Wilson’s Disease (WD) is a liver disease caused by mutations in a protein responsible for copper excretion. That protein, ATPase7B (ATP7B), is a pump that helps move copper in liver cells, and mutations in it cause it to lose its ability to function. The impaired excretion of copper from the liver leads to the accumulation of copper in the organs such as the liver and the brain. There are some treatments for this disease such as zinc supplements and drugs to absorb the excess copper, but those often are not sufficient, and the only curative treatment is a liver transplant. Donor livers are not particularly abundant to say the least, so alternative therapies, such as gene therapy, are being explored as treatments for WD.
In this study, the researchers looked at never before studied approach for repairing a specific mutation in the ATP7B gene in human cells. Using CRISPR/Cas9, a protein (Cas9) that can cut DNA paired with a guiding RNA molecule to target a specific DNA sequence, the researchers sought to first induce a mutation in the ATP7B gene and then repair the mutation. The guiding RNA molecule is highly specific, and even one incorrect letter will lead to the CRISPR/Cas9 system not working. When using CRISPR, there are two possibilities for what happens after the Cas9 protein cuts the DNA. One mechanism, called non-homologous end-joining, is very error-prone and is used to “knock out” a gene, inactivating it. The other mechanism, called homology-directed repair (HDR), uses a DNA template to repair the cut by copying the template. In their experiments, the researchers wanted to find a way to induce the second mechanism, HDR, in order to copy in a non-mutated DNA sequence to repair the mutation in the ATP7B gene. Using human embryonic kidney (HEK) cells and the CRISPR/Cas9 system, the researchers first deleted a single DNA nucleotide from the ATP7B gene, which resulted in a loss of the protein’s function similar to WD. To make sure they induced a mutation in the right place and got the results they wanted, the researchers added copper to the cells, which should not impact normal cells but will kill cells with a mutation in ATP7B. Sure enough, the mutated cells were not able to survive copper treatment, indicating that the CRISPR mutation succeeded.
Next, the researchers wanted to try to use CRISPR again, this time to repair the mutation and return the cells to normal ATP7B function. Using a short sequence of DNA know as a single-stranded oligo DNA nucleotide (ssODN) as the template, the researchers corrected the mutation. They used a couple ssODNs, each with a different number of “blocking mutations”, which are important to prevent recutting of the DNA by Cas9 after the initial cut and increase the accuracy of HDR. To confirm that they did indeed repair the gene, they treated them with copper under a couple different conditions as well as sequenced the cells. The researchers wanted to select for cells that had a functioning ATP7B as well as determine which conditions were the most efficient for gene repair, so they treated them with copper under 3 conditions. One condition was copper treatment 24 hours after CRISPR treatment, another was treatment 72 hours after, and the third was no copper treatment. The results of this test were that the copper treatment 72 hours after CRISPR was the most effective, resulting in 100% CRISPR activity with ~60% repair efficiency. The sequencing confirmed that the repair was in the right place in the cells that did have CRISPR activity, and then they treated the cells with copper again to determine whether or not the gene repair restored the activity of ATP7B. The cells that were repaired in either one or both copies of the ATP7B gene, as well as the wild type (normal) cells, all had the same survival in copper treatment, compared to greatly decreased survival in cells without the ATP7B gene, indicating that the repair was successful in reactivating ATP7B. Finally, to confirm that the ATP7B protein was present, the researchers used a method called a Western Blot, which allows them to visualize the amount of protein in cells. Consistent with the results of the copper treatment, the cells that were repaired as well as the wild type cells all had the ATP7B protein present, although the cells with only one copy had less protein. The researchers were successfully able to both induce a mutation and then repair that mutation, all using a novel CRISPR/Cas9 system.
Gene therapy is very promising in treating a number of diseases, but it is not without its drawbacks. In this experiment, the researchers only look at a single point mutation, meaning only one DNA base is switched or missing, in the ATP7B gene. Within just a small segment of the ATP7B gene, there have been 122 mutations associated with WD. Interestingly, the specific mutation the researchers analyzed is very similar to an identified mutation in some WD patients, meaning that this mutation that they repaired may have clinical significance. Given the wide variety of mutations in WD, any gene therapy would need to be personalized and specific for each patient, making it difficult to scale. As well, delivery of this CRISPR/Cas9 system to the liver, where the mutation has the greatest impact, also poses a difficulty. Other research has shown a number of successful methods of gene therapy delivery in animal models, some using virus and others using a method known as hydrodynamic delivery. Although successful, viral delivery poses some issues, such as an immune response to the treatment, which would render the therapy useless. The other method, hydrodynamic delivery, shows promise as a non-viral delivery method for gene therapies, and it utilizes blood circulation to insert the therapeutics into liver tissue. Specifically, for WD, the copper accumulation in the liver may increase the efficiency of CRISPR gene therapies, per the results above. Gene therapies do have the risk of off-target effects, and possible immune responses to the Cas9 protein, which is bacterial in origin, but the promise of curing diseases like Wilson’s Disease gives hope to many.