2936 - Designing a universal genome editing strategy to remove PLP1 duplications in Pelizaeus Merzbacher Disease patient cells.
Pelizaeus-Merzbacher Disease (PMD) is a rare pediatric disorder that affects around 1:100,000 boys at birth. The disease is caused by mutations of the PLP1 gene, encoding Proteolipid Protein 1, a key structural protein of the myelin, which is an insulating layer that wraps around nerve fibers to facilitate the flow of information in the nervous system. Mutations in PLP1 result in abnormal myelination, which ultimately leads to severe locomotor and cognitive impairment, and limited life expectancy. In 70% of cases, PMD patients carry duplication mutations of various sizes of the X chromosome region containing the PLP1 gene. Currently, there is no cure for PMD, with treatment options limited to symptom management. In a previous study, we successfully tested a novel CRISPR/Cas9 genome editing strategy that removed the Plp1 duplication mutation and that improved the disease’s manifestations in a PMD mouse model. This project aims at translating the genome editing strategy to individual affected by PMD caused by duplication mutations. Our goal is to test a universal CRISPR/Cas9 strategy to precisely correct the duplication mutations in cells derived from 3 PMD patients having PLP1 duplications. We will assess the nature of the duplications through Whole Genome Sequencing, then we will design and test a universal approach that could be applied to all PLP1 duplication mutations. Successful completion of this project will lay the foundation to support the development of novel therapies for the majority of PMD patients and open novel potential avenues for the treatment of genetic disorders caused by genomic duplications.
2903- Towards a Non-Invasive Gene-Therapy Delivery System for the Blindness Aniridia
Aniridia is a rare eye disease that reduces vision from birth and causes a lifetime of visual impairment. Aniridia is caused by defects in the paired box 6 (PAX6) gene, which is important for the development and function of the eye. Carrying defective PAX6 results in the malformation or absence of the iris (the colored portion of the eye), clouding and blood vessel growth in the cornea (the clear surface of the eye), and other severe eye problems (1). The poor vision in children progresses to blindness by their mid-20s to 30s. Hence, before vision deteriorates, there is an opportunity to intervene, and prevent blindness. Current treatments are unable to preserve vision long term, and thus new treatment strategies are needed. One possible approach for treating aniridia is gene therapy, where DNA is introduced into cells to alter their behavior, with the aim of preventing blindness (2). Common gene therapies for the eye are delivered in a safe virus, but by injections with needles (3). In contrast, topical application would be a non-invasive and thus more patient-friendly administration route (4). However, before such treatments are ready to help patients, we need to show that viruses can be delivered effectively in an eye drop, by doing research in cells and animals. Fortunately, mice with a defective Pax6 gene have an eye disease that resembles aniridia. We hypothesize, an eye drop formulation can be discovered that will allow viruses to survive and transfer their therapy to the corneal cells in such mice. We will use a fluorescent marker for assessing the success of our topical delivery in aniridic and wild type control mice. Although beyond the scope of this application, once an optimized topical route is found, we will deliver PAX6 by gene therapy in eye drops to increase the amount of PAX6 in the aniridic mouse cornea, and thereby improve vision. The experiments proposed here are important first steps towards developing a new non-invasive treatment for aniridia.
2932 - An efficient CRISPR/Cas9 knock-in system to restore full-length dystrophin expression in a Duchenne muscular dystrophy deletion background
Duchenne muscular dystrophy (DMD) is the most prevalent X-linked neuromuscular disease, affecting 1 in 5000 boys3. DMD arises from mutations in the DMD gene that renders dystrophin non-functinoal4. This protein is essential to muscle integrity, and without it, progressive muscle wasting occurs, causing loss of independent mobility and cardiorespiratory failure early in life1. Medical interventions can only extend life expectancy by 15 years at best8. There is a dire need for a cure, as current and proposed treatments are not feasible, ineffective, or produce the milder variant, Becker Muscular Dystrophy6,10. Around 68% of DMD mutations are the result of DNA deletions3. Recovery of dystrophin could be achieved if the lost DNA is added back (or knocked-in) into the muscle cell genome. I aim to cure DMD at its genetic source by knocking-in the deleted DNA to restore fully functional dystrophin. This will be accomplished using the gene editing tool CRISPR/Cas9 and a new DNA repair pathway, homology-mediated end joining (HMEJ)12. Previously, knock-ins on non-dividing cells like muscle and neurons were difficult, with efficiencies less than 4%12. Efficiencies greater than 50% were achieved in the neurons of live mice when CRISPR/Cas9 was paired with HMEJ 12. HMEJ may be a viable knock-in method for treating DMD deletions if this can be achieved in muscle. Our lab has a DMD mouse model with deletions in DMD exons 52-54. I will first knock-in exons 52-54 into the muscle of our live DMD mouse using CRISPR/Cas9 and HMEJ delivered on locally injected plasmids. Following this, the same system will be tested across the entire body of DMD deletion mice by delivering the required components with injected adeno-associated viruses. If a suitable recovery of dystrophin is achieved in these mice, we may open the door to a gene therapy cure for DMD deletion patients and a knock-in system for other rare disease deletion mutations.
2919 - Next generation antisense oligonucleotides to treat cardiomyopathy in Duchenne muscular dystrophy
Duchenne muscular dystrophy (DMD) is a devastating genetic disease affecting approximately 1 in 5000 boys worldwide. An exciting new approach to treating DMD is DNA-like molecule-mediated molecular therapy called exon skipping. These molecules act like a stitch or Band-Aid to skip over the region in the genetic code that blocks the normal process of protein synthesis and mitigate the effects of mutations. Our group previously demonstrated that these molecules effectively restore protein synthesis and improve muscle symptoms in various cell and animal models. Although the first drug of this class, called eteplirsen, was conditionally approved by the FDA in late 2016, it is highly controversial as exon skipping faces major challenges including the limited efficacy, especially in the heart.
We will address this issue using two approaches using new types of DNA-like molecules. First, we will employ new cell membrane-penetrating molecules attached to DNA-like molecules called antisense oligonucleotides (AONs). We developed these new molecules in collaboration with Dr. Hong Moulton (Oregon State University). These molecules are designed to penetrate the cell membrane in skeletal and heart muscles. Second, we will employ muscle/heart-targeting antibodies attached to AONs. We developed these molecules in collaboration with a biotech company Avidity Biosciences. We will test the effects of these new AONs in a DMD mouse model. Our study will facilitate the identification of a next-generation drug for DMD therapy, providing the potential for developing a cure for this devastating genetic disorder in the future.
2928 - Identifying the Genetic cause for Familial Abdominal Aortic Aneurysm
An abdominal aortic aneurysm (AAA) is an enlarged area in the lower part of the aorta. AAAs are often without symptoms and difficult to detect. The aneurysms grow slowly and eventually rupture, a serious complication and often fatal. AAAs are mostly sporadic caused by genetic and environmental risk factors. Some families suffer from the inherited form of AAA. The precise genetic cause of inherited AAAs is still unknown, making it hard to predict the risk and detect the aneurysm prior to the aortic rupture. In this study we aim to identify the causative gene in a large family affected by this disorder. This will be done by analyzing the entire genetic code for affected individuals. If the responsible genetic change is identified, other members of the family would be screened to determine their risk. Such information is crucial to implement preventative measures in individuals at risk and to elevate the enormous stress and uncertainty in members who do not carry the genetic defect. We intend to investigate how such a mutation produces this condition. Understanding the mechanism will help us to explore possible treatment options.
2892- Analyzing the UK Biobank to provide a comprehensive review on genetic signatures for rare diseases
Rare disease projects are often limited in sample size and comprehensiveness, due to funding or organizational challenges, and as a result many rare diseases are underrepresented in research. Over the past five years, large biobanks are being established, which instead of focusing on a specific disease, invest vast effort to collect detailed clinical and molecular information from the general population. Although rare diseases are defined has affecting fewer than 1 in 2,000 people (RDF, 2018), there are >7,000 rare diseases (CORD, 2015) and they affect ~3 million Canadians (1 in 12 people), as well as over 350 million people worldwide (Global Genes, 2015), so population-level biobanks can significantly extend the sample size for rare conditions, especially those which are underrepresented. Furthermore, the diverse range of information provided by biobanks is useful for understanding the overall burden of rare diseases. We will analyze data available from the UK Biobank (~500,000 samples) and present our results online for patients, clinicians and researchers to utilize. Patients in the UK Biobank have a wide range of rare diseases (including at least 49 with Stevens-Johnson syndrome and 148 with myasthenia gravis); the sample size for seven of these diseases is shown in the attached figure and we will consider many other rare diseases as well. Our aim is to provide detailed population characteristics for rare diseases in the UK Biobank, along with information about newly identified environmental factors and genetic risk variants.
2916 - Engineering protein-protein interactions to probe and rewire the Fanconi anemia pathway
Fanconi anemia (FA) is a rare inherited disease affecting 1 in 130,000 births. Mutations in any of the 21 genes in the FA signalling pathway lead to FA or FA-like syndrome, including bone marrow failure and increased likelihood of cancer. During cancer treatment, FA patients are extremely sensitive to radiotherapy or chemotherapeutic agents that cause breaks in the DNA as the FA pathway is essential to repair these DNA lesions in both healthy and cancer cells. Therefore, targeted therapeutic strategies that are less toxic to healthy cells and precisely target cancer cells are in urgent need. To tackle this problem, we plan to exploit the abnormally regulated nature of cancer cells by inducing “synthetic lethality”, a natural off-switch used by cells to prompt cell death when two highly important genes are dysfunctional. This approach has been recently successfully employed at the clinic in the treatment of triple negative breast cancer and ovarian cancer. Rather than targeting the DNA like wide-spectrum chemotherapeutic agents, we plan to leverage synthetic lethality by utilizing our recently developed protein engineering and synthetic biology platform. Our platform develops potent and selective inhibitors for selected target proteins by modulating protein-protein interactions (PPI) that modern drugs have been unable to target. We propose targeting an enzyme (RFWD3) crucial for the protein modification and function of the cell replication protein RPA, important for cell survival. We will model RFWD3 and RPA structure and characterize their interaction to understand the mechanism behind DNA repair and further improve our inhibitors to target the interaction between RFWD3 and RPA at unprecedented precision. Our approach should impact cancer cells without causing lasting DNA damage observed with current treatments. We hope our work will shed light into more effective and less toxic treatment routes for FA individuals diagnosed with cancer.
2918 - Defining the Pathogenesis of a Novel CARD11 Mutation Causing Profound Combined Immunodeficiency and Inflammatory Gastrointestinal Disease
We have recently identified an infant and 17-year-old boy with severe recurrent respiratory tract infections, lung disease, and inflammatory gastrointestinal disease requiring hospitalization and removal of the colon. Using sophisticated next-generation sequencing, we discovered some of the first patients (4th and 5th) in the world to have damaging alterations in a gene called CARD11 causing profound combined immunodeficiency. This gene is crucial for normal immune system function and development. Without it, certain immune cells develop abnormally and cannot respond efficiently to microbes, leaving patients more susceptible to infections. However, the specific mechanisms by which genetic changes in CARD11 impair these immune cells and cause disease is not fully understood. In the proposed research, we aim to carefully characterize the impact of this CARD11 alteration on immune cell activation and development. By focusing on these aspects of patient disease, this study will directly inform the treatment strategy of the current patients as well as future patients with similar CARD11 genetic changes.
2930 -Evaluation of high resolution peripheral quantitative computed tomography for bone health characterization and evaluation of pediatric patients with growth hormone deficiency
Growth hormone deficiency (GHD) is a rare disorder that results in people having a shorter height and overall poor growth. Research has shown that those with GHD also have a low amount of bone (i.e., bone quantity), as well as a higher risk of having a broken bone. Another important part of bone health is bone quality; some studies in animals have found that the quality of the bone is worse with GHD, but this has not yet been shown in humans. HR-pQCT is a non-invasive and safe way to image bone that can tell us about bone quantity and quality. HR-pQCT provides us with a detailed picture of bone health, and this information might allow us to better understand changes to the bone over time in a children/adolescent with GHD. There are currently no research studies that look at bone quality in children/adolescents with GHD. We will use HR-pQCT to measure bone quantity and quality in the lower leg and lower arm in 20 children/adolescents with GHD, who have not yet started growth hormone treatment. One year after beginning treatment, we will use HR-pQCT to measure bone health at the lower leg and arm again, to see if there are any changes to bone quantity and/or quality. We believe that the use of HR-pQCT will tell us more about bone health in children/adolescents with GHD, and will allow us to better monitor changes to the bone in response to treatment. This is a small pilot study that will be used to support a future larger study looking at how HR-pQCT technology can diagnose and assess changes to bone health (both in the short-term and long-term) in pediatric GHD patients.
2905 - International registry for congenital pseudarthrosis of the tibia
Congenital Pseudarthrosis of tibia (CPT) is a rare orthopaedic condition in children. It can present as a fracture of the tibia which may be present at the time of the birth or during the first decade of life. CPT is considered to be one of the most challenging conditions to treat in orthopaedics due to the difficulty in achieving bone healing. The fracture does not unite with standard methods of treatment adopted for fractures in children and the tibia can re-fracture even after satisfactory union of fracture There is a high risk of post-treatment complications such as re-fracture, nerve compression, limb shortening and even limb amputation. Currently, there is no standard surgical treatment and management protocol in orthopaedics that can successfully treat CPT. The reasons for this include the rarity of the condition, the lack of high quality research studies and the paucity of studies with an adequate follow-up till skeletal maturity. In order to plan meaningful studies it is important that multicenter studies are designed to recruit adequate numbers of children for statistical analysis. Hence we propose the establishment of a multicentre registry for congenital pseudarthrosis of the tibia. With a previous rare disease grant (#2290), our team at BC Children’s Hospital has developed the international database for CPT in REDCap. We are requesting for funds to start recruiting international sites. Several international investigators have shown keen interests to join this registry.
