Catherine Verfaillie

Catholic University of Leuven, Belgium

Genome editing for Wolfram syndrome:

Although rare individually, genetic disorders collectively constitute a common health problem. As the cause of these diseases is a defective gene, gene therapy would be able to resolve all of these disorders. Wolfram syndrome is a genetic disorder, with the ultimate symptoms of Diabetes, blindness and deafness in young kids. The most recent method of gene therapy is gene editing. Gene editing is repairing the defect of the gene by using molecular scissors such as CRISPR/Cas9. CRISPR/Cas9, which is the most commonly used type of this system is, composed of two components Cas9 protein which can make a cleavage in DNA and a guide RNA that is a RNA molecule which binds to Cas9 and guide it towards a specific target sequence of 20 bases. CRISPR/Cas9 allows us to cut the human genome by generating DNA double-strand breaks (DSB) close to the mutation. Such DSB is sensed by two main cellular repair pathways, nonhomologous end-joining (NHEJ) and homology-directed repair (HDR). While NHEJ is generally known to be an error-prone pathway causing insertions and deletions (indels) of DNA bases, which occurs independently of a repair template, HDR relies on the presence of a repair template to refill the correct bases and is considered an error-free pathway. Therefore, HDR has been extensively tested and used for precise gene editing.  Given that HDR is restricted to the S and G2 phases of the cell cycle, only present in dividing cells, this approach might not be suitable for editing and repairing the genome in non-dividing cells. However many of monogenic disorders, including Wolfram syndrome cause disease, because the function of terminally differentiated non-dividing cells in different organs is impaired. Therefore, to reverse the defect caused by monogenic diseases, therapies that can correct the genetic defect in non-dividing cells are required. One very recent tool that is made based on CRISPR/Cas9 system is called Base editor. Base editing is a direct replacement of a single DNA base with the correct one without making a DSB and therefore not relying on HDR. Base editor (BE) is an engineered fusion enzyme consist of Cas9 and cytidine deaminase (CD) that enables a C-G to T-A conversion in an activity window that can be as narrow as 1-2 bases. Since the initial description, BE has been extensively improved and expanded. Another important base editing tool is adenine base editors (ABEs) contains a DNA adenosine deaminase instead of CD and enables a T-A to C-G transition. ABE is a major milestone in base editing considering that the target mutations (C-G to T-A ) account for half of the pathogenic known point mutations in human. Some of the mutations causing wolfram syndrome are targetable by ABE (e.g. c.2002 C>T or c.1620 G>A). ABE has been proven effective in mice on different non-dividing cell types including the cells in retina. However, due to differences between mice and human in many aspects and specifically on DNA repair system, before moving forward to clinical application of ABE, it is important to evaluate this tool on human cells in vitro (in a dish). Therefore, in our study we are planning to evaluate the efficiency of ABE on human derived non-dividing cells. Since these cells are not accessible from human, we will generate them in the lab from Wolfram patient derived stem cells. We are focused on two cell types; oligodendrocytes and retinal ganglion cells as two non-dividing cells leading to eye and brain related symptoms of Wolfram syndrome. We successfully differentiated our patient derived stem cells to oligodendrocytes and retinal ganglion cells. Next, we tested the efficiency of the ABE tool on the stem cells of patients to confirm that the tool is generated correctly. Our result showed highly efficient correction of the mutated DNA base in the stem cells of patients (around 90%). Therefore, we proceed with making delivery vectors to be able to get the ABE tool to oligodendrocyte and retinal ganglion cells. Our vectors are successfully generated, and we are currently performing delivery of the tools to our target cells. We hope to release the result of this part in near future.

–Catherine Verfaille