Variant RNAi Scaffolds for Alpha-Synuclein Silencing and Neuroprotection
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Solution Overview
Problem
Current RNAi therapies for neurodegenerative synucleinopathies face challenges due to off-target silencing and neuronal toxicity, limiting their effectiveness in reducing alpha-synuclein levels, which are neurotoxic and contribute to diseases like Parkinson's disease and multiple system atrophy.
Innovation Solution
Development of variant RNAi molecules with specific guide and non-guide regions, linked by a loop structure, targeting alpha-synuclein (SNCA) expression, delivered via recombinant adeno-associated virus (rAAV) vectors, to reduce off-target effects and enhance neuroprotection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional RNAi molecules are used to target alpha-synuclein, then alpha-synuclein expression is reduced, but off-target silencing occurs and neuronal toxicity increases
Solution Approach 1:
The RNAi molecule is divided into distinct functional regions: a guide region (nucleotides 1-20) responsible for target recognition and binding, and a non-guide region (nucleotides 21-30) that does not contribute to target specificity. This segmentation allows optimization of the guide region for high-specificity alpha-synuclein targeting while the non-guide region can be designed to minimize off-target effects. The patent applies this by providing specific guide sequences (SEQ ID NOs: 24, 8) that are highly specific to alpha-synuclein mRNA, thereby reducing off-target silencing while maintaining effective target knockdown.
Solution Approach 2:
Different regions of the RNAi molecule are assigned different functional properties: the guide region is optimized for high-affinity binding to alpha-synuclein mRNA with minimal homology to other mRNAs, while the non-guide region is designed to avoid complementarity to off-target sequences. The patent implements this by selecting guide sequences with high specificity scores and designing non-guide sequences that do not create unwanted binding interactions, thus achieving localized optimization of specificity throughout the molecule.
2Reliability
If RNAi molecules are introduced to reduce alpha-synuclein levels, then neuroprotection is achieved, but neuronal toxicity from the RNAi itself increases
Solution Approach 1:
The patent extracts and eliminates the harmful component by removing the toxic effects of siRNA/shRNA through the use of an artificial miRNA scaffold (shmiRNA). This scaffold is processed by the cell's endogenous Dicer enzyme to produce functional miRNA molecules that interfere with alpha-synuclein expression without the neuronal toxicity associated with direct siRNA or shRNA introduction. The guide region (SEQ ID NOs: 24, 8) is integrated into the shmiRNA scaffold, allowing the beneficial neuroprotective effect while avoiding the harmful toxicity through molecular redesign.
Solution Approach 2:
The artificial miRNA scaffold acts as an intermediary between the therapeutic goal (reducing alpha-synuclein) and the delivery mechanism. Instead of directly introducing toxic siRNA/shRNA, the scaffold serves as a carrier that is processed into functional miRNA molecules, mediating the gene silencing effect while the scaffold itself and its processing intermediates avoid direct neuronal toxicity. This intermediary approach allows the therapeutic function to be achieved through a safer molecular pathway.
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 variant RNAi molecules effectively inhibit alpha-synuclein expression, demonstrating significant knockdown in vitro and in vivo, providing neuroprotection and potential therapeutic benefits for neurodegenerative diseases.
Implementation Method 1
RNA interference (RNAi) is a mechanism whereby target mRNAs are reduced by introducing RNA (siRNA) that is complementary to the target
Implementation Method 2
The siRNA sequence can also be inserted into an artificial miRNA scaffold ('shmiRNA') which allows for constitutive, polymerase II-based expression
Data Source
AI summary
Provided herein are RNAi molecules for treating neurodegenerative synucleinopathies. In some embodiments, the RNAi molecules target expression of alpha-synuclein (SNCA). Further provided herein are expression constructs, vectors (e.g. rAAV), cells, viral particles, and pharmaceutical compositions containing the RNAi. Yet further provided herein are methods and kits related to the use of the RNAi, for example, to treat neurodegenerative synucleinopathies including Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies.