heading:"Shaping the Future of Genetic Therapy: An interview with Prof. Dr. Zoya Ignatova",
interviewtabid:"ignatova2",
cardtext:"",
quote:"",
aimofcontact:"",
insights:"",
implementation:"",
summary:""
quote:"Precision is key to minimizing side effects and ensuring the safety of your therapy.",
aimofcontact:"We conducted the interview with Prof. Dr. Zoya Ignatova and Dr. Nikhil Bharti from the Institute of Biochemistry and Molecular Biology at the University of Hamburg, seeking to deepen our understanding of their research on cystic fibrosis (CF) and explore additional CF mutations, as well as to learn more about cell culture techniques specific to CF research, since they send us the CFBE41o- cell line. Our aim was also to gather more information about their approaches in CF research, particularly their focus on treating genetic mutations like nonsense mutations, which are highly prevalent in CF.",
insights:[<p>We were struck by Ignatova’s story about founding the iGEM team in Hamburg. Her passion for fostering creativity and innovation in science was inspiring. On a technical level, their advice on cell culture was incredibly practical and immediately useful. Dr. Nikhil Bharti explained how they handle CFBE41o- cells and ALI (air-liquid interface) cell culture. This advice directly addressed the challenges we’ve faced in our own lab, giving us a method to improve our cell culture success rates. During our interview with Prof. Dr. Zoya Ignatova and Dr. Nikhil Bharti, their innovative approach to cystic fibrosis (CF) therapy, particularly "read-through" and tRNA-based therapies, stood out. "Read-through" therapies aim to bypass premature stop codons that prevent full protein production, offering a way to restore the function of critical proteins like CFTR in CF. This approach has the potential to treat a broad range of genetic diseases caused by similar mutations. The tRNA-based therapy is even more precise, targeting mRNA to correct faulty codons without altering the DNA, making it safer for long-term use. This flexibility, along with the ability to apply these therapies beyond CF, broadened our understanding of how such strategies can revolutionize treatments for genetic disorders. A key focus throughout the discussion was safety. Prof. Ignatova emphasized the importance of ensuring that the therapies are highly specific, targeting only the defective codons while avoiding natural stop codons to prevent uncontrolled protein production. Moreover, their careful consideration of delivery systems further demonstrated their commitment to minimizing risks like toxicity in unintended organs. Their meticulous approach to safety has influenced how we think about developing and applying these therapies, making it clear that ensuring patient safety is as critical as achieving therapeutic success.</p>],
implementation:"Prof. Ignatova's practical advice on cell culture had a transformative impact on our project. By adopting her method for CFBE41o- cells and improving our lab's sterilization protocols, we successfully established the cell line and significantly reduced the risk of contamination. In addition, her emphasis on safety in gene therapy guided us to review our Prime Editing construct and lipid nanoparticle (LNP) design. We focused on minimizing toxicity and off-target effects while ensuring precise targeting of lung tissues and the F508del mutation of the CFTR gene, making our approach safer and more efficient",
summary:"",
interview:
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<QaBoxq="We have heard you are passionate about iGEM. What inspired you to get involved, and what has your experience been like with the competition?"
a="My journey with iGEM began when I moved to Hamburg in 2014. Back then, Hamburg did not have its own iGEM team. Despite Hamburg lacking an iGEM presence, there were motivated students who were eager to establish a team. We started quite late with me as a principal instructor, around April, with the competition scheduled for October, so we had limited time. However, we managed to form a team and participate. Fortunately, we were successful in convincing the university administration to establish a steady support for the initiative, which ensured stable funding, including covering registration fees early on. This financial and logistical support gave the team the security to focus on their projects. Over the years, the Hamburg iGEM team has become a well-known and respected group at the university. It is a creative environment where students can push the boundaries of science through interesting and impactful projects. I moved on to other duties after several years of supervision, but I am proud to have played a role in its foundation. The university has recognized iGEM within the curriculum of Molecular Life Science, allowing students to earn credit points and have their work reflected on their transcripts. This acknowledgment further incentivizes students and ensures that their efforts are formally recognized."/>
<QaBoxq="We have been having trouble with CFBE41o- cells not adhering well. Any advice?"
