<QaBoxq="Do you think there has to be more effort concerning diagnostics?"a="Early diagnosis is covered by the screenings."/>
<QaBoxq="Since you almost had to sue for your medication, do you know if there are any lawyers specializing in cases like this? "a="No, I don’t. "/>
<QaBoxq="Are most of the other patients you know in good health like you?"a="No. Another boy my age got a fungal infection and does not have long time left to live. "/>
affiliation:"Intern at Harvard/ Boston Childrens Hospital",
pictureurl:pics['placeholder'],
tag:"Academia",
heading:"Discussion on optimizing our pegRNA Design to improve precision in prime editing",
interviewtabid:"JPpegRNA",
cardtext:"",
language:"de",
quoteNachname:"Lenger",
quoteVorname:"Malte",
quote:"The interview proved invaluable in gaining an initial understanding of the principles of pegRNA design and optimisation, particularly in the context of silent edits.",
aimofcontact:"The aim of the contact was to engage in a discussion about prime editing and pegRNAs, as the Jan-Phillip Gerhards had used these technologies in his internship. We sought to exchange ideas, gather insights, and explore potential improvements or strategies for our project, leveraging his experience with prime editing tools. His practical knowledge in this field was very valuable for refining our approach and ensuring we were aligned with the latest advancements and methodologies in prime editing. ",
insights:"During our discussion we gained valuable insights that had a significant impact on our project. One of the most important findings was the effectiveness of silent edits, which will enable us to make our PrimeGuide safer. Silent edits changes the sequence of bases in the DNA in such a way that the resulting protein remains unchanged, because the genetic code is redundant. This means that different codons can code for the same amino acid. By making silent edits in addition to correcting the CFTR gene, we can prevent the pegRNA from rebinding. We have also learned that the length of the primer binding site (PBS) plays a crucial role in determining optimal results and that it is recommended to keep the PBS temperature close to 37°C. Specifically, PBS lengths of 17nt (38.3°C) and 16nt (36.4°C) were found to be ideal options. For our planned set of 12 samples, it was recommended to use three different PBS lengths (differing by +/- 1nt from that close to 37°C) in combination with four RTTs to achieve the best result. Another important finding was the use of non-annotated regions with overhangs for cloning, which could give better results in our experiments. However, we also encountered concerns that circRNA, a covalently closed circular RNA molecule, might be sterically hindered by Cas9, which we need to investigate further. When discussing cloning overhangs, we learned that a base-pair length close to 60°C is optimal. However, the use of a 15nt PBS was not recommended as it has a lower temperature range which could affect performance. Although we still need to confirm the oligonucleotide delivery time, these findings will help us to refine our cloning strategy, optimise PBS selection and improve our overall approach to primer editing, especially in terms of the pegRNA design.",
implementation:"We incorporated the lessons learned from our discussions on prime editing and silent editing directly into our project by refining our approach to gene editing. Based on feedback about the optimal length of primer binding sequences (PBS) and RTTs, we adjusted the design of our pegRNAs to ensure greater precision and efficiency in our experiments. In particular, we learned that using PBS lengths close to 37°C melting temperatures (e.g. 16-17 nucleotides) increased stability, which led us to fine-tune these sequences for improved editing results. The concept of silent editing became an integral part of our safety strategy, allowing us to make changes to the DNA more precise. We also revised our cloning strategies by considering the appropriate overhang length, targeting a base pair length near the melting temperature of 60°C to improve cloning efficiency. We also reassessed the practicality of ordering shorter PBS sequences, concluding that lengths shorter than 15 nt were less advantageous due to reduced efficiency. By integrating these findings, we optimised our experimental workflow and made informed decisions about the tools and methods for our prime editing experiments. ",
quote:"For most families, it’s a shock. Cystic fibrosis still has a strong association with being a life-threatening disease, despite the fact that we now have good treatments, and many patients can live healthy lives. The diagnosis puts a huge psychological strain on the family, especially when dealing with very young children.",
aimofcontact:"To gain a deeper insight into the path to diagnosis, we invited pediatrician Dr. Cristian-Gabriel Olariu from the University Department of Pediatrics and Adolescent Medicine to share his experiences with cystic fibrosis (CF) patients with us. We interviewed him because of his expertise in the effects of diagnosis on the patient and the family members, but also on daily life. Additionally, we want to close the gap and create a bridge between academic research and clinical applications. Therefore, Dr. Olariu gave insights about the clinical perspectives on CF patients.",
