Last post on Split-SCN1A

The idea for split-intein based SCN1A reconstitution started when I first learned of the split-Cas9 system. Long story short, packaging Cas9 with a guideRNA sequence into an AAV vector is pushing the packaging capacity of AAV vector. To circumvent this issue, the authors design a split-Cas9 system where the coding sequence for Cas9 is split into two fragments, each fused to a split-inteins sequence. When co-expressed in cells, the split-inteins can function as glue joining two protein fragments together.

In an attempt to “shrink” SCN1A to be packageable in AAV vectors, I ran into several design and cloning issues, ultimately leading to the decision to terminate this project. Cas9 is a well-studied cytoplasmic protein with structural information while SCN1A is a large multi-pass transmembrane protein that weaves in and out of the plasma membrane. While success packaging and application of the split-Cas9 system for gene editing is encouraging, my attempt to translate similar design to SCN1A, without structural information, proves to be an oversight. Design aside, the coding sequence for human SCN1A is difficult to maintain. The SCN1A cDNA sequence tends to accumulate deletions and rearranges when transformed and propagated in E. coli. One way to improve plasmid stability and reduce unwanted rearrangement is by propagating plasmid in E. coli strains with recA1 genotype at room temperature. recA1 confers a loss of function in RecA1, a protein that is essential for homologous recombinational DNA repair in E. coli. While this strategy helps to alleviate the problem, it is time-consuming and eventually, I decided to make use of a codon-optimized human SCN1A cDNA as the cloning template for split-SCN1A constructs. While this solves the plasmid stability issue, the codon-optimized cDNA has not been well characterized and while the proteins expressed reconstituted, they failed to traffic to the correct cellular compartment. These raise an issue that needs to be addressed – how to propagate mutation-free gene therapy vectors with SCN1A sequence in large scale, have any of us managed to move the project to treatment pipeline.

Thank you all for reading. While this project didn’t work out as planned, I truly believe that this is an exciting time for Dravet Syndrome. With recent positive Fenfluramine clinical trial results, exciting new drugs in the pipeline, and cross-continental efforts in pushing various gene therapy approaches for treating Dravet, I am hopeful that we can see these exciting results moving into medical treatment in the foreseeable future.

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