Gene Replacement Therapy
Using an AAV9 vector for replacing a mutated gene that causes disease with a healthy copy of the gene.

Spliceosome-Mediated RNA Trans-Splicing
Using ASOs to correct mutations at the post-transcriptional level by modifying the mRNA.

Targeted Augmentation of Nuclear Gene Output
Using ASOs that target RNA splicing to increase protein production from the healthy gene.

CRISPR Genome Editing (prime editing)
A “search and replace” gene editing method that can correct mutations in a precise way.

Gene replacement therapy is single dose treatment in which a patient receives a new, working copy of the missing or nonfunctional gene. The new gene copy is able to give the body instructions to produce a particular protein that the person is missing.
Gene therapy is delivered via vectors that carry a new, working copy of the gene into the right cells inside the body. The most commonly used vector is AAV (adeno-associated virus). Viruses are used because they are very good at transporting cargo into cells. The original, viral DNA, is replaced with DNA encoding the gene of interest, and the virus is therefore no longer considered a virus, but solely a transport vector, unable to propagate or cause disease.
The CTNNB1 gene is a very good candidate for gene replacement therapy since CTNNB1 Syndrome is caused by a loss-of-function mutation (one copy of the gene does not function properly), and also because this specific gene is small enough to fit into the vector of choice.
The program “Accelerating Development of gene therapies for CTNNB1” has already started. This program is led by the University of Sydney and Children’s Medical Research Institute in a collaboration with the National Institute of Chemistry Slovenia. Read more HERE.

The Spliceosome-Mediated RNA Trans-Splicing (SMART) approach relies on correcting the mutation at the post-transcriptional level by correcting the mRNA sequence. With this approach, the gene retains its natural function, meaning the expression of the protein is self-regulated. This is important for the CTNNB1 gene, because an appropriate level of the gene product is critical. An important challenge in this approach is to achieve maximal efficiency, optimally resulting in a 100% repair of the endogenous mRNA target. Although this approach has been established in several models of genetic diseases, it appeared to be more efficient in recessive mutations (where only half or even less of the mutant mRNA target can be enough to see a significant effect). In the context of dominant negative mutations, a highly efficient trans-splicing reaction is needed.
Our researchers are investigating several strategies to improve the efficiency of RNA trans-splicing. Initially experiments will be performed in HEK293 cells. An additional RNA-level approach includes exon-skipping, however based on the evaluation it is not applicable for the CTNNB1 Syndrome.

Targeted Augmentation of Nuclear Gene Output is one of more promising approaches. This is an antisense oligonucleotide (ASO) technology developed by Stoke Therapeutics. This approach relies on targeting naturally occurring, non-productive RNA splicing events to restore proteins to a normal level. Our researchers are currently screening the CTNNB1 gene to identify non-productive alternative splicing events.

CRISPR/CAS9 Prime editing offers incredible promise to reverse genetic diseases, however, it is still at an early stage. Our researchers will work on the development of prime editing variation for genome editing for delivery via viral or nonviral (RNP, RNA) delivery. Initially this will be tested on a reporter and if successful on relevant cells harboring the specific CTNNB1 mutation.

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