August 12, 2022
Unlike traditional methods for developing therapeutics for common and (relatively) rare diseases, ultra-rare disease drug development requires a different approach and mechanism in light of a limited ability to commercialize and even reach BLA/NDA approval once clinical trials are complete.
This document serves as an overview for the methodology that Cure Rare Disease and our collaborators pursued to develop a First-in-Human CRISPR transcriptional activator for a single patient and offers a potential pathway for the lean development of therapeutics for other ultra-rare diseases amenable to gene replacement, gene-editing or antisense oligonucleotide (ASO) technologies. Please note that this overview references examples using Duchenne muscular dystrophy and CRISPR primarily.
In characterizing the mutation, it is essential to collect inputs from the patient or patient population. Under CRD’s research study, the team will enroll patients of interest in the study to allow for collection of relevant biological materials such as skin, muscle and blood.
One of the first steps in the therapeutic development process is establishing a cell line, which is a culture of cells that are genetically identical to one another. In this case, the cells will have the mutation or disease that is being targeted for treatment. As long as they are given a growth medium (this can be thought of as “food” for cells) and have adequate space, the cells can continue to reproduce, in-vitro, for an extended period of time. The term “in-vitro” means “in glass” and simply refers to any studies that are conducted outside of the body. This is different from the in-vivo studies that are conducted in animal models in later steps.
To obtain the cells to establish a patient cell line, a tissue biopsy is performed. In the case of neuromuscular diseases like Duchenne muscular dystrophy, muscle tissue is utilized. The muscle is chosen on the basis of clinical presentation of the disease in question (the type and location of the muscles most commonly affected by the disorder), as well as patient symptoms. If possible, the surgeon will choose to take the biopsy from a muscle that the patient is experiencing some symptoms in, but not one that is significantly atrophied. Atrophy is the wasting away of muscle cells that occurs in diseases like Duchenne, so if the biopsied muscle exhibits severe atrophy, it will not be usable. The biopsy is an outpatient procedure that can be performed with a local anesthetic to numb the specific location that the biopsy will be taken from. For younger children, though, general anesthesia is often given so that they will be asleep throughout the entire procedure. At this time, the surgeon makes a small incision and extracts a portion of muscle tissue. Once the sample is collected, the tissue is immediately placed on ice to keep the cells chilled and alive, and the sample is sent to the lab at UMass for processing. If general anesthesia was used, the patient will wake up shortly after the procedure is finished. Sometimes, but not always, they may feel groggy, but this should wear off within a few hours. Patients may experience soreness at the site of the biopsy for a few days following the procedure, but doctors will provide guidance on pain management.
In addition to the biopsy, the collection of blood and serum allows for whole genome sequencing (WGS) as well as neutralizing antibody analysis against AAV vectors. When designing a therapeutic for a small group of individuals, or even an individual, it’s important to know the challenges that may be ahead including immunity to AAV vectors. This information will be important later when designing the clinical trial and the immune suppression protocol, if relevant. Whole genome sequencing, for CRISPR-based therapeutics, clarifies the break points of the mutation in question. From time to time, WGS will also identify a potential inconsistency within the clinical diagnostic report.
The major goal of this step is to understand in full detail what genetic mutation the patient has as well as how it is affecting production of RNA and the protein that the gene codes for. Knowing this information is vital so that a therapeutic can be developed that targets the specific mutation that the patient harbors. Of course, some components of this step may be avoided depending on the indication and complexity of the diagnosis present in the patient(s) intended for treatment.
After the cell line is established, the therapeutic is prototyped. For CRISPR, therapeutic prototyping is done by selecting the ideal guide RNA that bind to the mutation site; the ideal promoter to improve upon the safety of the therapeutic; lastly, is the selection of the AAV serotype. Based on the results of the neutralizing antibody assessment conducted in Step 1, and based on the target tissue, an informed selection can be made. It’s important to understand that ultra-rare disease drug development programs are urgent – therefore, utilizing technologies which are, or already have been, used in clinical trials will speed the path to FDA approval since FDA has previously been exposed to the components through other trials. Since this overview is focused on the development of a CRISPR therapeutic, the content will be based on that technology.
