Featured Provider: Theresa A. Grebe, MD with guest speaker Vinodh Narayanan, MD
This presentation by Dr. Terry Grebe and Dr. Venode Narayanan outlines the multidisciplinary clinical management of Rett Syndrome (a neurodevelopmental disorder typically linked to MECP2 mutations) and explores emerging research, including the FDA-approved treatment trofinetide and experimental gene editing therapies.
All right, good morning, everyone. It is 7 a.m. and we are going to start bright and early and on time, and I have the great pleasure to introduce two speakers today, um, Doctor Terry Grebe and Doctor Vinod Narayanan, and these are our, you know, OG leaders in genetics and child neurology. Um, they were sort of the neurogenetics before neurogenetics was even a, uh, a term. now let's see. Wonderful thing. So, Doctor Grebe, many people know her. She's a clinical geneticist here in the division of Genetics and metabolism at Phoenix Children's, and she's served on many teams, the craniofacial Center team, multiple specialty clinics. Um, she worked, she focuses on craniofacial and neurogenetic disorders including autism and epilepsy, and she's a fierce advocate for all of these children with, uh, complex diseases and their families, um, and especially has interest in Rhett syndrome. And so I've been very excited that she's going to join us along with Doctor Vinod Narayanan, who is a child neurologist with special interest in the genetic basis of neurological disease, and he um has been at Barrow Neurological Institute after he was on faculty for many years at the University of Pittsburgh, and he founded the pediatric neurology and neurogenetics Practice, his neuro neurology and neurogenetics practice in 2011. And he serves as medical director at the Center for Rare Childhood Disorders at TGI. And he also has been um monumentally important in um learning more about Rett syndrome. And so I'm very excited to have these two people, and I will stop talking and start by letting uh Doctor Greby and Doctor Narayanan take it away. Thank you. Thank you and good morning, everyone. It's a pleasure to be here. I wanna thank Doctor Lewis for inviting me to speak to you today about multidisciplinary care for RET syndrome. And I'm very happy to share this talk with my friend and colleague, Doctor Narayanan. These are our educational objectives. Uh, I'm going to speak to the 1st 3 which are mainly clinical and then turn it over to Doctor Narayanan to talk about the latest developments and new treatments and research in RET syndrome. Uh, our disclosures are that we are genetics advisors for Acadia Pharmaceuticals. Uh, one of the most common reasons for referrals to both genetics clinic and neurology clinic is the child with developmental delay and a concern for neurodevelopmental disorders. So just how common are they? Well, among the approximately 20,000 protein coding genes in the human genome, more than 1300 causative genes and 1100 candidate genes have been implicated, uh, and that number is rising as of 2025. I've really seen in my practice an explosion of new disorders over the last 5 or so years, and I can estimate, I see 1 new gene approximately every 1 to 2 weeks in my clinic. So when we consider both established and proposed disorders, estimates suggest that there's over 3000 different potential genetic neurodevelopmental disorders. And among these, red syndrome was the first described, uh, likely the most common and the best studied. So Rutt's syndrome was first described by Doctor Andreas Rett, an Austrian neurologist and author who in 1966 published a paper on 22 girls that he followed who developed a very distinctive and progressive neurologic disorder after a period of apparently normal early development. So he described these girls in detail who developed developmental stagnation and regression after 18, 6 to 18 months of age. Followed by loss of motor skills and language skills and the emergence of certain characteristic features. He also noted the development of seizures in the majority of his patients, and he described the progressive nature of this disorder in 4 distinctive clinical stages, which I'll go through. So how common is RET syndrome? Well, the worldwide prevalence is estimated at approximately 1 in 10 to 23,000 female births. So in Arizona, we currently, uh, as of 2020, had 360,000 births. So we may have as many as 18, uh, children born each year, uh, with RET syndrome. I know they're out there and I think some maybe are going undiagnosed. Um, a systematic review estimated a pooled prevalence at 7.1 per 100,000 females. So we know that because REDD syndrome is X-linked, it almost exclusively affects females, uh, but not always. There are some very rare males with RT's syndrome and I'll talk about them in a bit. They typically present with much more severe manifestations. Uh, and finally, there's several phenotypes of RET syndrome which we now call MEC P2 related disorders, and I'll talk about them briefly as well. Um, this slide shows the RET syndrome diagnostic criteria that have been described and revised. On the left, you see the um main criteria for classic or RT typical RET syndrome. So there must first be that short period of normal development followed by a period of regression, followed by recovery or stabilization. Uh, the main criteria are partial or complete loss of acquired purposeful hand skills, partial or complete loss of spoken language, gait abnormalities, and stereotypic hand movements. Um, and these include, include most classically hand washing or hand wringing, but I have patients who do hand clasping, some finger crossing, and a lot of mouthing of their fingers. So atypical Goret's syndrome, the other phenotype, um, is similar. It also follows a period of normal development followed by regression and then followed by recovery or stabilization. Um, this diagnosis requires 2 of the 4 main criteria on the left plus 5 of 11 of these supportive criteria, uh, many of which are very classic Barrette's syndrome. So those include breathing disturbances while awake, which is really striking when they're in clinic with you, um, Bruxysm, teeth grinding while awake, uh, sleep disorders, hypotonia, peripheral vasomotor disturbances, scoliosis and or kyphosis, uh, growth retardation, short stature, uh, small cold hands and feet. Inappropriate, sometimes prolonged laughing or screaming spells, diminished response to pain, and a very um specific um behavior Tourette's syndrome called eye pointing. And this is an intense form of eye communication where the patient, the individual stares intently at a person or object that they want. It's a very purposeful, um, nonverbal form of communication which really indicates that the individual's cognitive function is