a="CFBE41o- cells can be tricky when it comes to adhesion, but you do not necessarily need to coat your cell culture vessels with fibronectin unless you are doing very specific studies, such as primary culture comparisons. For seeding, we simplify the process by skipping the PBS washing step. Instead, we seed the cells directly into DMEM supplemented with 10% fetal calf serum (FCS) and streptomycin. These cells may take a few days to recover and begin adhering properly, that usually works without requiring extra coatings."/>
<QaBoxq="How do you manage fungal contamination in ALI cultures?"
a="Fungal contamination is one of the more frustrating challenges in cell culture because it is difficult to eliminate once it takes place. In cases of contamination, the best course of action is to shut down all ongoing cell culture work and clean everything thoroughly. You should start by running a sterilization cycle in your incubators, which ideally should reach around 180°C. This should kill any fungal spores. If your incubators do not have that capability, you will need to autoclave everything and clean all surfaces and equipment multiple times with ethanol. It is crucial to remove all traces of contamination, as fungal spores can spread rapidly. The key is prevention through rigorous cleaning and maintenance protocols, and unfortunately, sometimes the only solution is to start fresh with new cultures after a full decontamination round."/>
<QaBoxq="What are you currently researching?"
a="Our primary research focus is on genetic diseases caused by nonsense mutations, also known as premature termination codons (PTCs). While cystic fibrosis (CF) is a major area of interest due to its high prevalence and the impact of specific mutations like the F508del, our research extends far beyond CF. We are targeting a broader category of genetic diseases that share a common feature—early stop codons that lead to production of truncated proteins, which are non-functional. In CF, for instance, our main goal is to restore full-length CFTR protein production in primary patient-derived cells bearing various PTCs. One approach we are exploring is known as a 'read-through' therapy, which involves bypassing the premature stop codon so that the cell can continue producing the full protein. This strategy is applicable not only to CF but can be used in many other genetic disorders caused by nonsense mutations. Briefly, the read-through therapies we develop are tRNA-based therapeutic approaches, in which we design suppressor transfer RNAs (sup-tRNAs) to selectively target and read through PTCs, restoring the production of full-length disease protein without altering the natural termination codons. It is a highly specific and safe method, and because we are targeting mRNA rather than DNA, it allows for terminating the therapies by any unforeseen side effects."/>
<QaBoxq="What are your downstream validation methods?"
a="After we have developed a therapeutic approach, the first step is to validate whether it works at the protein level. First, we check whether the full-length protein is being produced. For CFTR, for example, we look at whether the protein is being correctly synthesized. We also conduct functional tests to ensure its functionality. For CFTR specifically, we test the activity of the ion channel by measuring ion flow through the cell membrane. Another test involves monitoring the height of the air-liquid interface (ALI) cultures, which reports on the ionic balance across the membrane. These functional tests are crucial for confirming that the therapy is not only leading to a production of the protein but is also restoring its function."/>
<QaBoxq="How often would patients need to undergo this therapy?"
a="Since our approach is designed to correct nonsense mutations during translation the therapy would need to be administered periodically. Based on our current understanding, we anticipate that patients might need treatment every three to four weeks, but this has to be determined in clinical settings."/>
<QaBoxq="How does your tRNA-based approach address safety issues?"
a="Safety is the top priority of our tRNA-based therapeutic approach. At molecular level, we ensure that the suppressor tRNAs we use are highly specific—they are engineered to target only PTCs without affecting natural stop codons, which are essential for terminating the synthesis of every protein. In addition to the specificity, we address the immune response that can be triggered by any nucleic acids, including tRNA. Generally, tRNA has a lower immunogenicity than other molecules, such as mRNA, because of its partially double-stranded structure, which reduces the activation of the innate immune reaction. Another critical safety aspect is the safety of the delivery system. We need to ensure that the tRNA reaches the right type of cells without causing toxicity or accumulating in untargeted tissues like the liver, which is a common issue with many gene therapies. We are also working on optimizing our delivery methods. This precision is key to minimizing side effects and ensuring the safety of our therapy."/>
<QaBoxq="Why focus on CF research?"