For CRISPR, which stands for ‘clustered regularly interspaced short palindromic repeats,’ is a system that is naturally found in bacteria. They use it to recognize and defend themselves against pathogens if they are infected a second time by the same or a similar virus. Using this as a model, scientists have manipulated the system to be able to target specific lengths of DNA, allowing for gene editing techniques to be applied to treat genetic disorders like Duchenne muscular dystrophy, which results from a mutation in the dystrophin gene. A CRISPR therapeutic is broadly composed of 4 pieces: Guide RNA (gRNA), Cas9, a promoter and AAV. gRNA will bind itself to the section of DNA that needs to be modified. An enzyme such as Cas9 (CRISPR-associated protein 9) can then cut the DNA at that location like a pair of scissors so that gene editing can occur. The adeno-associated virus (AAV) is used as a vector to deliver the DNA that codes for the production of guide RNA and the enzyme that cuts the DNA, like Cas9. The muscle promoter is another component that ensures that the correct tissue (muscle tissue, in the case of neuromuscular disorders) is being targeted. In a process called CRISPR-mediated exon skipping, this technology is leveraged to delete one or more exons, which are coding portions of DNA. In doing so, the gene will now be able to be read and translated into a full or near-full length functional protein.
The goal of the first therapeutic that CRD developed to treat its first patient is to upregulate an alternate dystrophin isoform. An isoform is a protein that has an extremely similar, but not entirely identical, amino acid sequence as another protein. Amino acids are the building blocks of a protein.
Before applying the system to in-vivo studies, the cell line that was created from the patient’s muscle biopsy is used to test the prototyped CRISPR therapeutic to measure for dystrophin upregulation on a DNA, RNA and protein level. By testing the therapeutic in the cell line, efficacy can be evaluated and changes can be made to optimize it in-vitro before moving on to the in-vivo studies, such as changing gRNA to target a different part of the genome.
The next phase marks the transition from in-vitro to in-vivo testing. In-vivo studies are most often conducted using rodent models. Rodents are used for a variety of reasons: they are small, reproduce relatively quickly, and are genetically similar to humans, meaning they can develop similar diseases. CRISPR-based therapeutics are both mutation-specific and human specific, so the mice are genetically engineered to have a humanized version of the gene with the specific mutation being targeted on that humanized gene. The dystrophin gene is one of the longest known human genes, and there are many different mutations within this gene that can lead to Duchenne. This means that for each therapeutic developed to target a specific mutation, a different mouse model will need to be generated, a process that often takes 2 or more years and is done in parallel with cell line and therapeutic development and optimization. Moving forward, one key goal is to reduce the need for animal models to show pharmacology once a better understanding of CRISPR is achieved.
The purpose of in-vivo testing is to show that not only does the treatment work in a cell line, but also within a living organism. At this stage, it is extremely important to determine to what degree is efficacy maintained (durability) and to what degree is target upregulation achieved at different doses. The mice are observed at different doses of the drug to determine protein expression levels, mortality rates, and any possible reactions that result from dosage. At intervals over a period of 4 weeks to 6 months, quantitative PCR (qPCR) tests are conducted to measure the production of the protein in the target tissues such as heart and skeletal muscle. This confirms that in the mice, gene upregulation is maintained by the therapy over an extended period of time. It is also important to understand what can be reasonably measured as far as pharmacology goes. In our first endeavor, we attempted to use a mass spectroscopy assay to measure the Dp427c protein, though given the high degree of similarity between the Dp427m and Dp427c isoforms, this approach proved challenging. In engaging with the FDA during a preIND meeting, we expressed this and FDA found it suitable to measure mRNA levels. Again, as is innate to ultra-rare drug development programs, time is of the essence and must be balanced with the reasonable ability to measure various endpoints. In a perfect setting, additional resources and effort would be applied to better measure efficacy.
Once the in-vivo mouse model studies are successfully completed, the team begins to prepare a pre-IND briefing for use in the pre-IND meeting with the FDA. Before a clinical trial can be conducted, an Investigational New Drug (IND) application must be submitted to the FDA for review. During the pre-IND meeting, the research that has been done up to that point is presented and comments and feedback are offered. The logistics of the meeting include submitting a meeting request which is then followed by a briefing document from the sponsor. Once the meeting request is submitted, 30 days later the FDA will communicate the meeting date which will be roughly 60 days after that period. Excluding preparation of the documents (which take 1-2 months), the preIND process is roughly 3 months long. The guidance provided gives a template and timeline for the current therapeutic’s development as well as for future onesassuming a high degree of similarity. For CRD’s first patient, this meeting took place in the Fall of 2020. This was a great opportunity to present the case to the FDA, including the story of the program, the intended patient(s) for treatment and to get feedback from the FDA to guide the rest of the program. For our program, FDA provided very helpful comments just before the meeting which answered the majority of our questions. A few remained and so we took advantage of the opportunity to have a teleconference with the FDA. Following the preIND meeting, we began GMP plasmid production though engaged a number of times, over email, with our Regulatory Program Manager with questions.
With pre-IND feedback in hand, the next phase is focused on producing the human grade (GMP:good manufacturing practices) therapeutic. Producing the GMP material includes the production of both the plasmid (AAV helper, Ad helper, GOI plasmid) along with the AAV component. This AAV manufacturing and testing process, once started, will take roughly 6 months until full release. To expedite timelines, CRD made the decision to take the GMP material prior to QC into our toxicology study. Although at-risk, this allowed us to save 3 months worth of time while the material was going through release testing. It’s also important to remember the supportive assays needed – a titer assay, stability studies and preclinical / clinical device compatibility studies. These assays, especially the device compatibility study, can result in a clinical hold if not conducted. While there is a degree of leeway with what type of material can be used for the supportive assays/study, it’s best to use GMP material to reduce risk. If there is limited GMP material due to poor yields, it is critical to communicate this to the FDA and possible to make an argument, where appropriate, to use toxicology or research grade material if it is the same product (serotype, plasmids) made in the same way (adherent, suspension methodologies for instance). For each drug, manufacturing costs are roughly $2+ million, so this phase can often be a challenge due to the extremely high price tag.
Identifying the CMO to work with is critical. CMOs come in a variety of levels of sophistication. CRD felt it was best to use a US-based supplier to reduce transit risk and conflicting regulatory requirements (USP vs. EP) and language barriers. Moreover, it’s important to talk to customers who have worked with the CMO of interest before, understanding how long the team has worked together, and the number of failed runs. CMC consultants often have a great perspective having worked with multiple clients before and offer a good source of input prior to engage in a large contract. Another point to consider is simplifying the plasmid and AAV production into one provider so that the program is more seamless and potentially lower risk.
With the GMP material completed, toxicology studies are conducted to determine the safety risks associated with administering the therapeutic to the patient(s). For CRD, we elected to pursue acombined pharmacology and toxicology study as previous pharmacology studies were based on smaller volumes of animals. It’s important to note that most, if not all drugs, have some degree of side effect or risk. The purpose of the toxicology study is to understand to what degree those risks present themselves at different doses. The FDA considers the risks in light of the disease the drug is intended to treat. Duration of the GLP study depends on the intention of the program and severity of the disease. One strategy to consider is running a multiple timepoint study - a shorter, agreed upon timepoint to dose a single patient, potentially 4-12 weeks in length – and then a longer timepoint to allow for the potential future dosing of additional patients such as a 26-week study. Working with the ideal CRO is critical in this step as the reporting post-takedown can take several months. With time being critical, Charles River Labs acted as an excellent partner to expedite the study reporting so that it did not become a bottleneck.
Submitting an Investigational New Drug (IND) application for FDA approval is the final preclinical step before conducting a clinical trial. A series of in-vitro and in-vivo pharmacology and toxicology studies are conducted in partnership with academic and industry collaborators to determine that the CRISPR-based therapeutic is not only effective in upregulating dystrophin production, but also does not cause unacceptable side effects, such as altering the expression of other critical genes. The in-vitro studies involve the use of a patient-derived cell line, while the in-vivo studies utilize a mouse model. The goal of the pharmacodynamics (study of impacts and mechanisms of drugs), toxicology, and biodistribution studies conducted with the mice is to determine that the therapeutic maintains activity over an extended period of time and that no adverse effects result from dosage. Details must also be provided on whether the drug has previously been dosed to any humans, and what the effects of dosage were.
The clinical trial protocol covers in extensive detail the plan for conducting the trial, justification for the methods that will be used, and the care that will be taken to minimize risks to the patient. The protocol also discusses the role of the Site Investigator, whose job is to monitor the patient over the course of the trial to ensure that everything is going as planned. The Site Investigator is responsible for reporting any adverse drug reactions, including those that may require termination of the trial.
The manufacturing information details the development of the therapeutic, including drug composition, storage, and production. This is done to determine whether the drug can be reproduced with consistency, if necessary.
Once the IND has been submitted, the FDA has 30 days to review all of the documents and make a decision. In time sensitive cases, this process can be expedited to 2 or 3 weeks or less. If they find that there are no unnecessary risks to the patient’s health and they have no additional questions, the therapeutic is approved and the clinical trial can begin. If they are not satisfied with any component of the IND, they will come back with specific concerns that must be addressed and handled properly before approval can occur.
The hospital that dosing occurs at, which in this case is UMass Medical School, has an Institutional Review Board, or IRB. The IRB, in addition to the FDA, is responsible for reviewing the documents submitted before approving the clinical trial.
For CRD, each team was responsible for preparing their study reports with guidance from our regulatory lead. With templates in hand, the teams compiled reports and data and then we held roundtable reviews so ensure that the IND submission process was a smooth one. The process took roughly two months and then an additional 30-days, once submitted, to get IND approval from the FDA. Please note – during this 30-day period, CRD and FDA engaged a number of times to discuss questions both over email and phone as needed.
In the final stage, the clinical trial is conducted at UMass Medical School. This drug involves the use of the adeno-associated virus (AAV) as a vector to deliver the CRISPR therapeutic. Many people have naturally occurring antibodies against AAVs called neutralizing antibodies (NAbs), which can make dosing and redosing a challenge in patients who have high levels of NAbs.From our work in the initial Step 1, we knew that the patient’s NAb levels were relatively low which made immune suppression a lesser challenge. However, given the advantage of dosing small groups or even an individual, immune suppression protocols can be geared toward the patient(s) and their respective titer levels. This is a juxtaposition to standard drug development wherein the clinical trial picks the patient vs. the patient picking the clinical trial.
Prior to giving the patient the therapeutic, therefore, they are placed on immunosuppressant drugs to minimize the risk of an immune response to the AAVs. The therapeutic, which is administered in a single dose, is given intravenously in the hospital. The total amount of time the infusion takes is dependent on the patient’s body weight and the exact dosage (gene therapies are generally dosed on the basis of viral genomes/kg) but a break is taken after the first few minutes to confirm that there will be no acute reactions such as anaphylactic shock. Afterwards, the patient remains in the hospital for several days to be monitored. Learnings from other gene therapy trials help to inform and shape the clinical plan. For gene therapy especially, it is critical to have a multidisciplinary clinical team active and on stand-by for the trial should intervention become necessary.
Once the hospital confirms that the therapeutic is not causing any severe immune reaction or other adverse reactions, the patient is released. Patient monitoring post-therapeutic administration is different depending on the technology used. For CRISPR, the FDA has shared that the patients should be followed for 15 years. Over this period of 15 years after dosage, the clinician will follow up with the patient to monitor their progress. For other treatments, such as gene replacement therapy, the length of time the physician is required to follow up with the patient is shorter.
As the last three years culminates in our first-in-human dosing, the entire team at Cure Rare Disease, our patient community and collaborators are cautiously excited at the prospect of using CRISPR to stop DMD. Cure Rare Disease intends to share the clinical findings with the community so that others can learn from this accomplishment. We are forever grateful to the community who helped make this vision a reality and for the framework by which other ultra-rare disease patients can find treatment.
As we move forward in developing and treating other patients, a critical challenge that remains is the financial mechanism for payment. It is not within the current structure of private insurance companies to pay for therapeutics which do not have a BLA or NDA. Many ultra-rare interventions may never be commercially approved but rather remain in perpetual IND. Therefore, we, as a society, must incorporate a new mechanism for reimbursement. For without, the very nature of ultra-rare, non-commercial drug development is inaccessible to a large majority of families who do not have the means or network sufficient to raise the $3M+ required to advance a program.
Based on our conversations with private and public payers, it appears as though one potential avenue to move forward is to leverage the Centers for Medicare and Medicaid Services as the US lacks a single payer. A significant amount of effort will be necessary to improve the inequity that exists.
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