higher than their expressive language abilities. How common are these classic features? Uh, as you can see on this slide, the vast majority in classic RE syndrome occur in over 99% of individuals, uh, with the exception of deceleration of head growth, still high in 80% and seizures in 60 to 80%. As I said, Doctor Rhett described a timeline of progression, which he divided into four stages that still applies. So I'd like to go through those for a minute. Uh, starting on the left, there must be a period of normal prenatal and perinatal Development up to about 6 months of age. And then stage one, the early onset stage goes from about 6 months to 18 months. This is characterized by a slowing of head growth with microcephaly, hypotonia, and delayed development. Stage 2 is called the rapid deterioration stage, and this goes from about 18 months to 4 years. And during this stage, there's loss of motor and communication skills. The individuals become nonverbal. That's when they develop these stereotypic hand movements and breathing irregularities. Seizures are likely to start at this time. Microcephaly worsens, and they may have features of autism. Then in stage 3, between age 4 and 10 years, uh, there might be actually a period of stabilization. It's called the pseudo-stationary phase and there's actually potential for some improvement in cognitive and social skills. Scoliosis can rapidly worsen during this phase. Seizures may continue or start and autonomic dysfunction might appear. And then the last stage, uh, the late motor deterioration stage begins at about 10 years and can last either several years to decades. And it's during this phase that the patient really becomes much more debilitated. They're likely eventually to lose ambulation and become wheelchair-bound. They may have dystonia and Parkinsonian features. So I do wanna briefly mention um a couple other uh variant types of RET syndrome which we now call MECP2 related disorders. It's important to mention these because uh these may be conditions you see where you weren't initially suspecting RET syndrome as a diagnosis. So the first is, is variant RET syndrome. So females with this can have a very broad spectrum of symptoms, uh, uh, which overlap those of classic RET syndrome. So at the mild end, they, um, may have, um, less regression and intellectual disability is, is less severe and they may have some preserved speech. Well, at the severe end, Um, they might have developmental delay beginning very early in infancy, uh, hypotonia and even infantile spasms. Then there are some girls with MEC P2 variants who have only mild learning disabilities. Um, they may have no hand stereotypies or regression. Um, and this we feel is due to a favorable skewing of X in activation. If you remember with two X's, one is inactivated, so instead of the 50/50, it might be skewed toward the normal X. And finally, some MECP2 pathogenic variants have been identified in individuals with just autism and schizophrenia. Now, historically, RETT syndrome in males was felt to be incompatible with life, but we do know that there are some males uh with RETTs syndrome, although it's extremely rare. Um, in survival is improved if the male has somatic mosaicism, i.e. the mutation not in all the cells of the body or the germline, or they have a sex chromosome abnormality such as Klinefelter where there's an extra copy of the X chromosome. But typically, males with RT's syndrome are gonna present with a very severe neonatal encephalopathy. They may be, they'll be normal at birth, but there will be a really relentless clinical course with severe seizures, breathing abnormalities, and typically death before age 2 years. And finally, one unique phenotype called X-linked intellectual disability and pyramidal signs, Parkinsonism and macroorganism syndrome is caused by one specific variant in MCP2. I've never seen that one that I know of. And I do wanna mention an allelic disorder which is quite distinct from RAT syndrome and that's called MECP2 duplication syndrome. So not to be confused with rat syndrome. This is a a condition that affects males who have an extra copy or duplication of MECP2. Uh, and sometimes they have a much larger visible, uh, duplication on XQ28, the uh, the, the chromosome, and in that case, they have a much poorer prognosis. So this syndrome is a bit different. Uh, males present typically, uh, in early infancy at birth with hypotonia, delayed development, severe intellectual disability. They might be minimally verbal, but a characteristic is they develop progressive spasticity, so might be diagnosed with cerebral palsy. They're at very high risk for recurrent respiratory infections, which is a frequent cause of death before age 25, and they also develop seizures. This little guy here on the left is actually one of my patients here at PCH. I'm happy to report he's doing very well at age 6 years. This is his uh lovely mom who lobbied, um, the legislature in Arizona and was able to have the governor um announced a MEC P2 duplication syndrome Awareness Day. So she's been very active for her son. I want to spend a few minutes talking about the discoveries in in RT syndrome. So as I said, the syndrome was first described in 1966, but the protein, the MECP2 protein, was identified in 1992. It's called the methyl CPG binding protein 2, and it's a nuclear chromatin-bound protein that binds specifically to methylated DNA. Here's a little uh model of the protein in green bound to double stranded DNA. Uh, down here, I'm showing you the components of the protein that I know Doctor Narayan will talk about as well. Um, there's the, in blue, there's the N-terminal domain, and at the other end, the C-terminal domain. The active parts of the protein are here in green, the methyl CPG binding domain. This is a less functional area of the intervening domain, and then the transcription repression domain is also uh very important. So there are two main transcripts of the MECP2 protein that lead to two isoforms, 1 and 2. They uh result from alternative splicing. Um, isoform 1 includes exons 13, and 4, while isoform 2 includes exons 23, and 4. So the one that's most prevalent in the brain is the uh first isoform which represents more than 90% of the MCP2 protein. And we do know, of course, that the protein is most highly expressed in neurons and it plays critical roles in brain function and development. Just a brief um diagram here to show you uh kind of the different roles MECP2 plays in the, in the cells in the brain. Um, we knew initially that MECP2 is involved in transcriptional uh repression. Um, it binds methylated CPG uh dinucleotides. And then recruits various suppressor complexes such as MSIN 3A and HDX whereupon it um condenses the nucleosomes