insights:[<p>We invited Max, our CF patient contact, to join Dr. Olariu in discussing the intersection of academic research, clinical application, and patient needs. Through our connection with CF Vests Worldwide (link zu deren Website? https://www.cfvww.org), an organization dedicated to providing life-saving therapy vests to cystic fibrosis patients globally, we gained insights into the challenges faced by CF patients, particularly in regions like Thailand, where access to advanced treatments and medical devices is limited. The conversation highlighted the critical role of early diagnosis and intervention, as well as the quality-of-life challenges many patients endure due to conventional treatments that may not be effective for everyone. Innovative approaches, such as our SORT LNP (lipid nanoparticle) delivery system, present promising alternatives for CF therapy. This system, which allows for RNA encapsulation and administration via dry spray inhalation, could revolutionize treatment by targeting lung cells more effectively, particularly in resource-limited settings. Dr. Olariu underscored the need for psychological support and coordinated care for CF patients, emphasizing that novel therapies like LNP-based gene treatments have the potential to improve treatment efficacy and accessibility, ultimately reducing the lifelong burden of care for patients and their families. </p>
<ol>
<li>Diagnosis:</li>
<li>Detection through newborn screening.</li>
<li>Further tests (including sweat tests) are conducted if results are abnormal.</li>
</ol>
<ol>
<li>Early Treatment:</li>
<li>Begins with inhalations, physiotherapy, and medications.</li>
<li>Aim: Prevention of severe complications and organ protection.</li>
</ol>
<ol>
<li>Challenges:</li>
<li>Some patients do not respond well to conventional treatments.</li>
<li>Significantly impacts quality of life.</li>
</ol>
<ol>
<li>Family Burden:</li>
<li>Medical challenges create a significant burden.</li>
<li>Psychological stress due to lifelong treatment.</li>
</ol>
<ol>
<li>Importance of Support: </li>
<li>Psychological support is crucial.</li>
<li>A well-functioning treatment team is essential.</li>
</ol>
<p>We have jointly weighed up the extent to which an early diagnosis is always an advantage, as some parents perceive an early diagnosis as an additional burden and would prefer to experience the first years of their child's life without constant medical intervention. Especially when there are cases in which patients only show a clear clinical picture at an advanced age. The psychological burden also lies with the children, who often experience medical trauma because they are involved in such intensive medical care from birth. Additionally, the treatment of cystic fibrosis is very expensive, and the costs are covered by health insurance companies to varying degrees. In some countries, such as the USA, Ukraine or Developing countries, many families cannot afford the necessary treatments. Dr Olariu drew our attention to another problem in the treatment of cystic fibrosis. Infections, especially with bacteria such as Pseudomonas spcc., are difficult to treat and often lead to long hospital stays. Max, our patients’ representative, who knows Dr. Olariu through his treatment, talked about his infections with Pseudomonas spcc., illustrating the reality of an invisible danger that determines a patient's everyday life. Strict hygiene measures are required to prevent infections, such as wearing face masks in hospital and careful handling of potential sources of infection. The clinics where cystic fibrosis patients are treated work closely with a multidisciplinary team of doctors, psychologists, physiotherapists and nutritionists to ensure that patients receive holistic care. At the same time, research is constantly being carried out and new therapeutic approaches developed, such as the use of nanoparticles to improve drug delivery. Former patients are also involved in research and provide valuable insights and advances. </p>
<ol>
<li>Pros of Early Diagnosis and Treatment</li>
<ol>
<li>Timely Intervention: Prevents severe organ damage and improves long-term outcomes.</li>
<li>Holistic Care: Involves a multidisciplinary team for comprehensive patient support.</li>
<li>Access to Innovations: Allows patients to benefit from advancements like nanoparticle drug delivery.</li>
<li>Family Support: Provides education and resources for effective management from the start.</li>
</ol>
</ol>
insights:"",
implementation:"",
<ol>
<li>Cons of Early Diagnosis and Treatment</li>
<ol>
<li>Psychological Burden: May cause stress for parents and children due to constant medical interventions.</li>
<li>Cost Implications: Treatments can be expensive, with varying insurance coverage, leaving many families unable to afford care.</li>
<li>Infection Risks: Patients still face risks from infections like Pseudomonas spp., leading to potential hospitalizations.</li>
<li>Over-medicalization: Continuous focus on treatment can overwhelm families, affecting the quality of early childhood experiences.</li>
</ol>
</ol>
],
implementation:"In summary, our project greatly benefited from the conversation with Dr. Olariu. His insights into the complexities of cystic fibrosis treatment, particularly the significance of early diagnosis, were invaluable. Max’s personal experiences added a crucial human perspective, illustrating the medical and psychological challenges he faces, including infections with Pseudomonas spp. Dr. Olariu emphasized the importance of a multidisciplinary approach, involving not just medical professionals but also psychologists, physiotherapists, and nutritionists for holistic care. This discussion helped us appreciate the balance between timely interventions and the emotional burden on patients and their families, guiding us to develop a more empathetic understanding of living with cystic fibrosis. ",
interview:<>
