Dup15q Alliance Professional Advisory Board Member
University of Connecticut Health Center, Farmington, CT, Associate Professor, Genetics and Genome Sciences
Associate Director, Graduate Program in Genetics and Developmental Biology
CRISPR is a great tool for the lab, but it’s use as a therapeutic for Dup15q is likely far off and not ideal. Although CRISPR cutting is extremely efficient, using CRISPR to delete the duplicated chromosome or chromosome segment altogether is incredibly inefficient–even in the lab. We start with 1 million cells and only sometimes get the idic chromosome deleted. We haven’t ever gotten the interstitial chromosome deleted. One would need this inefficient process to work just right in many individual neurons in the brain without any mistakes. Did I mention it’s inefficient?
Couldn’t we delete/change one gene at a time? Yes, except we still don’t know which gene that would be–and it’s likely more than one. We also can’t control how many of the 3-4 copies of a gene it would hit. Even to change one gene, CRISPR would be in a neuron for a long time, efficiently cutting DNA it shouldn’t be cutting. The repairs of these cuts are full of errors, which can lead to dangerous situations such as mutations and cancer. There are better ways than DNA cutting CRISPRs to reduce gene production, such as ASOs, siRNAs, miRNAs, epigenetic regulation, etc…. These are likely to be safer, more effective, and quicker to be developed as a therapeutic than CRISPR.
That said, there are other CRISPR approaches that may be more likely to be developed as a therapy for Dup15q. These use a “dead” CRISPR that doesn’t cut DNA. Instead this dead CRISPR can be used to bring regulators to specific genes to decrease their production. There are also CRISPRs that cut RNA rather than DNA and can reduce the production from specific genes. We heard about these at our scientific symposium in 2018. There are some risks to RNA-cutting CRISPRs, too, but not as much as DNA-cutting CRISPRs.
In the context of the recent publication about the use of CRISPRs being injected directly into the eye, (https://www.nature.com/articles/d41586-020-00655-8) the researchers stated in the press release that “The gene editing tool stays in the eye and does not travel to other parts of the body, so “if something goes wrong, the chance of harm is very small,” he said. “It makes for a good first step for doing gene editing in the body.”
The reason they can do this in the eye is that they can remove the eye, if anything bad/dangerous (I.e. extra mutations leading to cancer) happens. The eye is also immunoprivileged—which means there’s very little immune reaction in the eye. Elsewhere in the body, the immune system will react to the CRISPR protein, which is yet another ginormous hurdle for CRISPR to overcome before it is used as a therapy in humans. Blood cells are readily CRISPRed because they can be removed, edited by a brief exposure CRISPR, and then confirmed for intended edit and lack of dangerous mutation. The correctly altered cells can then be expanded to produce more of them and replaced into a human. This is called ex vivo editing and cannot be done for neurons. Brain cells cannot be removed from the body, expanded, and replaced.
Finally, one scientist recently publicized her work delivering CRISPRs to the brains of mice with Fragile X syndrome using nanoparticles (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544395/). They used CRISPR to mutate the gene encoding mGluR5, which has been shown to help with Fragile X syndrome in mouse models of the disorder. Although they showed that they could use CRISPR to mutate a single gene in the brain, they used the dirty/dangerous cutting followed by the error-prone repair process to do this. They did not look at what else was mutated and did not follow the mice for long term consequences.
Importantly, they did not change the genetic problem that caused Fragile X, but rather a down-stream target that may have other unintended consequences. This target, mGluR5, can also be efficiently inhibited using traditional small molecule drugs, and there are ongoing clinical trials to test these drugs in humans. It doesn’t seem wise to use a permanent and dangerous CRISPR to accomplish a simple goal that isn’t curative—it’s like using a sledge hammer to perform delicate brain surgery. Sure, the patient might not have the brain problem they had before, but now there are potentially many other problems, and you didn’t even fix the primary problem.
The Dup15q Alliance is dedicated to supporting the evaluation of existing gene therapy technologies, and driving the development of novel technologies forward. We are incredibly proud that this is the primary purpose of our recently awarded CZI grant that focuses on complex copy number variants like dup15q where lots of genes are affected.
CRISPR is an exciting technology that has already revolutionized laboratory science and will likely revolutionize medicine in the future. CRISPR-based approaches can be divided into two major themes: genome editing, which changes the actual DNA base pairs in the genome of a cell and alteration of gene expression, which changes how much of a gene or protein is produced, but does not change the sequence of the DNA itself.
In the lab, we are using the genome editing version of CRISPR to mutate individual genes, to delete an entire copy of the 15q11-q13 region, or to get rid of the isodicentric chromosome in Dup15q induced pluripotent stem cell lines. These are terrific lab tools, but the technology is not efficient enough or safe enough to use as a therapeutic approach just yet.
We are also using the other version of CRISPR to activate or repress genes specifically. In this version, the Cas9 protein, which usually cuts DNA is changed so that it cannot cut the DNA. However, this protein can still be targeted to a specific place in the genome. Groups have attached different cargo to this protein to activate silent or quiet genes or to dampen highly expressed (loud) genes. We have been developing this approach to turn UBE3A up or down in our induced pluripotent stem cell lines. We haven’t yet applied this to Dup15q cells, but if it works well, we will look to do that in the future.
Therapeutic approaches using CRISPR are still a ways out in the future. While some groups have used them therapeutically, this has thus far been limited to blood cells, where they can take a cell out, use CRISPR in a dish, and then return the cell to the patient. This is not really feasible for disorders involving the central nervous system. For therapeutic approaches, the CRISPR components will have to be delivered using a virus, such as AAV. Labs are working on this, but the CRISPR machinery is a little too big for the current approaches. There are also worries about off-target effects—CRISPR can edit the DNA in places that it is not supposed to—which can increase risk of cancer and other issues. Finally, some worry about accidentally editing the germline—eggs and sperm—which could make unintended changes to future generations. This is a major ethical dilemma facing CRISPR therapeutics.
Nonetheless, progress will continue to be made to develop therapeutics using both types of CRISPR approaches. They will likely take longer to develop and vet than some other promising therapeutic approaches. For instance, antisense oligonucleotides (ASOs) and short hairpin RNAs (shRNAs) can all currently be used to alter gene expression in a targeted and reversible fashion. Both of these types of approaches are already used in the clinic for other disorders. If we know which gene(s) to focus on, these might be more promising approaches for Dup15q syndrome.