to this condensed um formation which um uh prevents RNA polymerase 2 here from binding and then blocks transcription. But we also know that uh MECP2 is involved in in transcriptional activation in other settings. So, in this um role, it binds to 5 hydroxymethylcytosine here uh and recruits different activators including the KREb protein and the various activators and then um modifies the um nucleosomes into the open configuration you can see here so that RNA polymerase can bind and transcription can proceed. So, it's interesting, I think uh about this protein is just that it's, it involves many of functions and even beyond a DNA methylation, um, but it has this dual functionality of both activator and repressor which is really context-dependent, uh, and it varies with chromatin modifications and the cellular environment. So, I'm sure Doctor Narandon will talk more about its role in the brain and uh how therapy can um correct deficiencies. So it wasn't until 7 years after the protein was discovered in 1999 uh that Doctor. Hutazobi and her colleagues discovered that mutations in MECP2 cause RET syndrome. And I remember being at that meeting, uh, when it was discovered. Uh, and this published, was published in Nature Genetics. So she and her team used systematic screening to identify MECP2 mutations in, um, 21 uh sporadic cases and they found 5 mutations. And they um included multiple types of mutations including frameship, nonsense, and missense uh variants. Um, her study also identified, interestingly, a pair of affected half-sist which suggested, uh, that their mother had germline mosaicism. This study was really groundbreaking cause it revealed uh that RET syndrome results from abnormal epigenetic regulation and it was the first of its kind. We now know of many other conditions uh with similar mechanisms. So, I wanna mention just briefly a few genotype, phenotype correlations, but to state, of course, um that most pathogenic variants are de novo, so new mutations in the affected uh child. And for MECP2, pathogenic variants are loss of function, which is opposite of the MECP2 duplication syndrome that I mentioned. There have been more than 620 mutations identified to date. And most of them occur in the active region in the methyl binding domain, but some um do occur in the transcriptional repression domain. Uh, and there are a large number of frame shift variants at the end of the protein in the C terminal domain. Uh, this is a busy table, but it just, uh, wanted to illustrate that, uh, 5 of the most common recurrent pathogenic variants as well as several others that cause milder phenotypes and non-classic RET syndrome and one that's been described in some males, uh, uh, with ID and features of, of MECP2 disorder. But again, sequencing will allow us to identify any of the 620 or more uh mutations. Um, this is just again a diagram of the MECP2 protein showing you the hotspots for the mutations here in the methyl-CPG binding domain and the transcriptional repression domain. OK. How should we test for RET syndrome? Well, there's multiple testing options available. Um, if you're absolutely sure your patient fits the classic phenotype of RET syndrome, you can just do sequencing of MECP2 which would detect, um, all different types of variants and deletion testing can also be done. Um, this is offered, of course, by multiple labs including our own Phoenix Children's Genomics Lab as well as outside labs. But more typically, uh we do multi-gene panels because there are several other conditions that overlap with RET syndrome. So there are uh epilepsy panels um offered here at PCH as well as, uh, outside labs, um, panels called RET Angelman syndrome panels because of the overlap there. But if your patient really has a phenotype that resembles REDD syndrome but not classically and overlaps with other uh disorders that are characterized by intellectual disability or neonatal encephalopathy, and again, 3000 of them, it's better to do whole genome or whole exome sequencing. Um, right now, we're sending ours to Gene DX but there are many labs all over that do this, uh, and Um, our genomics lab is offering that. So basically, multiple labs around the country can offer this for you. So how is RTD syndrome inherited? Um, almost 100% are due to de novo pathogenic variants. So as I said, new mutations. But even still, we do recommend testing the, uh, both parents for the female proband and if we have a male, testing is recommended for the mother. And as I mentioned before, um, occasionally a mother, um, may have germline mosaicism or have favorably skewed X inactivation, so she might be mildly or, or minimally or, or completely unaffected and unaware that she has the mutation. So if parental testing is negative, we assume then there's either a de novo variant in the proband or germline mosaicism in the parent. And we typically will counsel for those um families that their recurrence risk is approximately 1%. And for couples who are still um anxious, which is understandable, prenatal testing for RT um and pre-implantation genetic testing with IVF is possible for all MACP2 related disorders. So just quickly, what if it's not Rhett's syndrome? A few conditions to keep in mind in your differential, um, I've shown here. So on the left, this little guy has Angelman syndrome. Of course, you're all familiar with that. You know, in the early months, it, it is difficult to distinguish Angelman from Rhetts syndrome, uh, although Angel of course, occurs frequently in males. Um, they have intellectual disability, severe speech impairment with almost no language development. They have that very happy demeanor, but they have a gait ataxia and even an upper truncal ataxia with some tremulousness. That's not typical Frette's syndrome. Um, their seizures are often earlier at 6 months of age. And for the epileptologists, I know, uh, there's a characteristic EEG pattern in Angelman's syndrome that's somewhat specific to the disorder. Uh, the most typical finding is high amplitude rhythmic delta activity, 1 to 3 Hz, uh, with a bifrontal predominance. And I'm not an epileptologist, so don't ask me about that. Um, also to keep in mind is FOXG1, which is on, often on the RET panels. This is sometimes called congenital RET syndrome. Um, these children, um, are all de novo mutations. They're more severely affected than a RET, typically don't learn to sit or walk, have severe intellectual disability, uh, very severe seizures, spasticity, and movement disorders. They have multifocal, uh, spike in waves and may have delayed myelination or a simple gyro pattern on their brain MRI. And then finally, CDKL5 deficiency um also is in that differential. It's an early infantile epileptic encephalopathy, also