SMN2 Base Editing to Stabilize SMN Protein in SMA
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Solution Overview
Problem
Current treatments for spinal muscular atrophy (SMA), such as antisense oligonucleotides, provide only transient upregulation of SMN protein and do not offer a lasting cure, as they do not address the stability issues of the truncated SMN2 protein.
Innovation Solution
Employing base editing strategies to target the SMN2 genomic locus, specifically editing the C840T mutation and removing or inactivating degrons like the EMLA sequence to enhance SMN protein production and stability, using base editors and guide RNAs to correct the nucleobase edits and modify the SMN2 gene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If base editing strategies are employed to target the SMN2 genomic locus and remove degrons, then SMN protein stability and functional activity are enhanced, but the device complexity and difficulty of gene editing increase
Solution Approach 1:
The base editor system is segmented into distinct functional modules: a guide RNA component for target recognition, a Cas9 nuclease component for DNA binding and cleavage, and a deaminase component for the actual base conversion. This segmentation allows each component to be optimized independently and facilitates the removal of specific degrons like EMLA from the SMN2 gene without affecting other regions.
Solution Approach 2:
The patent employs an intermediary base editing approach rather than direct gene replacement. The base editor acts as an intermediary tool that facilitates precise nucleobase conversions (C-to-T or A-to-G) at specific positions in the SMN2 gene, enabling the removal of degrons and stabilization of SMN protein without requiring complete gene replacement or complex viral vector systems.
2Duration of action of stationary object
If base editors are used to correct nucleobase edits in SMN2 gene, then lasting cure for SMA is achieved, but the manufacturing precision and editing accuracy requirements increase
Solution Approach 1:
The base editing approach applies local quality changes at specific nucleobase positions within the SMN2 gene rather than affecting the entire gene. The guide RNA is designed to target specific regions containing degrons like EMLA, and the deaminase is positioned to convert only the necessary nucleobases (C-to-T or A-to-G) at those locations, achieving precise local modification that stabilizes SMN protein without altering other functional regions.
Solution Approach 2:
The invention changes specific nucleobase parameters (C to T or A to G) at targeted positions in the SMN2 gene through base editing. This parameter change approach allows for precise modification of the gene sequence to remove degrons and stabilize SMN protein, creating a lasting therapeutic effect without requiring complex gene replacement or affecting non-targeted regions of the genome.
3Productivity
If base editing targets C840T mutation and removes EMLA sequence, then SMN protein production is enhanced, but the treatment duration and complexity of the procedure increase
Solution Approach 1:
The base editor system performs preliminary action by pre-targeting and pre-modifying the SMN2 gene sequence before SMN protein production occurs. The guide RNA and Cas9-nuclease complex first bind to the target region containing the C840T mutation and EMLA sequence, and the deaminase then performs the nucleobase conversion to remove the degron. This preliminary genetic modification ensures that subsequent SMN protein production is enhanced without requiring repeated treatments or prolonged procedures.
Data Source
Figure 1A~1B
Figure 2A~2B
Figure 2C
AI summary
The disclosure provides methods, base editors, vectors encoding base editors and cognate gRNAs, and compositions and kits comprise said components, for installing nucleobase edits to the SMN2 locus to increase the activity and/or amount and/or stability of SMN2 protein in a cell, thereby treating Spinal Muscular Atrophy. In certain aspect, the disclosure provides compositions and methods to edit C840T of exon 7 of the SMN2 gene, or installing another one or more nucleobase edits which have the effect of removing or inactivating a degron, such as the C-terminal portion of the region encoded by exon 6 or the 4-amino acid region encoded by exon 8 (i.e., the EMLA (SEQ ID NO: 466) -tail) so as to remove or limit their degron activity to reduce, mitigate, or eliminate the intracellular degradation of the SMN2 protein.