2922 - What makes a peer mentoring relationship powerful? A qualitative exploration of the iPeer2Peer Program for youth with chronic pain
Chronic pain, or pain lasting more than 3 months, is a symptom of various rare diseases (Chronic Regional Pain Syndrome (CRPS) affecting 1/3,800 people; Ehlers-Danlos syndrome (EDS) affecting 1/10,000-1/25,000 people). It is critical that youth with rare diseases learn how to manage chronic pain symptoms to avoid long-term negative effects on their wellbeing and development. Connecting people with shared lived experiences is both a unique and high-priority source of meaningful social support that can improve illness management and quality of life. The iPeer2Peer Program is a Skype-based peer mentoring program that offers social support and disease self-management skills by connecting a young adult (mentor) who is successfully taking care of their chronic pain with a newly diagnosed younger person (mentee). Each mentor-mentee pair have 10 weekly-bi-weekly Skype calls to talk about disease and life topics they want advice on. Exploring the content of calls revealed that pairs talk about the impact of chronic pain on daily life as well as managing pain and other non-pain related issues. Adolescents who took part in iPeer2Peer reported better and more efficient self-management of pain symptoms. It is therefore crucial to explore mentoring relationships across pairs to understand what makes a relationship most likely to motivate change. This project will evaluate 8 mentor-mentee pairs with a total 10 calls each. The selected pairs include 3 different mentors (18-25) and 8 mentees (13-17 years) with a range of diagnoses (e.g., CRPS, EDS, and neuropathic pain). All mentoring calls will be transcribed to conduct a deep analysis of recurring content and emotional bond formed by each pair and how this content and bond develops over the course of 10 mentoring calls. A better understanding for the development of mentor-mentee relationships will allow us to provide better, more efficient individualized care through improved pairing strategies and mentor training.
2913- Do Patient Specific 3D Heart Models Improve Outcomes for Children Undergoing Interventional Cardiac Catheterization?
The aorta is the main blood vessel that carries oxygen rich blood from the heart to the rest of the body. Coarctation of the Aorta (CoA) is a type of heart disease that people are born with, where a part of the aorta is much narrower than normal. This narrowing prevents blood from flowing properly to the rest of the body and causes the heart to pump much harder (1). CoA is a rare condition, it is estimated that it affects about 1 in every 2500 live births (2). CoA can be treated with a procedure called cardiac catheterization. During cardiac catheterization, a cardiologist threads a long, thin tube, called a catheter, through the person’s blood vessel to their heart. Using the catheter, the cardiologist can place an expandable metal tube, called a stent, into the person’s narrowed aorta to hold it open. Cardiac catheterization is highly effective and is less invasive than surgery. However, cardiac catheterization utilizes radiation to be able to see the location of the catheter and it is known that greater exposure to radiation is associated with increased lifetime risk of cancer. Cardiac catheterization can also be technically challenging because the size and shape of each patient’s heart is slightly different (3,4). 3D-printing is the process of making a 3D model from a digital file. Using 3D printing, it is possible to make an exact replica of a person’s heart using images from a CT scan (5). In this study, we want to determine if simulating the catheterization procedure using 3D models of patients’ hearts will improve cardiac catheterization. For half of the patients in our study, we will make exact an 3D model of the patient’s heart and use this model in a simulation of the procedure, before the actual procedure. The other half of the patients will be treated according to standard of care. We will compare to see if the group of patients who had 3D models printed, had a lower radiation dose and better blood flow after the procedure.