X-linked. Um, it's, uh, they also have very early onset severe seizures that are difficult to control. Some of them have some minor dysmorphic features. This is a little cutie here showing the broad forehead and large eyes. OK, for the last part of my talk, I'm gonna focus on the appropriate medical management for RET syndrome. So there are many guidelines uh published in the genetics literature and I'm sure in the uh neurology literature, but probably the most comprehensive guidelines have been published by the International RET Syndrome Foundation shown here. This is, uh, the cover of their uh book. So I'm gonna go through what initial evaluations uh should be done for a child with RET syndrome after diagnosis. And it is a very multi-system disorder as I've mentioned, so, uh, there are a number of uh evaluations that should be done initially. Um, so, um, it goes without speaking that a neurologic evaluation should be your initial, uh, appointment. Uh, it, it should include a brain MRI and consideration of an EEG and video monitoring, uh, if seizures are a concern, which they likely will be. Uh, a developmental pediatrics assessment is also critical at any age to include motor adaptive cognitive and speech, uh, skills, and they should be referred for early intervention as soon as possible. A neuropsychiatric eval is also really helpful for children aged greater than 12 months, uh, because they have frequent sleep disturbances, uh, anxiety, ADHD, and many have findings of autism. Uh, due to hypotonia, musculoskeletal issues are quite common, so orthopedics should be involved as well as physical medicine and rehab and our therapy teams. Uh, many develop scoliosis, so they need monitoring for that. They really benefit from, uh, a lot of mobility training and physical therapy, and many will need adaptive devices. So the longer they can, um, remain ambulatory, the better the prognosis. Uh, our GI partners are crucial for managing RET syndrome. Um, individuals with this have a very high likelihood of having GI dysmotility, uh, which can result in aspiration and feeding issues, so they should have a feeding eow. Nutrition is a great concern. Uh, they're usually underweight. Uh, constipation is pretty classic as well as reflux, and many later on will need a G tube placement. I mentioned breathing abnormalities early on, so pulmonology uh is a um indicated referral to analyze for abnormalities of breathing regularity. Overnight sleep studies also uh should typically be done. Um, sleep medicine is also beneficial to assess, um, both nighttime breathing and sleep disorders which are common. And every girl with REDD syndrome should have an EKG to assess for prolonged QT syndrome, which is a known risk factor for early death, and that should be repeated regularly every few years. Um, bone density studies are important, especially later when they become non-ambulatory to assess for osteopenia. I refer all my patients to ophthalmology. There's a higher frequency of vision problems including uh low, low vision and strabismus uh in RT syndrome, and then a yearly audiology evaluation should also be done. And finally, attention should be paid to, to the skin as they tend to have decreased profusion of their hands and feet, which goes along with a lot of autonomic abnormalities uh in this cohort. Uh, so, what treatments are there specifically for RET syndrome? Um, starting with seizure management, there is no single RET syndrome specific anti-epileptic drug, but valproate, carbamazepine, and lamotrigine are, are frequently the, the first line, uh, drugs used and Uh, are typically effective, but of course, drug selection should be guided by the patient's age and seizure onset and timing. Again, I'll stress that approximately 60 to 80% of girls with RTT syndrome will develop seizures, uh, typically around age 4 years. For behavior management, those crying spells and anxiety and agitation that we see in a lot of the girls, low-dose risperidone or SSRIs have been somewhat successful. Um, our sleep medicine doctors, uh, are very helpful in treating sleep disorders. Um, several that have been looked at, but really studies are early on include gabapentin, clonidine, trazodone, and mirtazapine. I will say that antihistamines and benzos should be, uh, used cautiously due to potential side effects in these girls. And then refinnotide has been shown to help somewhat in nighttime behaviors, but it hasn't been specifically approved for sleep. I did mention the risk of cardiac arrhythmias, so VTC um can arise from long QT and interestingly, uh, sodium channel blockers such as phenytoin and others have really shown significant benefit in treating this symptom in RET syndrome over the usual use of beta blockers. Um, avoidance of QT prolonging drugs is really essential. And there have been some small studies looking at acetyl L-carnitine. For the breathing abnormalities, um, which are quite distressing when the child is awake, um, we do know that they result from, um, abnormal gabiergic inhibition and serotonergic dysfunction in the brain stem. Um, several of our patients, we've used acetazolamide off-label, but it really hasn't shown too much efficacy. So, um, more recent studies are looking at serotonergic agents, uh, specifically 5-HT1A receptor agonists and and SSRIs as well as gabaergic modulators. So I'm hoping we'll have more progress in that area. And then, as I mentioned, GI problems are extremely common and this is probably the, the most common non-neurologic feature of RET syndrome in about 92% of our girls. Uh, there is no specific treatment. They do need, um, reflux medications for frequent vomiting. They may need G tube for dysphagia, feeding therapy, and standard constipation treatments. And finally, targeted MEC P2 treatments, I'm going to leave to Doctor Narayinan. So because RA syndrome is a multi-system disorder, it really requires a multidisciplinary team. And this team is typically led by neurology in close partnership with developmental pediatrics, sometimes genetics, uh, physical medicine and rehab, GI and pulmonology are close partners, as are our therapy team, our psychiatry and psychology teams, and I want to mention research too. It's very important for the treatment of RET syndrome, both the natural history study and keeping abreast of all the new, uh, treatments coming down, uh, the, the road. So, um, just wrapping up, what, what support do we have for families with RET syndrome? Well, we have great specialists here at PCH and I think I'm hopefully safe in saying we are working on starting a REAT syndrome multidisciplinary clinic here to be led by Doctor Agapan, working with Doctor Narayanan, uh, Doctor Rass from Developmental Pediatrics, and myself. Um, we are going to be on the team and we are, we have recruited some of our GI and pulmonology and physical and medicine and rehab partners. But we did learn that some of our Phoenix families are traveling up to Colorado to uh the University of Colorado's International uh Rat Syndrome Center of Excellence, and there is a support group up there. So we hope to bring them back to Arizona as soon as possible. Um, overall, the largest support group for, uh, families with threat syndrome is the International RET Syndrome Foundation or IRSF, which is a very large, wonderful organization that provides education, uh, support, and, uh, supports and funds research, uh, for RET syndrome. Uh, so I'll just leave you with the thought that RET syndrome is not just a pediatric disorder, but it's a lifelong condition. Um, the RET syndrome Natural History Study, which ran from 2006 to 2015 and followed almost 1200 individuals with Rhett syndrome, found that among the 51 deaths in that cohort, uh, the age range of death was from 3.9 to 66.6 years. So survival into the fifth decade is really typical now, um, with recent data showing more than 70% survival at 45 years of age with classic and atypical RETT syndrome. So this is a very dramatic improvement from Doctor Rtt's original uh cohort in 1966, which had only a 21% survival to age 25. So cause of death in RET syndrome is typically cardiorespiratory compromise, uh, and often pneumonia. I mentioned cardiac arrhythmias, particularly thet associated with long QT is another risk factor for sudden death. Um, and there's several, um, modifiable, so treatable risk factors that influence survival. One is disease severity, um, but the second is ambulation status. So the longer we can, um, Continue ambulatory status for our patients, the better survival, um, good nutrition and weight and, uh, and seizure control. They're all associated uh with increased mortality if they are not well treated. So I'll say that multidisciplinary care for R syndrome with emphasis on nutrition, weight gaining, maintaining ambulation as long as possible, seizure control, and also surgical correction of scoliosis have contributed to the extended life expectancy. So this suggests that we can continue to improve care uh for our patients with RET syndrome over the course of their lifetime and also stresses the need, I should say, for our transition program with our adult medicine partners. So finally, this is Anne Charlotte. She's 73 years old and lives in Sweden and believed to be the oldest patient living with RT's syndrome. So I'm over time. I'll stop there and turn it over to my friend, Doctor Narayanan. Thank you. I hope. OK, now. PowerPoint share. And OK. How does that, does that look OK to everybody? Yes. OK, great. Uh, first of all, uh, thank you, Kara, and, uh, uh, Terry for inviting me to present what I've learned over the last 40 years about Rhett syndrome. Uh, I'll skip over this because Terry already mentioned this. Uh, some of our laboratory work, uh, has been supported by, uh, really intramural donations from the Dorn's Foundation and from TGen, and, uh, we were recently successful in getting one of our projects funded by the NIH, and I do, I have served as a consultant to Acadia Pharmaceuticals on their genetics advisory board. So I thought I would put this in at the beginning rather than waiting till the end, just in case uh we're short of time. And I want to acknowledge. Almost 40 years of collaborative work with Doctor Sakua Naidu, who is a pioneer in the field of, uh, RET syndrome. Uh, I met her in 1987 when she came to our lab and I was working at Ghan Tennequi at Hopkins. She wanted to, uh, isolate mitochondrial DNA from brain tissue of postmortem tissues from patients with RET syndrome to look for mitochondrial DNA mutations. Um, and I'd like to also acknowledge Doctor Sampa Rangasami, my colleague who's, uh, here now at ASU. He worked with me at TG and at the Barrow from, uh, 2014 till now, who was very, a key person in all of our RE syndrome studies. And most recently, uh, collaboration with Doctor, uh, Chatterjee, who's at the Beckmann Research Institute at City of Hope, and her team that includes Aravind, who was, who used to be in our lab, and Swati, who has, uh, been there. So I'll start by reviewing a few things, some things that, um, that Doctor Grebe has already mentioned. By the way, uh, what I'm going to do today is really focus on, on kind of two things. One is a little bit of the biology and science that led to, uh, the identification of, uh, IGF-1, you know, insulin-like growth factor 1 as a key kind of signaling pathway. And that led to the FDA approval for refinitide. The second is to review pre-clinical, uh, studies on gene therapy, which led to the two, human clinical trials that are ongoing right now with a gene replacement therapy product. And lastly, a few slides about our own work on gene editing. So this cartoon, which you've seen already before, shows a diagram of what McP2 gene looks like. There are 4 exons and 2 isoforms generated by alternative splicing. And as Terry had already shown you, I think maybe my pointer will work. 99% of the variants that are seen in human cases of REDD syndrome are localized to these coding regions of Exxon 3 and 4, and in genomic DNA distances that accounts for less than 2.5 kilobase pairs of DNA, which means that whole thing can fit into the AAV virus as a single stranded genome. Uh, here, you know, again, uh, something that Terry had already showed you, the key domains. And, uh, you know, a lot of work has been done on the biology of REDD syndrome, but still comes down to two areas, the methyl binding domain or MBD, uh, which is the part of the protein that, uh, um, recognizes and binds to, uh, methylated CG dinucletides, as well as other, uh, methylated, uh, cytosine residues. And then the transcriptional repressor domain. That binds to protein complexes that are involved in transcription modulation. Um, down here at the bottom, I, I've, I've shown a picture taken out of a 2001 paper by Adrian Byird. Adrian Bird was the one who first identified MECP2 way before it was linked to Red syndrome, and this shows, uh, the, the survival curves for in a mouse model in which MCP2 has been knocked out. So Exxon 3 and 4 were deleted. And you can see this is the, this is the survival of the knockout mice, this is the wild type mouse, and roughly about 9 weeks is how long a male animal that carries the null allele lives. All of the early studies, uh, once people started making mouse models were all geared towards uh phenotype characterization. Did it reproduce what we see in humans, and what does this teach us about the biology of MCP2 and its function. So that was really the focus for a very, very long time. Um, knockouts, knock-ins, you know, uh, uh, because, of course, none of our human patients have a deletion of exon 3 and 4. Most of the mutations are, are either point mutations leading to truncation, missense mutations, or, uh, C terminal deletions. Uh, so, but you know, the null mouse was a very, very useful model that led to a lot of insight on the biology. Um. But, um, the key, I would say, here is really a landmark discovery that came out of, again, from Adrian Bird's lab in Edinburgh, uh, 2007 paper demonstrating that if you were, you, you know, through again, uh, genetic technology, if you're able to reactivate expression of MGP2 in a null mouse model. That you could rescue the phenotype, uh, that means survival would be close to normal, and many of the symptoms, the motor impairment, the breathing issues could all be corrected, and also that, uh, the cellular pathology, you know, the neuronal, uh, the size of the neurons, the dendritic uh, uh, arborization. All of the pathology was also corrected in these mice, even when you reactivated the gene at a fairly late stage in disease. So this was really amazing because it it gave us hope that, uh, you know, you could really treat this condition. That the effect of the mutation was not a permanent thing, that it did not, uh, you know, it was not irreversible, so that was, that set us off to finding, trying to find treatments, as well as genetic and non-genetic, you know, small molecule therapies. So the, you know, there are a lot of things that, uh, are, are challenges for RET syndrome therapy development. And first is based on, on what we knew about molecular mechanisms. You know, MEC P2 as a transcriptional modulator is modulating the activity of thousands of genes. This is in contrast to a condition like tuberous sclerosis, where there's a single pathway, you know, the MTOR pathway is a key pathway in TSC, so you could potentially tackle that with small molecules. Here you have to somehow restore expression to normal of thousands of genes, so difficult task. The second problem is that MEC P2 is a highly regulated molecule itself. It is, its expression is controlled, and it is, its structure is altered by phosphorylation and other post-translational modifications. And so these transcriptional modulation complexes are assembled in a very dynamic fashion. And this is very important for learning and synaptic plasticity, and so that tells you why MG P2 is critical for normal brain development. So the dynamic aspect of it has to be tackled. The third thing, which is really the major problem, uh, is what we call the Goldilocks problem, a gene dosage sensitivity, and we know now from both transgenic mouse models where, you know, 1.5 to 2 times gene dosage can cause a neurological syndrome, and then the identification of the MCP2 duplication syndrome, you know, again, confirmed that this is really a human disease. Too much is as bad as too little. And the major problem is, of course, 99% of our patients are female, and females, because of the pattern of Xin activation, are mosaic, uh, so that means they have normal cells and mutant cells in their brain, and, uh, you would like to somehow target only the mutant cells without disturbing the normal cells, and that is a big challenge. Now, years and years of studies of biological mechanisms, you know, the thousands of genes that are altered. Uh, led to focus on two things. One is brain derived neurotrophic factor, a growth factor that's very important in brain development and plasticity, uh, was one of the first things that was discovered to be modulated by MECP2, and increasing expression of BDNF can rescue the phenotype in MECP2 null mice. The second thing is IGF-1, which also works through similar signaling pathways, you know, AKT PI3 kinase, AKT, and MTOR also promotes, uh, neural development and synaptic maturation. BDNF does not cross the blood-brain barrier and so it was not possible to develop as a therapeutic agent, although there are efforts at identifying small molecule ligands ligands that would activate BDNF. Uh, so using kind of molecular mechanism-based strategies, people have tried many, many things, including track B ligands, fingolimod, which is used in multiple sclerosis, known to increase BDNF levels, neurotransmitter receptors, and of course, genetic therapy, small molecules that could, uh, target specific subclasses of mutations. And maybe even reactivation of the silent X chromosome, which is a strategy that has been tried in in cellular and mouse models, but the focus really is on IGF-1, which has, uh, you know, been developed into an FDA approved drug right now. So how did this come about? So IGF-1 was known to be a very important molecule in the development of cortical synaptic plasticity. This is work from many, many years ago from basic neuroscientists who were studying, uh, you know, ocular dominance cortical plasticity in a mouse model, and one of the genes that showed up was uh were IGF and IGF binding proteins. And in a completely different pathway of investigation in people who are looking at genes by RNA sequencing that were affected by MECP2 mutation, they found that IGF binding proteins were also regulated by MCP2. So putting these two things together, there's a group in Boston, you know, uh, the laboratory of Dr. Mirgan Kasur, who published his paper in 2009. Showing that in. A MGP2 null model, they use the exon 3 deletion model that you could treat with the active N terminal tripeptide. This is the IGF-1 gets cleaved off from a pro molecule into the N terminal tripeptide and the rest of the molecule, and the IGF tripeptide is active in, in all of, in the cortical plasticity model. And they found that if you, you could treat these animals to rescue the phenotype in, in the McP2 null mouse, motor function, survival, cellular pathology, as well as physiology. Uh, of course, you know, IGF-1 is already FDA approved for treating, uh, endocrine disorders and growth failures, and so this was a natural thing to test, and it turned out that it was safe, but, uh, did not have any efficacy in, in placebo controlled crossover studies in humans. And, uh, the problem also is that the IGF-1 tripeptide, this is the glycineroline glutamate tripeptide, has to be given parenterally. So, in mice, you can give it intraperitoneally, in humans, you can give it IV, but it has an extremely short half-life, lasting only a couple of minutes. So, for therapy development, um, uh, this was altered to get a synthetic form of this, uh, tripeptide. Which is what is now called trofinitide. They added an alpha-methyl group to the prolyl group, and this improved the stability, the half-life in the terms of hours, and it's also orally bioavailable, so you could have an oral drug, and for many years this drug was being tested in many different neurodevelopmental disorders, including brain injury and stroke. Um, So this was uh tested between 2017 and 2019 in safety trials in RE syndrome, uh, led by a group that included Daniel Glaze, Alan Percy, and Jeff Newell, and they found that it was safe to use in various doses. A dose escalation study suggested that even up to 200 mg per kilogram BID was tolerated in patients with RET syndrome, with the main side effects being diarrhea and vomiting. And in these preliminary studies at the highest dose, there was evidence of some improvement in the phenotype as measured by a caregiver rating scale called the RSBQ and the clinician's gauge of clinical improvement, the CGII. Um, this led to a phase 3 study, uh, that was gone between, I think, 20 2019 to 2023. Uh, our own clinic at TG was, uh, one of the sites for this study, and so we were participants in this study, and based on this, 187 patients were recruited, and the primary endpoints were these two behavioral, um, measures, RSBQ and, uh, CGII. And based on the improvement in the score, as well as, uh, for RSBQ and CGI, it did get approved by the FDA. The only thing is that I would say, you know, there was a drop off in the number of patients that went on into the open label studies, both the LAC 1 and the LAC 2 studies. So I think I'm, I'm going to go a little bit faster because we're running out of time. Uh, I'd like to show this picture because this is a female mouse in which, uh, you know, this is a hippocampus, dentate gyrus in a mouse where, uh, the, the paternal X has been marked with green and the maternal mutant X, uh, mutation carrying X is not marked with green. It shows you in graphic pattern, the X in activation in the brain. So, in the last few minutes, I'm going to talk about the gene replacement strategies. Uh, these are the problems, you know, especially the Goldilocks problem. It needs to be present throughout the brain. It has to be lifelong, you know, it's not enough to just have this gene active for a few years, because adults need this too. from between 2013 and 2024, there were a number of pre-clinical gene therapy studies in, in mouse models. Almost all of them started with the male null animal because it eliminates the confounding factor of Xin activation, and then these were also tested in the females. And the kind of the uniform finding was liver toxicity when you give it systemically, this was less of less of a problem and the vector was given intrathecally or into the intracerebral ventricle, and then overexpression toxicity in both female mice and in wild type animals. So this is a big problem, and uh these two labs, led one led by Stephen Gray, and the other one by Stuart Cobb, were trying to get around this problem by introducing um regulatory elements into their uh vector. And then another, uh, Stuart Cobb's lab also found that you could drastically truncate the MEGP2 protein. Eliminate, you know, 80% of the protein and just include the MBD and a portion of the transcriptor repressor domain, we call it the mini gene that could, uh, uh, those animals, mutant animals that carry that mini gene where near normal, and this thing could rescue the phenotype in a null mouse. And so that led to the development of the mini gene as one potential gene therapy agent and then. MicroRNA elements were included to regulate the problem of overexpression. So, these are the two, phase two clinical trials that are going on right now. One that's, uh, using this, uh, agent called TSHA 102 or TCA 102. It's a self-complementary AAV that means the package that you can only carry about 2 KB of DNA. It includes microRNA binding elements that that have a feedback loop, keeping things under control and controlling overexpression. So this vector, at least in early studies, the outcome measures are regain or gain of developmental milestones, as well as the clinical global impression of improvement and a motor behavioral assessment, and results are promising. A second, uh, uh, trial that's also ongoing right now is sponsored by Neurogene and their product is called NGN 401. It's a different strategy, but they don't, they use a full-length human, uh, MCP2 cDNA, but it's driven by a modified CMV enhancer, chicken beta actin promoter. And includes microRNA elements that are non-mammalian to regulate the overexpression problem. Again, early results are promising for both of these things. Now, lastly, let's see, I think I have 2 more minutes to tell you about what we have been doing for the last 5 years. Um, in 2018, I read a paper written by Doctor Chatterjee on her discovery of a new, uh, clay, uh, type of AAV virus. These were isolated from human stem cells that they were harvesting from cancer patients at the City of Hope. And she discovered that this clade of AAVs mediated high efficiency, high fidelity, uh, gene editing using the traditional homologous recombination machinery of the cell. So when I saw this, I said, oh, great, this is the answer to all of my problems. We don't have to worry about including, um, heterologous promoters or microRNA elements. We can retain all the control elements of the normal gene. So I, I finally talked her into helping me and we designed an editing vector that would potentially correct all, you know, more than 95% of the mutations. We tested these in patient derived cells, you know, fibroblast cell lines that we had maintained here, and showed that a single virus had the potential to correct all of these mutations. This was published already. And here it shows that, uh, this panel shows that in a male cell, this is the male deletion cell, it actually restores uh expression of the protein, which is again, to, to us earth shattering that you could do this. The vector does not contain any promoter itself, so it's using the native elements to drive the protein expression. And the only way you can get a re-expression of protein is if the, the homologous recombination editing event has actually occurred. Uh, we in our, our lab here at TG, uh, again with Sampath and Arvind, we tested this editing vector for Exxon 4 mutations using one very common model, the R306C. This is in the top five of the mutations in humans, and this is really unique because it is not a null model, but the point mutation completely disrupts the function of MCP2. That means The mutant protein is made in these mutant cells. It may occupy the, the methyl DNA binding sites, but it doesn't do its job. The effector component is gone, and we showed that by edit by injecting the editing virus, uh, systemically, you know, through an IV injection, we were able to, uh, rescue the survival problem, you know, R306C die off just like the null mice. And we were able to rescue the phenotypic severity, and here it shows that at a at a certain time point, there's a dramatic difference between edited and unedited, unedited mice. Um, Dr. Chaterjee has now got a new editing vector. We call it the universal editing vector. That will take care of everything else, including deletions, and she has tested this in the same MEGP2 knockout mice. and here again, this is data from Doctor Chatterjee. There's a micrograph showing the untransduced null male. There's uh red staining would be MEGP2. This is a wild type animal where MGP2 is in the nucleus. These are examples of edited male MGP2 mice showing that you can get re-expression of the protein in there. Now with the R306C moss, we cannot use antibodies because the antibodies will cross react to the mutant protein. So I think I'm going to finish there and just uh conclude by summarizing that, uh, over the last 20 years, uh, work has led to one FDA approved drug, that's trophinitide, the IGF pathway. Two human gene therapy trials are currently underway, and something that we have been working on is still in a pre-clinical stage, uh, MCP2 gene editing system using homologous recombination. Where a single vector can treat more than 99% of all the mutations that causeretts syndrome, high fidelity, high efficiency. And this is, this can be used for practically any genetic disease, not just REI syndrome, and we are testing it now in Usher type 2 cell models. I'll stop there. I'm sorry I've gone a little bit late and uh. See, open to questions and discussion. That was excellent from both of you. I really appreciate it. It is a wide-ranging improvement that we have and so I'm glad to hear all the information. There is a question, looks like from Doctor Creer. You can unmute yourself if you want, Michael. Yeah, fantastic talks, uh, Terry and Vod. I, I had a question in particularly in regard to the, the gene editing. Um, you know, Dr. Narayanan, if you could talk a little bit more about what the next steps are in that particular process, I'd, I'd be really interested to hear them. Right, so that, uh, gene editing vector that we made that we call the universal vector, it actually deletes the intron 3, that's between exon 3 and 4. We've just realized that there were no important elements in that intron, and, uh, by joining exon 3 and 4 together with a what we call a codon altered sequence without changing the protein sequence, this forces recombination to occur outside of. Uh, the exon 3 exon 4 area. So this again, we have tested in male animals and it seems to rescue phenotype and, and, uh, expression of MCP2. Uh, we have to do, we have started the testing on females because that's the most important thing to make sure it's not toxic in females, and I think with all the experience using AAV viruses, we know how to handle, uh, the immune responses and how to pre-treat human beings. I think, uh, going to human clinical trial will be, will be probably 2 years away once we get all of the female studies done. Great. Thank you. Yeah, and the, the other, as a corollary to that, you know, the, uh, Usher 2A, we, this, that work started off because of a patient that came to see me in my clinic. Uh, the US2A gene is humongous. I mean, it, it's, uh, it's quite, it's near, near, nearing the size of the DMD gene, and, uh, there's a recurrent mutation in Exxon 30 or 13 of the Usher 2A gene that causes, uh, retinitis pigmentosa, and the biggest main cause of Usher type 2. Uh, but that, uh, you know, the usual AAV strategies just will not work just because of the size of things, um, and so it seems ideal, and so we are working on, uh, again, gene editing strategy just to take out, replace Exxon 13 mutation with a wild tap Exxon 13. Again, very promising, uh, we'll, again, a few years away from seeing that in human clinical trial. Yeah, I might mention one more thing, uh, please, yes. Before, before, uh, the gene was discovered. Uh, we were collecting, you know, families. There were, there were probably about 10 familial cases that were known, uh, where a mother had a child who was affected, and some of them, uh, were, you know, and, uh, an auntie passing and things like that, just, just a handful about every day, everybody else was de novo. But there was one family from Brazil that Doctor Naidoo invited up to, up to Kennedy Krieger in Baltimore. This is a mom that had 4 affected children. 3 were alive, 1 had died away. And, uh, she told me about this, and I put her in, I, you know, I connected her with Eric Hoffman, who was then at the, the Children's National Medical Center in DC, uh, again, expert in, in gene mapping. And they use this family to really localize the gene to XQ28. So that was a real breakthrough because they had pretty much ruled out the entire X chromosome from consideration by exclusion mapping, but this was a key family, and then it was just a matter of slogging through the end of the X chromosome and you know, the Baylor Baylor program had were sequencing the X chromosome anyway, so it was just a matter of time before they, they localize the gene. I mean, Doctor Naidoo's work was very critical in identifying the gene. The note, could I ask you how many adult patients you have? Um, well, you know, since I've been working here for, for 20 years, many of my patients are now adults, um. I would say about 50% of them are in their over 20. Probably, you know, um, I mean, I can think of 5 or 6 of them off the top of my head, but probably more than that around here. Mhm. OK, thank you. You guys both highlight the importance of the collaboration and just knowing what's going on around the country and around the world. There's nothing that, that takes the place of that and the research and the research funding, the research dollars, so I appreciate all you do. It's amazing. You know, we have been, uh, Terry and I have been talking about, uh, Red Cent clinic at CRS for, I don't know, at least, ages. It never happened, so I'm just really, really happy to, to see that Terry and, and Doctor Ungapan have, uh, are planning this, and I think it's just wonderful. I think this is where, how it should go because, uh, you'd be ready for all the clinical trials that are coming down the pipe. Yep, exactly. Yeah, I appreciate you guys. Thank you so much. Thank you. There's a lot of good comments in the, in there. No other questions, but just, you know, everybody's very excited to. People to provide this here and maybe they don't have to go to Colorado. Hope not. Yeah, exactly. You guys have a good rest of your Monday. Thank you everybody and if v was still on, if you didn't see the text that I think there's the uh CME system is down, so as long as the business names are in there or if they just reach out, so.
