Development of a PRIME editing therapy for Ataxia-8 due to the c.121 A to T point mutation
Principal researchers: Prof Jacques Tremblay, Department of Molecular Medicine, Faculty of Medicine, Laval University (Canada)
Scientific summary
The researchers aim to develop a therapy for Spastic Ataxia Type-8 by correcting mutations in the NKX6-2 gene by PRIME editing. All point mutations can in principle be corrected by this technology. The team have successfully used PRIME editing to modify the EMX1 gene in human cells with Duchenne muscular dystrophy as well as insert point mutations in exons 9 and 35 of the DMD gene. The team will focus on the c. 121 A-to-T mutation. They will demonstrate that this mutation can be reversed in human cells by transfecting them with a plasmid coding for the PRIME editing (PE2) enzyme (SpCas9 nickase fused with reverse transcriptase) and for a pegRNA (prime extended guide RNA). This pegRNA will contain a sequence complementary to a 20-nucleotide sequence preceding the SpCas9 PAM (photospacer adjacent motif) located close to the point mutation. The pegRNA will also contain a 3' extension containing a primer sequence for the reverse transcriptase and a reverse template specifying the nucleotides to be modified.
The researchers will determine by PCR and Illumina deep sequencing the percentage of corrected NKX6-2 genes and confirm by western blot the expression of NXK6-2 full-length protein. The researchers will test in normal mice the delivery of the SpCas9-RT, the pegRNA and if necessary the sgRNA by both the AAV and the EV delivery method. Two weeks later, the mice will be sacrificed. DNA will be extracted from the brain and the NKX6-2 gene will be PCR amplified and the amplicons will be deep sequenced. The resulting sequences will be computer analysed to determine what percentage of the sequences have been modified. Mutations in off-target sites will also be investigated by PCR amplification and deep sequencing.
Lay summary
The DNA present in each human cell contains all the genetic information that we have inherited from our parents (e.g., eye and hair colours) including mutations responsible for hereditary diseases. This DNA is formed by only four molecules called nucleotides (A: Adenosine, T: thymidine, C: cytosine and G: guanine). The DNA is a double helix formed from pairs of these nucleotides, either A:T, T:A, G:C or C:G. Half of our genome originates from our mother and the other half from our father. We have received 3.2 billion nucleotide pairs from each parent. Genes carrying the instructions for proteins that make our cells are sequences of these nucleotide pairs.
Patients with Spastic Ataxia Type-8 (SPAX8) have a change in one of these nucleotide pairs in a gene called NKX6-2 leading to problems making proteins. The researchers aim to develop therapies targeting NKX6-2 mutations in SPAX8. Since 2019, a new type of gene editing that originates from CRISPR technology called PRIME editing has been developed. This PRIME editing technique allows for a nucleotide pair to be changed to any other nucleotide pair. They have been able to successfully use PRIME editing to correct mutations in human cells with muscular dystrophy. The team will use this method to correct NKX6-2 mutations in SPAX8, using human cells in the lab. The researchers hope this method will be able to treat those with SPAX8 who have mutations in other parts of the NKX6-2 gene, as well as those with other neuromuscular disorders.
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Ataxia UK, 12 Broadbent Close, London, N6 5JW
Website: www.ataxia.org.uk.
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Email: helpline@ataxia.org.uk.