2911 ITG - Investigator-initiated Trial of MPAN 1-H9 in patients with mitochondrial membrane protein-associated neurodegeneration (MPAN)
MPAN is a very rare autosomal recessive progressive neurological disorder with iron deposition in the brain. It belongs to the group of syndromes with neurodegeneration with brain iron accumulation (NBIA). Patients develop early-onset motor dysfunction, movement disorders, cognitive decline, psychiatric symptoms, and visual disturbance. Brain imaging shows the eponymous brain iron accumulation in the basal ganglia, a central region of the brain. On brain pathology features related to Parkinson’s disease and dementia have been observed. MPAN is an inherited disorder caused by mutations in C19orf12. The exact pathophysiology is unknown. Recently , a drug screening study in MPAN ﬂies demonstrated positive effects of the drug MPAN1-H9, which is approved for other indications. So far there are no controlled data of its use in human MPAN patients. We thus propose a pilot trial to investigate the tolerability of MPAN1-H9 and its positive effects on symptoms and disease course. Aim is to repurpose MPAN1-H9 for its new use in MPAN. In our database more than 60 MPAN patients are registered. The study will be performed at the Neurology Clinic of the University Hospital in Munich, Germany. We plan to treat 15 MPAN patients with a genetically -conﬁrmed diagnosis in a monocenter openlabel single arm pilot trial over a period of 6 months (3 visits, baseline plus at month 2 and 6) and compare the data with natural history data pre treatment. Clinical outcome measures will include clinical rating scales of disease severity (such as the Uniﬁed Parkinson`s Disease Rating Scale, Dystonia rating scales, Quality of life measures, functional independence measure, Beck Depression Inventory), functional tests (gait studies) and imaging parameters to assess cerebral atrophy and MRI-connectivity.
2914 ITG - Does strength-training program can slow muscle weakness of women with DM1
This study aims to determine if muscle strengthening can diminish muscle weakness experienced by women with myotonic dystrophy type 1 (DM1). Loss of skeletal muscle mass (also known as muscle atrophy) leads to muscle weakness. Individuals with DM1 frequently report muscle weakness as a major symptom. Muscle weakness and fatigue are two major causes of disrupted social participation in DM1. To help these people, rehabilitation interventions must be developed to target muscle weakness and fatigue. Muscle strengthening is safe in DM1 population. However, only few studies have addressed its efﬁciency to increasing maximal muscle strength and physical performance. Moreover, to this date, no studies have been speciﬁcally conducted in women with DM1. While there are no signiﬁcant differences in the responses to exercise between men and women in healthy subjects, this question remains unknown in DM1. We recently developed a 12-week training program that has led to spectacular improvement in men with DM1. Indeed, lower limb muscle strength and power as well as walking speed were improved while fatigue and apathy were decreased during and after the training program. Participants have also reported to feel in better physical shape, have a better mood and more physical capacities, and be able to walk more and to climb stair more easily. All those changes can have a positive impact on patient’s lives by attenuating restrictions to social participation and improving patient’s autonomy. This study would be the ﬁrst one to evaluate women with DM1 exclusively. This is of great importance if we want to develop rehabilitation interventions that are suitable for both men and women. From a broader perspective, the difference between men and women to muscle strengthening remains unknown in many rare diseases. This study could constitute a starting point to evaluate this effect among them.
2933 ITG - Harnessing Exercise in the Diagnosis and Treatment of Catecholaminergic Polymorphic VT: A Case Cross-Over Design Trial
The tragic, unexpected death of a young person can be caused by undetected heart rhythm disorders. Finding and treating rhythm problems prior to unexpected death is our focus. One dangerous and rare heart problem is catecholaminergic polymorphic ventricular tachycardia (CPVT), which is caused by dysfunction of heart cells during exercise. Thus, it is missed when tests occur at rest in the clinic. To ﬁnd CPVT, an exercise stress test (EST) is needed, which involves monitoring the patient while they jog on a treadmill. The treadmill setting used is called the Bruce protocol (ie. Bruce-EST), which is an endurance program designed 70 years ago to detect heart artery blockages in older people, not CPVT in the young. We now think that CPVT happens mainly during sudden sprints. Therefore, the Bruce-EST may miss CPVT, even if the condition is present. Thus, treatment is not started, leaving a patient at risk of a dangerous rhythm. Our ﬁrst goal is to design a new “Burst-EST” to see if sprinting on a treadmill is more likely to ﬁnd CPVT. Our second aim is to make sports safer in CPVT. Because rhythm problems happen during exercise, doctors advise against athletics. This is unhealthy and reduces quality of life. New research suggests that most CPVT patients who take medicine (called a beta-blocker), can safely go back to sports. A newer medication option also exists called ﬂecainide. Right now, ﬂecainide is only used when dangerous rhythms occur despite using a beta-blocker, and the extra pills must be taken 2-3 times per day for life. In our study, we also want to determine if CPVT patients could exercise safely if they take a ﬂecainide pill before planned exercise, but not regularly. To study this, we will ask patients to do our new Burst-EST on a beta-blocker, and then do another Burst-EST after taking ﬂecainide. We will compare the results between the two tests to see if there were fewer dangerous heart rhythms on the second Burst-EST compared to the ﬁrst.