a="Our involvement with CF research emerged somewhat by chance. Initially, we were deeply interested in understanding the variability in disease, specifically why individuals with the same genetic mutations show different symptoms or present different disease severity. Even siblings or twins with usually similar genetic makeup exhibit different disease outcomes. CF became a focus as we delved into the molecular mechanism of CFTR biosynthesis. However, our work is not confined to CF—we are using the knowledge we gain from CF research and our expertise in protein synthesis and translation to develop treatments for other genetic diseases caused by nonsense mutations. The mechanisms behind these diseases are often similar, so the therapeutic strategies we are exploring can potentially be applied to a range of conditions."/>
<QaBoxq="What do you see as the biggest challenge in translating your research to real-world applications?"
a="Safety is the most critical hurdle in translating our research from the lab to clinical applications. Before any therapy can be considered for human use, we need to ensure that it is both safe and effective. In terms of efficacy, we have specific targets we need to meet for each disease. For CF, for example, you only need to restore about 10% of normal CFTR protein function to alleviate the symptoms. However, in other diseases, the therapeutic threshold is much higher, sometimes requiring near-complete protein restoration. Another significant challenge is the small number of patients affected by many rare genetic diseases, which requires regulation bodies to consider this and redefine conditions for clinical trials."/>
<QaBoxq="What are good preclinical models for CF research, in your view?"
a="Preclinical models are essential for testing the safety and efficacy of any new therapy. For CF research, one of the most reliable models is the patient-derived air-liquid interface (ALI) cultures, which replicate the lung environment and are mutation-specific. Primary cultures are available through the CF Foundation (USA) and allow researchers to test therapies in a context that closely mimics the human lung. While ALI cultures are excellent models, they are also challenging to grow and require about two months to be set up properly. For earlier-stage experiments, we often use simpler cell lines that are easier to handle. These lines allow us to perform studies at molecular level, such as testing how well a therapy restores protein production. While they do not fully represent the primary epithelial environment of the lungs, they are useful for initial validation steps before moving on to more complex models like ALI cultures."/>
<QaBoxq="What are your thoughts on using lipid nanoparticles (LNPs) versus other delivery systems, like AAV vectors?"
a="Lipid nanoparticles (LNPs) are a promising delivery system for many genetic therapies, but they have limitations. While LNPs can effectively target certain organs, such as the lungs and liver, they cannot cross the blood-brain barrier and thus unsuitable (for now) to target neuronal pathologies. For these conditions, adeno-associated viral (AAV) vectors may be more effective, as they exhibit an inherent ability to cross the blood-brain barrier. For CF specifically, we have used LNPs to deliver sup-tRNAs directly to the lungs. We teamed up with an US company that develops safe LNPs used also for vaccines. Delivery methods like intratracheal instillation—where the LNPs are introduced into the trachea—allow for targeting the lung tissue more directly, which is critical for treating CF."/>
<QaBoxq="How do you view prime editing compared to other gene editing technologies?"
a="Prime editing is an exciting development in the field of gene editing, but it is important to recognize that no single approach is universally superior. Technologies like prime editing, CRISPR-Cas, and our own tRNA-based therapies each have their strengths and limitations. For instance, prime editing offers a highly precise method for correcting mutations directly at the DNA level, potentially providing a one-time, lifelong cure. However, our approach, which focuses on restoring mRNA translation, does not introduce permanent changes to the genome and unforeseen, also individuum-specific side effects, can be counteracted by immediate termination of the therapy. In turn, it requires continuous re-administration over time. Ultimately, the safety and efficacy of any approach must be carefully weighed. We are not yet at a point where we can definitively rank these technologies because the field is still evolving. Each approach has potential, and the choice of which to use will likely depend on the specific disease and mutation being targeted."/>