<QaBoxq="Could you please tell us about the journey that parents go through with their CF-sick children from the first visit to diagnosis and treatment?"a="Since 2016, cystic fibrosis (CF) diagnosis has been part of newborn screening. This means that we receive many children right after birth whose screening results were abnormal. These children are then sent to us for further clarification. Not every child with an abnormal screening result is sick, so we perform a sweat test, and about one-third of the children are diagnosed with the disease. The advantage of early diagnosis is that we can intervene and start treatment early to prevent organ damage. However, there are also rare mutations where the course of the disease is difficult to predict."/>
<QaBoxq="What are the pros and cons of newborn screening for cystic fibrosis?"a="From a medical point of view, it’s beneficial that we can catch many of these cases early, allowing us to act swiftly. There are even medications for small babies, and early intervention can protect organs, preventing conditions that would require transplants later on. On the downside, because of the wide variety of genetic mutations, some cases we identify may not show significant symptoms until adulthood. This creates a dilemma, as we can’t predict how their condition will progress, but we still start treatments early, which can be stressful for families."/>
<QaBoxq="Can you give us an example of how this stress impacts families?"a="Yes, I’ve been caring for a patient from birth who is now five years old and doing very well. However, from the beginning, she had to undergo physiotherapy, regular check-ups, and blood tests, even though she hasn’t shown any major symptoms. Her mother once told me she wasn't sure if she would make the same decision again, as the early intervention caused a lot of stress. She wondered if she might have enjoyed the first year of her child’s life more if things had been more relaxed. Now, at age five, nothing significant has changed in her condition, and they’ve decided against starting modulator therapy for the time being."/>
<QaBoxq="How do families typically react when a CF diagnosis is confirmed?"a="For most families, it’s a shock. Cystic fibrosis still has a strong association with being a life-threatening disease, despite the fact that we now have good treatments and many patients can live healthy lives. The diagnosis puts a huge psychological strain on the family, especially when dealing with very young children. The most important factor in managing this, aside from medical treatments, is the support from the medical team. It’s critical to have a team that works well together, not just a single doctor calling all the shots. Families often need much more psychological and nutritional support early on than medical intervention, and this is where having a multidisciplinary team becomes essential."/>
<QaBoxq="What is the process for diagnosing and treating older patients who haven’t been through newborn screening?"a="Older patients who come to us with complaints may not have undergone newborn screening, so they are diagnosed based on their symptoms. These complaints can range from mild to severe and are often non-specific, like chronic cough or failure to thrive. When the cause of these symptoms isn’t immediately clear, we do a sweat test. Once diagnosed, we can start treatment, which often involves working with a psychologist to help the family process the news."/>
<QaBoxq="How do you support families during the initial shock of diagnosis?"a="When the diagnosis is particularly difficult for families to process, we sometimes have the patients stay in the hospital for up to a week. This gives us time to meet with them daily, answer questions, and provide guidance. During the first consultation, families often fall into a state of shock, and no matter how carefully the doctor explains things, it’s hard for them to absorb all the information. Meeting with them again over the following days helps, and we have specialists in hygiene, physiotherapy, and social counseling on the team to offer holistic support."/>
<QaBoxq="What happens if a child gets infected with Pseudomonas or another bacterial culture in the lungs?"a="Pseudomonas is one of the most feared infections for CF patients. It’s a common environmental bacterium that is difficult for CF patients to clear from their lungs. Once we detect it, we treat the patient with specific antibiotics, often through intravenous delivery over two weeks in the hospital. After the initial treatment, patients may continue with inhaled antibiotics for several months to prevent further infection. It’s a very intensive process, taking a lot of time and energy, and even though we may get rid of the infection a few times, eventually the germ can become resistant and stay in the body."/>
<QaBoxq="Are there any preventative measures to avoid Pseudomonas infection?"a="Yes, there are hygiene measures. For example, CF patients always wear masks in the hospital to avoid infection from other patients. But it’s difficult to avoid Pseudomonas entirely since it’s found in stagnant water and other places in the environment. We advise patients to be cautious with water sources like sinks or ponds. However, we need to balance strict hygiene with quality of life, especially for children, as being overly strict can lead to obsessive-compulsive behaviors without necessarily reducing the risk of infection."/>
<QaBoxq="Do some families resist the medical advice on preventing infections?"a="On an emotional level, I feel that families who take calculated risks to improve their quality of life tend to cope better. Overprotection can lead to greater psychological stress. However, I don't have enough experience to say for sure whether those who don’t protect themselves as strictly get infected earlier or suffer worse outcomes. It’s also worth noting that new therapies are now available that help reduce infection risks, allowing for a bit more freedom, especially for children."/>
