Testing CRISPR Therapeutics in DMD Patient Cells: Process and Timeline

Establishing a cell line for Duchenne Muscular Dystrophy (DMD) is very important for several reasons: it helps to understand how the mutation impacts the dystrophin messenger RNA (mRNA) and protein, and it also allows for the testing of different therapies including genome editing treatments such as CRISPR. Establishing these key tools involves several key steps and can take a considerable amount of time. This is a critical step in our process to gauge efficacy of the approach before moving into testing in mouse models.

Here is an overview of the process and a rough timeline:

1. Cell Line Selection & Preparation (3 months)

A cell line is a culture in which all of the cells are genetically identical. In the case of DMD, all cells will have the same mutation in the dystrophin gene that is being targeted for treatment. 

When establishing a cell line for neuromuscular diseases, a muscle tissue biopsy is performed. The type of muscle chosen depends on the disease and patient symptoms. Surgeons aim to biopsy muscle tissue in which patients are experiencing some symptoms, but are not significantly atrophied. If the muscle is too atrophied, meaning most muscle fibers have disappeared, it may not be usable. To perform the biopsy, the surgeon makes a small incision and extracts a portion of the muscle tissue. Then, it is placed on ice with cell culture media to keep the cells alive and is sent to the proper facility for processing. 

From this muscle biopsy, myoblasts are isolated, which are the precursor cells to mature muscle. The cells are cultured in a laboratory, ensuring they are suitable for investigating the outcome of the mutation on the dystrophin mRNA and protein as well as for testing therapies such as genome editing. By providing a cell culture media for cells to grow, the cell line can be properly maintained and will reproduce, in-vitro, what happens in the muscle of the DMD patient. 

It is also very important to collect blood and serum, both for whole genome sequencing and to test for neutralizing antibodies (NAbs). Neutralizing antibodies are naturally produced by the body against foreign antigens. Some patients have high levels of NAbs against what most researchers use to deliver the treatment: a small virus referred as the adeno-associated virus (AAV). This small virus, which does not become integrated into the genome of the patient, has been shown to be safe and to target most of the muscle affected in DMD patients. However, humans could have already been in the presence of AAV as it is a natural virus. Therefore, It is essential to evaluate NAb levels in the blood and serum of patients prior to performing clinical trials because of the risk of an immune response. 

2. Designing CRISPR Components (2-3 months)

Next, researchers use the patient cell lines to develop a genome editing strategy using the CRISPR approach. CRISPR is a system naturally occurring in bacteria that is used to defend against pathogens if they are re-infected by a same or similar virus. Bacteria use this approach to cut foreign DNA.

Researchers have modified this system to apply it to the human genome, using it to edit disease-causing genetic mutations. It can cut the DNA of the DMD patient, thus removing the mutation or replacing it with a normal version of the gene. To accomplish this, a guide RNA, or gRNA, is used. Guide RNA is a molecule that binds specifically to the segment of DNA that is being modified. Many gRNAs need to be tested during this phase in order to identify the most efficacious and safest one. This gRNA is delivered in combination with molecular “scissors” referred to as Cas9, a protein that can cut the DNA when it binds to the gRNA.

This approach (gRNA and Cas9) is being delivered by AAV which, when injected systemically, can target DMD muscles, including the heart and the diaphragm.

3. CRISPR Delivery & in vitro Testing (2-3 months)

Next, the myoblasts are cultured into myotubes. Myotubes are mature muscle cells that no longer divide. Myotubes produce dystrophin protein, so levels of expression can be measured. The AAV CRISPR approach is then delivered to the myotubes to evaluate the effect of the genome editing therapy and its effect on protein restoration and correction of the mutation.

Because the cells have the same genome as the patient, they can also be analyzed to assess off-target effects (when a therapeutic has targeted a different location in the genome than the intended one). At this stage, the therapeutic is optimized by modifying the gRNA to minimize off-target effects and ensure proper targeting of the mutation.


Overall Timeline: Approximately 7-9 months

This timeline is an estimate and can vary based on various factors, including the complexity of the genetic editing, the growth characteristics of the cell line, and the resources available. It's important to note that this is only the preclinical phase. In-vivo, or animal model studies must be performed, and FDA approval must be obtained before proceeding to the clinical trial stage. 

Establishing and testing a cell line for rare and ultra-rare diseases like DMD is a meticulous and time-consuming process, but it is an essential step that must be taken to develop safe and effective treatments.