solution Overview
Problem
Loss of nuclear TDP-43 protein leads to aberrant splicing of cryptic exons in genes such as STMN2 and UNC13A, resulting in functional protein depletion and contributing to neurodegenerative diseases like ALS and FTD, with existing technologies failing to effectively target and correct this splicing.
Innovation Solution
A modified U7 snRNA construct with an antisense sequence complementary to TDP-43 regulated cryptic exons and a binding domain for hnRNP proteins is used to recruit hnRNPs, repressing cryptic exon splicing and restoring normal gene function.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If TDP-43 is depleted or lost, then cryptic exons are aberrantly included in splicing, but functional protein production is depleted and neurodegenerative diseases occur
Solution Approach 1:
The patent employs an engineered U7 snRNA molecule as an intermediary that binds to cryptic exon sequences and recruits hnRNP A1 protein to repress aberrant splicing. This mediator restores splicing fidelity by facilitating the interaction between the guide RNA and the splicing repressor protein, thereby preventing cryptic exon inclusion and restoring functional protein production without requiring TDP-43
Solution Approach 2:
The invention changes the splicing parameters by introducing exogenous U7 snRNA with specific antisense sequences complementary to cryptic exons. This alters the splicing machinery's behavior by providing new binding targets that redirect hnRNP A1 to repress cryptic exon inclusion, effectively changing the splicing outcome from aberrant to correct
2Manufacturing precision
If existing technologies are used to target cryptic exons, then some splicing correction may occur, but they fail to effectively repress cryptic exon inclusion in the absence of TDP-43
Solution Approach 1:
The patent creates a composite therapeutic molecule by fusing U7 snRNA (which provides sequence-specific targeting capability) with hnRNP A1 binding domain (which provides repressor recruitment capability). This composite structure combines the targeting precision of guide RNA with the repressive function of hnRNP proteins, achieving both accurate cryptic exon identification and effective repression that neither component could achieve alone
3Reliability
If hnRNP proteins are recruited to cryptic exons, then splicing repression occurs and cryptic exon inclusion is reduced, but the mechanism must work in the absence of TDP-43 binding
Solution Approach 1:
The patent extracts and separates the splicing repression function from TDP-43 by utilizing hnRNP A1, a different protein family member, to perform the repressor role. The engineered U7 snRNA is designed to recruit hnRNP A1 instead of relying on TDP-43, effectively taking out the dependency on TDP-43 while maintaining the essential splicing repression function through an alternative molecular mechanism
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 construct effectively represses cryptic exon inclusion, restoring functional protein production even in the absence of TDP-43, offering therapeutic potential for diseases associated with TDP-43 dysfunction.
Implementation Method 1
an antisense sequence having between 16 to 30 nucleotides which is at least 90% complementary to a TDP-43 regulated cryptic exon sequence
Implementation Method 2
a sequence comprising a binding domain for a hnRNP protein, wherein the construct is capable of modulating splicing of the TDP-43 regulated cryptic exon
Data Source
AI summary
A modified U7 snRNA construct, more particularly a U7 smOPT construct, is described having (i) an antisense sequence having between 16 to 30 nucleotides which is at least 90% complementary to a TDP-43 regulated cryptic exon sequence or flanking regions thereof, and (ii) a sequence comprising a binding domain for a hnRNP protein, wherein the construct is capable of modulating splicing of the TDP-43 regulated cryptic exon in a cell. Vectors and pharmaceutical compositions comprising the construct are also described, as well as constructs for use in the treatment of diseases associated with TDP-43 dysfunction. Example TDP-43 regulated cryptic exon sequences include TDP-43 regulated cryptic exons in UNC13A, STMN2 and INSR genes.