<QaBoxq="How often do patients need to be tested for infections like Pseudomonas?"a="The official guideline is every two months, but realistically we aim for every 3-4 months. Regular testing is important because Pseudomonas can be present without symptoms. If too much time passes before detection, it becomes harder to remove the infection."/>
<QaBoxq="How do you manage chronically infected patients?"a="Patients who are chronically infected with Pseudomonas don't stay in the hospital indefinitely. They usually remain at home, inhaling antibiotics daily and taking physiotherapy to help clear mucus from their lungs. Intravenous antibiotic therapy is reserved for more severe cases or during clinical deterioration."/>
<QaBoxq="Are chronically infected patients allowed to visit your practice?"a="Yes, chronically infected patients are allowed to visit the practice. We try to schedule them at different times to avoid contact between infected and non-infected patients, and we often use separate rooms to minimize risk."/>
<QaBoxq="How often do children and adults need to have lung function tests?"a="You can’t conduct a good lung function test until the child is around five years old. After that, it becomes part of the routine check-up because it’s non-invasive and provides a good indicator of lung health. We see children every three months, and I believe the protocol is the same for adults."/>
<QaBoxq="What do you think about support groups or health retreats for CF patients?"a="Support groups are extremely important. Although we are a good medical team, advice from peers often resonates more with patients. We’ve organized two parents' evenings recently, where parents can exchange experiences and support each other. Unfortunately, we can’t invite the children themselves due to the risk of infection, but in rehabilitation settings, they can meet in germ-specific groups and benefit from shared experiences."/>
<QaBoxq="Is there a risk of antibiotic resistance with repeated treatments?"a="Yes, resistance is a concern, especially with repeated antibiotic treatments. However, there’s often a discrepancy between what we see in lab tests and the clinical outcomes. Even if a germ shows resistance on paper, many patients still respond well to treatment. We base our decisions more on clinical outcomes than lab results, changing antibiotics only if the patient’s condition doesn’t improve."/>
<QaBoxq="Are there any side effects to the medications?"a="Yes, all medications have potential side effects, though many of them are minor, like rashes or stomachaches. One serious side effect of some antibiotics is hearing damage, which can lead to lifelong hearing loss. This is why we closely monitor patients in the hospital when starting treatments. The newer therapies, like modulators, can cause liver stress, so we regularly check liver enzymes in the blood. However, severe side effects are rare, and the drugs are generally well tolerated."/>
quote:"I believe the challenge lies in the process when your cell has to repair the new DNA strand",
aimofcontact:"Shortly after we decided to use prime editing as the gene editing method for our cystic fibrosis therapy, Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven and his colleagues published a paper directly related to our research1. In contrast to our approach, Bulcaen et al. 2024 targeted other, less common but drug-refractory CFTR-specific mutations (L227R- and N1303K). ",
insights:"The insights gained from this interview proved invaluable in shaping our subsequent mechanistic approaches. Besides his comprehensive perspective of the significant advancements in the field of prime editing, Mattijs engaged us in a constructive dialogue, offering a critical assessment of our own ideas and approaches. ",
implementation:"Mattijs explained why he did not try to target F508del. Of all the possible applications of Prime Editing in gene therapy, he told us, the CFTR F508del mutation is the most obvious target, but no substantial editing efficiency could be achieved at this target yet. He further elaborated that this might be due to DNA packaging, low cutting efficiencies at that locus, the AT rich region surrounding F508del and also possible intramolecular binding of the pegRNA, but we do not really understand the mechanistic reason yet. Additionally, he mentioned a talk given by David Liu,[Link] the principal investigator behind prime editing that helped us to consider further novel advancements in F508del-specific CFTR-treatment and include them into our project. Beside the clinical context, we discussed different methods of testing edited cells for CFTR reconstitution, such as measurements using the Ussing chamber, organoid swelling assays and halide sensitive eYFP fluorescence assays. Additionally, we discussed the use of stem loops for protection of the pegRNA, which we then included into our own pegRNA design. Lastly, we officially cooperated with the research group and the university: Mattijs was willing to share two different HEK293T cell lines [Link] stably overexpressing the CFTR wild-type and F508del proteins. We also visited Mattijs in his laboratory in Leuven to discuss this further. During our visit we discussed different downstream applications on proteomic, genomic and electrophysiological levels. Besides antibody staining of the CFTR-3HA protein, we asked for his insights on patch clamp measurements and DNA sequencing as well as the advantages and disadvantages of different delivery systems for prime editors. Additionally, Mattijs provided more details into the CFTR overexpressing cell lines, and we received guidance on handling of these kinds of cells. Until the end of the project, we maintained communication with Mattijs Bulcaen via email for further questions and gave updates concerning our project to implement the feedback loop according to our integrated Human Practice Framework.",
