NIMH Workshop: Gene-based Therapeutics for Rare Genetic Neurodevelopmental Psychiatric Disorders
NIMH Gene based Therapeutics for Rare Genetic Neurodevelopmental Psychiatric Disorders Caption Transcript January 21, 2021 * * * * * Geetha Senthil: Good morning. Everyone, I welcome you all to the NIMH virtual workshop on gene based therapeutics for genetic neurodevelopmental disorders. Today we have a panel of experts from academia, government and industry to share some success stories and their perspectives on challenges and opportunities in developing gene based therapeutics for individuals with rare genetic neurodevelopmental disorders. Specifically we will review the current state of science in gene based therapeutics including gene targeted approaches, vector design and delivery options, characteristics of suitable gene molecular targets and mechanisms amenable to gene based therapies. To set us off with overview talks, followed by a set of talks on key studies of gene targeted approaches that are currently in the clinic or in the development to highlight the scientific rationale for treatment options and also the milestones for reaching key stages of therapeutics development, as well as we will talk about the challenges of these individuals or experts faced while developing the gene based therapeutics and lessons learned along the way.
So I want to thank the members of workshop planning committee for their immense efforts in developing the program of the workshop. My special thanks to the chairs of the workshop for the relentless efforts in developing the agenda for this entire workshop. It's a two day workshop. Please note day two will be on the 29th. We will release a summary of that discussion following the meeting in a month or so. The chairs of the workshop I
want to thank are Drs. Mustafa Sahin from Boston Children's Hospital, Guangping Gao from the University of Massachusetts, Sitra Tauscher-Wisniewski from Novartis Gene Therapies, Beverly Davidson from Children's Hospital of Philadelphia, Cynthia Tifft from National Human Genome Research Institute and Terrance Flotte from University of Massachusetts. I also want to thank my NIH colleagues from the program. There several of them listed. They have played a key role in developing the goals of this workshop. I thank them all, especially Meg Grabb and Ann Wagner and David Panchision for the countless hours they spent with me discussing various strategies for this workshop. We have a five hour meeting today. So the first part of the meeting will be a set of
four 20 minute talks, these are overview talks, followed by a 10 minute break. Please stay on Zoom. Don't go anywhere. Commute yourself and join back on time. During the second part of the meeting, we'll have six talks on case studies focusing on rare genetic neurodevelopmental disorders to highlight some success stories and also the challenges for specific disorders. These include Rets syndrome, Angelman syndrome, MeCP2 duplication, and spinal muscular atrophy and giant axonal neuropathy. We will have a 40 minute time at the end of these talks for taking questions from the audience. So audience, please use the Q&A feature on Zoom
to type up your questions. And please add the name of the speaker to whom you're directing the question. And the audience have an option to vote up a set of questions to prioritize for the speakers to address them. We will adjourn at 4:00 p.m. and thank you all for joining the meeting. We'll now welcome Dr. Josh Gordon, director of NIMH to say a few words, opening remarks, for this workshop.
Josh Gordon: Thank you, Geetha. I want to thank you, I want to thank all the NIMH staff and the members of the organizing committee for putting together the program today. I'm really excited that we're hosting this workshop in an era where gene targeted therapeutics have begun to be applied in the nervous system and have already achieved FDA approval even in a couple of cases, and we need to think about approaches that will be appropriate to treat the disorders that NIMH is responsible for. We need to think about it from the perspective of the long term. In terms of developing platforms that will enable a precision medicine approaches
in the central nervous system in much the way that other Institutes are pioneering them for other organ systems. And we also, I think, have the right and responsibility to think about it in the shorter term which of our disorders are most amenable to this approach, to pioneering this approach, in as safe and ethically responsible a way as possible and in a way that's demanded by the tremendous morbidity and mortality that is conferred by some of the more severe neurodevelopmental disorders and other disorders that we care about. So I want to thank you for getting together as a group. I apologize, I'm going to have to leave to go back to another meeting. I'll be popping in and out the rest of the day. I hope you have productive discussions and I look forward to hearing more about the conclusions that arise from the day. Thanks. Geetha Senthil: Thank you, Josh. I will turn this now over to the chairs, Mustafa and Beverly
and Sitra and others to continue our first part of the meeting. Mustafa Sahin: Thank you very much. And good morning everybody. I want to thank Josh Gordon, Geetha Senthil and the whole NIMH team for sponsoring and organizing this workshop and the co chairs as well as all the speakers for their contributions in preparation of this meeting. We all think this is a very timely and impactful meeting. We have a packed
agenda today, an ambitious agenda, so to be able to stay on time as co chairs, we've decided to keep the introductions to a bare minimum, but I would urge you to look up the bios of the distinguished speakers that will be presenting today. Our first speaker and co chair of this workshop is Dr. Guangping Gao from the University of Massachusetts Medical School and he's going to be presenting about the gene targeted approaches and tools for CNS disorders. Guangping Gao: Can you see my screen? Mustafa Sahin: Yes, we can. Guangping Gao: Okay. Thank you. My name is Guangping Gao and first just like a mask update,
I want to thank our NIH colleagues and leaders for their tireless effort in organizing this timely and important workshop. I also want to thank our speakers and chairs as well as the panelists for their participation and important contributions to the workshop and thanks to all the attendees to the workshop. So my task today is give an overview on approaches and tools of genetic based therapy for rare disease. This is my disclosure. And human diseases can be categorized into two categories.
One is acquired disease and one is inherited genetic diseases. The causes for genetic diseases includes autosomal recessive mutations, autosomal dominant mutations, X linked mutations and haploinsufficiency. So the consequences of those genetic mutations include loss of function, gain of function or gain of toxicity, haploinsufficiency. So gene therapy actually is a highly attractive approach for rare genetic diseases, particularly neurodevelopmental and psychiatric disorders. This is because most of them caused by genetic defect and most of them are monogenic disorders.
And the gene therapy actually offers the most direct therapeutic potentials. Now I want to give an overview on strategies for gene based therapy. The first approach called the in vivo gene therapy, in this approach there are two categories of this disease and once viral gene therapy. And I used FDA approved AAV drugs as example. The second category is non viral and I use FDA approved nucleic acid drugs. As examples, this include ASO and SIRNA. The reason we call in vivo is because those drugs we give you just like any other
pharmaceutic drugs you directly give to human and treat the disease. The second category called ex vivo, where you take out human patient cells, genetically modified, expanded and infused back and those modified cells will function as living drug in patient to complete therapeutic outcome. And there are four drugs approved by FDA and EMRA. That's overall strategies and in terms of conventional approaches for gene based therapy I would like to summarize into the following categories. The first when you have a loss of function, then you replace these normal gene and accomplish gene therapy, that’s called gene replacement. The second category you may not have mutation on your gene, but you could get exogenous or endogenous gene through overexpression to accomplish gene addition therapy. The third is when you have a loss of function and the gene become toxic,
then you can deliver a molecule to silence those genes. And finally, you can modulate gene and its expression through gene editing. So those are the approaches, but what are the key components for gene therapy research? I will summarize into the four: therapeutic gene, vector, route-of-administration to target tissues safely and efficiently, and animal models to study efficacy and safety. And among them, vector is most important component for the gene therapy. As the gene therapist, our desired features of a viral vector for in vivo gene delivery are the following. First we want high efficiency just like adenovirus, we want long term stability like lentivirus, we also do not want immunogenicity and toxicity, and we do not want genotoxicity.
Actually, now all the viral vectors we have, Adeno-associated virus has it all. This virus is a single stranded adenovirus and is a protein capsid and is consisted of regulatory proteins and capsid proteins. In addition there is some accessory proteins such as AAP and MAAPs. Most importantly to date there is no direct evidence of a causative association base any disease in this virus.
How do we convert this virus into the vector? Basically you take out the viral gene and then you replace with your therapeutic gene cassette and then you provide a helper function and Rep/Cap function and put all those components into a cell that can be transient or stable transfection or to infection such as baculo-, adeno- or herpes virus. And then you generate recombinant AAV. The beauty of the system is you can simply replace the capsid, in this case we replace with AV9, and then become AV9 virus because the biology of the vector is primarily determined by the caps itself. So now AAV mediated gene therapy actually is a teamwork inside out. The Capsid is very important. How it dresses matters. It directs tissue tropism and intracellular trafficking, it dictates host immune responses, it delivers the therapeutic genes to the nucleus.
Because Capsid is most important for AAV vector, so vector development is centered at Capsid discovery. The first strategy is looking to natural reservoir. In this case you identify sequences, capture it, and then through high throughput in vivo evaluation. The second strategy is called directed evolution. There are three strategies, the first strategy is called capsid shuffling, and second called error prong PCR, by those means can generate the variants of the new AAV capsid. The third approach is peptide insertion, it basically conducts capsid surface panning of random peptides and you create new Capsid with those insertions. And third strategy is rational design. You basically, based on Capsid structure-function relationship or you redirect a tissue tropism receptor/ligand engineering. And the final
strategy is called in silico design or machine learning. This is based on the phylogenetic analysis and computer science. And the most popular current approach is really the pathway insertion, many vectors in the past several years have been generated through this pathway.
My AAV gene therapy career starts in 90s, when I joined Dr. James Wilson at the University of Pennsylvania searching for the next generation of AAVs. This strategy we started 2001. We designed primers, a neotube conserve reaching and the general library of the Capsids. And really I want to give you example, that's AAV9, which is the most popular AAV today for rare disease. This was discovering a human liver samples on January 21, 2003. As showing here, that's the PCR band that contains, we amplified on human tissue contains this AAV9 and this is the topochromic clones, and this marks the beauty of the virus at the strong transcytosis and across vascular structure spread transgene widely through muscle, liver and most importantly can cross blood brain barrier lead to widespread seeing this transaction.
We also recently discovered another AAV2 natural variant called a V66. As you can see, it's a very close relative to AAV2. And this virus only has 13 amino acid difference from AAV2. However this difference makes it 13 fold more neurotropic after intrahippocampal injection. And so that's the Capsid. However, as I said, AAV is teamwork, inside out. The genome itself is very important. This therapeutic payload carrier it mediates therapeutic benefit, achieves
long term stay in the transmuted cells, triggers innate and adaptive transgene immunities, if you do not have good design but it causes potentially other transgene-related toxicity. And actually engineering the vector could start from ITRs, which is terminal repeats, as showing here actually pioneer work by [name] and Dr. McCarty back in early 2000s, they have done this and created a potent vector itself called [Away from mic] vector. With single strand DNA we package that can go up to 5KB, if packed as single stranded DNA and once get into cells, you go through this second strand synthesize or self allele-ing, you can generate virus that is transductionally active. This is a two difference dose in mouse variants, however, those scientists, they modify the one ITR, make a mutant ITR, then when package, you package as a mirror image of two copies and folding back from double stranded. By doing so, as you can see here, you bypass the second strand synthesize, the
early onset of expression and high level of efficiency. As you can see here, there's at least 10 folds difference between single strand and double stranded. The second engineering could be a transcriptional regulation. When you use ubiquitous promoter single IV injection AAV9 it transduces many different tissues. If you add different tissue-specific promoter
you can limit transaction in that tissue. You could use cell-specific by using neuronal promoter or glial promoter accomplish cell specific single transduction. The beauty of the AAV is a single injection can maintain long time, this is ubiquitous promoter expressed, which is equal in monkey muscles for eleven years. However you could also add a regulated promoter, in this case erythropoietin you can have inducible promoter as the level as well as the intervals you want. Another engineering you can do is post-transcriptional regulation. In this case are you can add a microRNA binding site to the three prong UTR when you are getting the tissue down without this microRNA binding site and you will have a beautiful expression in CNS and peripheral tissue. If you get into tissue that express the microRNA binding site, what happens is
the messenger will be CLIP and you only express in the CNS. Another engineering can be done on the cDNA the other transcriptional accessory, this can be a Kozak sequence and the transgene code optimization to generate a much potent cDNA cassette and you can improve intron to get a higher expression. However, I have to warn you that when you are engineering cDNA, you want to sustain strong expression, you need to balance the CPG content and the codon optimization. This is because you have two opposite facts. That is, you have balance between expression and the DNA sending as well as stability of the expression when you have a reduction of the CpG islands this stability will be enhanced. And however, when you have completed codon
optimization, you will have the DNA sensing and your stability and your expression goes up but your stability will be reduced. The balancing is the key to accomplish high level but stable expression. That is the basics of AAV. I want to give you overview of the current landscape of rAAV gene therapy. We have done survey and found currently there are 13 different diseases and tissue targets. Among them, as you can see, CNS is the hottest target as you can see here. As the gene therapy target CNS has when we do gene therapy, we have several factors
we need to be considered for gene therapy. First when you think about distribution and molecular mechanism of disease pathology, that include localized versus global pan-CNS pathology. When you think about genetics versus epigenetics, gain vs loss of function vs haploinsufficiencies. Second factor is a cellular target. This could include neuronal such as neurodevelopmental, psychiatric and neurodegenerative disease vs glial such as leukodystrophy. Blood brain
barrier is the key delivery barrier and should have a different approach to overcome. First is direct CNS injection, as you can see, we have many different ways to deliver genes in CNS by direct injections. You could also use AAVs that can cross the blood brain barrier by intravascular/systemic injection. One thing I want to bring to your attention is haploinsufficiency because this is a common genetic cause for neurodevelopmental and psychiatric disorders. And the potential for approach for this therapy of this type of disease is first we can deliver cDNA gene augmentation. We can also repair mutant allele by DNA/RNA editing, we can also boost expression of the mutant free allele through either gene activation through CRISPRa activation or by ASO through productive transcripts and also enhancing transcript stability by ASO.
Another approach I want to indicate is readthrough therapy of nonsense mutation. As you may know, 11% of all pathogenic mutations are nonsense mutation. We could use small molecule drugs, such as G48 and ACT128, but we could also use suppressor tRNA. And the factors to be considered in this haploinsufficiency gene therapy is first need tight control of gene expression. This include level expression as we know many cases gene replication is toxic. Second it's anatomic regions and tissue cell type specific. The third window because the developmental disease you miss the window and you may have less therapeutic effect.
The second large category is promoter, self regulation and negative feedback and it includes microRNA regulated feedback. The third is sometimes mutant allele is not purely loss of function. In this case knock down mutant allele and the same time augment the normal gene.
Actually I want to introduce some concept at work by my colleague professor Dan Wang and his group. What he was trying to do is use AAV suppresser RNA for readthrough therapy. You have regular tRNA and regular stop codon but also could have premature termination codon. And suppresser tRNA only have one difference from the natural tRNA. So the group pick up this old concept pick up for new opportunities. We are doing research for treating Rett Syndrome,
FoxG1, CDKL5, Dravet Syndrome and others. The long term delivery is AAV advantage. Of course one concern is global readthrough and perturbating elongation and they are doing tRNA sequencing ribosome profiling. The reason Dan pick up this approach includes you can use one stone kill experts because a single suppresser tRNA can maybe attack different mutations and different target genes. Now you put a multiple expression cassette of a higher potency, you can see there's no exogenous protein or transgene immunity. And this is under control of the transgene regulation, no over expression. And this amenable for
engineering and transient delivery. And so – Mustafa Sahin: Two minutes. Guangping Gao: Yes. AAV by this method, Dan used a hurler model for concept. You can see through systemic delivery, in four weeks you can see partial restoration of IDUA and also normalization in 10 weeks of the urine GAG substrate. If you look at individual tissues,
you can see you have even higher expression or so you have almost complete normalization of the GAG. If you target the brain by systemic injection, this dose is not very effective. But if you do intracranial injection you can see you can restore partially brain activity. But that's showing the StRNA is quite effective. But whether this is safe in treating disease?
They also analyze whether this is safe by ribosomal finding. And in this case they show in patient fibroblast, G418 caused perturbation off target global rate, is true but not by StRNA. Also in mouse liver it's the same situation. If you look at also the elongation that G418 cause disturbance of elongation but not by this StRNA construct, in vitro patient fibroblast and in mouse liver. Thank you very much. I want to end here. I want to thank my colleagues. My group is a big group directed by four PIs, includes Dr. Xia and Wang and Tai. I want to thank our vector core. Thank you very much for the opportunity to present. Our next speaker is Dr. Sahin
from Boston Children's Hospital. He will give an overview of a rare genetic disease and neurodevelopmental disorder. Thank you. Mustafa Sahin: Thank you. My task is to give you an overview of rare genetic variants associated with neurodevelopmental disorders. I will start by acknowledging my disclosures because of my role at the translational neuroscience center in their hospital I work with a number of pharmaceutical and biotech companies. Let me start by defining rare diseases. In the United States rare diseases are defined
as conditions affecting fewer than 200,000 individuals at a given time. Taken together an estimated 300 million people are affected with rare diseases. They used to be referred to as orphan diseases. Due to the orphan drug act and better understanding of the biological mechanisms, many companies have been interested in these rare diseases over the last few decades. Here is a visual representation of the number of rare diseases increasing over the years as we identify genetic causes. As you can see, in 2020 now we're over 6700 rare genetic disorders identified and only about 500 of them have any type of therapy. Importantly,
for the purpose of this workshop, roughly 75% of rare genetic diseases affect the central nervous system. There have been a number of successes in this field, as mentioned earlier. One of the major success stories is the FDA approval of drugs for spinal muscular atrophy. This is an announcement
from the FDA around Christmas of 2016 when the first drugs was approved. Since then two other drugs have been approved and they have completely the changed the natural history of this disorder in child neurology. We’re going to hear much more about that from Jerry Mendell later today. While these success stories are occurring, there's a huge unmet need in the field, especially in mental health disorders. Here's another FDA announcement. This time warning about
potentially dangerous therapies and products in the field of autism. And this is due to the dire need of families dealing with this disorder. And since there are a limited number of scientifically proven therapies in this field, they are reaching out to therapies that may not have scientific basis and that might be potentially dangerous. So how do
we start to address this huge unmet need? One aspect that has come to the forefront is the role of genetics. Here are the heritability estimates for neuropsychiatric disorders, especially for disorders such as autism spectrum Disorder, the heritability is remarkably high. Here's a slide from NHGRI workshop back in 2009. This summarizes the role of rare and common genetic variants in the field of complex
disorders. For the purposes of neuropsychiatric disorders we really fall into the diagonal for the most part. There's a role in the population for common variants, but these common variants have a small effect size per individual. On the other side we have rare alleles, what we would call Mendelian diseases where a variant of this sort, while rare, would have a high effect size. And for the most part in the field of autism spectrum disorder, the main progress has been made in identification of rare alleles associated with autism spectrum disorder. Here's the progress that's been made over the last several decades from a slide from Tom Bergeron's paper. This has happened thanks to advances in the technology we're using,
we're moving from karyotype to chromosome microarray to identify deletions and replications to finally exome and genome sequencing that allowed us to identify sequence variants. And the variants that are associated with autism in terms of the type of variant really take place in the whole spectrum. There are structural variants with a large chromosome deletions or duplications that can encompass several genes. In several cases, either deletion or duplication of that chromosomal region can be associated with neuropsychiatric symptoms. Importantly, in these disorders which encompass several genes, often it is the case that it is hard to identify a single gene that's predominantly responsible for the phenotype. It might be
a combination of oligogenic effects. There's also trinucleotide groupings, missense mutations, nonsense mutations, indel splice site and possibly and likely intronic and intergenic variants as well. There was an NIMH organized workshop in 2017 that discussed the role of rare genetic disorders in neuropsychiatry and I won't have time to go into the details of this discussions but I urge you to take a look at this paper led by Stephan Sanders for some of the highlights of that discussion. What I'm going to do today is take a much more simplistic approach and focus on the heterogeneity of rare genetic variants associated with Autism Spectrum Disorder. In the field the estimates range from 400 to a thousand susceptibility genes. How do we approach treatments for this heterogenous condition? There might be a couple of scenarios. On one side one can think of a single broad spectrum
treatment that works for all types of genetic variants. But as we start to understand the genetic code of autism, it is becoming clear that certain genetic causes result in diametrically opposite changes in synaptic function, such that a treatment that works for one form of genetic cause of autism may actually worsen other genetic cause of autism. On the other side we may have to think of a scenario where we have to develop a treatment for every gene or every variant. This is very much the topic of our discussion that's going to take part today. I should highlight that several of us in the field believe that there might be subcategories of autism that share convergence either at the cellular, circuit level or maybe at the level of platforms used for interventions. And learning from one single variant or one single gene may have implications for related disorders.
So with this landscape at our institution and many other institutions around the world we have been taking this approach to try to develop new therapeutics for autism spectrum disorder, starting with our genetically defined autism patients at specialty clinics, we try to do phenotypic characterization of individuals affected if there is neurodevelopmental disorders as well as biomarker identification. In many cases we develop cellular and animal models to test for both efficacy and toxicity. And we finally move into clinical treatment trials. What I'm going to do in the next portion of the talk is to give examples from some genetic disorders that we have tried to approach from a mechanistic perspective and tried to address some of the issues that have come up as potential discussion points for the rest of the workshop.
The disorder I'm going to focus on is tuberous sclerosis which I have been working on for a number of years. There are a number of reasons we chose this disorder to focus on, roughly half of the patients with TSC are affected with autism spectrum disorder. But importantly, any of the specialty patients will be diagnosed very early in life, and sometimes even before birth. The cellular mechanisms of TSC have been studied from a number of different perspective from a large number of labs around the world. And importantly there are FDA approved specific
inhibitors of this pathway that can be repurposed to test hypotheses in clinical trials. The combination of these factors makes tuberous sclerosis a good model to study in neurodevelopmental disorder space. This is a simplistic point of view of what TSC1 and TSC2 proteins that are the causal genes in this disorder do in the cell. These proteins, encoded by TSC1 and TSC2, form a complex. When this complex is functional, it puts a brake on a protein called mTOR which stands for Mechanistic Target of Rapamycin, which controls protein synthesis and cell growth. Importantly, if you missing TSC1 or 2, you have up regulation of this pathway resulting in this disease. A corollary of the understanding of the genesis is if you
over express TSC1 or TSC2 alone, you actually don’t see much of an effect. The complex is such that you have to over express both of these genes to actually see a bio chemical effect. In terms of dosage there is a safety factor here that's inherent to the biology of the disorder. One of the most important aspects of this interaction between TSC genes and the mTOR pathway has been the recognition that we have naturally occurring inhibitors of the mTOR pathway that have already been in the clinic for various indications. These are molecules
such as rapamycin that we've known for several decades. I'll give you examples of how rapamycin and similar molecules such as sirolimus have been used in the clinic and clinical trials to address symptoms associated with tuberous sclerosis. I'm going to talk about three prelaminated symptoms of tuberous sclerosis, one is benign astrocytoma, the second one is epilepsy, and the third is autism and related neurodevelopmental deficits. First with astrocytomas, here's
a first study from David Franz, and colleagues at Children's Hospital, when he took a handful of patients with these benign astrocytomas shown here and treated with rapamycin, what he showed is when the patient is taking the drug, the tumor shrinks, if the patient is off the drug, the tumor comes back, when you put the patient back on the drug, the tumor shrinks again. This was a very strong effect, easily quantifiable. And this led to a Phase 2 trial of 28 patients led by Darcy Krueger and the effect was reconfirmed in this study and published in the New England journal that everolimus was effective towards this type of tumors in tuberous sclerosis. This led to approval of this drug a month later by FDA. The next aspect I want to talk about is epilepsy which is seen in about 90% of the patients with tuberous sclerosis. In this paper by Elizabeth Thiele and colleagues at MGH, they show that most of the diseases with tuberous sclerosis start early in life and it's thought these seizures have an important impact on neurocognition and development of the child.
There have been a number of mouse models developed in various labs around the world. Here's an example from Michael Wang's lab at Washington University. When the animals are developed these knockout mice have seizures starting early in life. If you treat the mice with
rapamycin early in life, you can stop the development of seizures completely. And you can also increase survival markedly. These are the vehicle treated knockout mice, dying by about 16 weeks, and these are the mice treated by rapamycin. Importantly, almost all the mouse of tuberous sclerosis developed in different labs show this very strong effect. We have been working with iPSC-derived human in the lab. We see a similar hyperexcitability
phenotype that could be associated with epilepsy. These black lines are showing firing of human neurons in culture. This is a wild type, this is a heterozygous TSC2, and this is a homozygous deletion of TSC2. As you can see, the cells that are missing TSC2 are firing much more
than the other two genotypes, and importantly you can treat these cells in culture with rapamycin, you can significantly reduce the hyperexcitability phenotype. These type of evidence from mouse and cell culture led to the hypothesis that you could use rapamycin type drugs in the caudate to reduce epilepsy in patients affected with tuberous sclerosis. Here's a Phase 3 trial that took place with individuals between the ages of 2 and 65 with refractory seizures. This enrolled 345 patients and the primary outcome measure was the frequency
and severity of seizures in the placebo group versus the everolimus groups. I'm showing you the results from this study. The response was defined as greater than 50% of reduction in the number of seizures from baseline. There was a 15% response rate in the placebo group,
30% response rate in the low everolimus group and a 40% response rate in the high everolimus group. This was not 100% response rate but this was sufficiently successful to lead to approval of this drug by the FDA in 2018. The third aspect is the neurodevelopmental and neuropsychiatric symptoms associated with tuberous sclerosis. We took the same approach and used Everolimus in a Phase 2 trial in patients with tuberous sclerosis between the ages of 6 and 21. This was a randomized placebo controlled double blind study with two sites: 47 patients in all. We did neurocognitive testing at baseline, three months and six months and also looked at autism symptoms.
We published it a few years ago, and the basic finding was that there was no significant signal in the arm tree of the everolimus compared to the arm tree of the placebo. There might be a number of reasons why we may not see an effect. Outcome measures is obviously a significant concern in this field. Another concern is the age of treatment. By the time a patient is 10, 15, 20 years old with these symptoms is a short treatment of six months sufficient to see the effect. We have turned to the preclinical model models and Peter
Tsai in my lab had developed this mouse model of tuberous sclerosis by deleting the TSC gene in the cerebellum and these mice have both social interactive deficits and repetitive behaviors and cognitive inflexibility. If you treat these mice at one week of age with rapamycin, you can rescue all of these deficits. If you wait six weeks to start treatment, you can only rescue the social interactive deficits but no longer can rescue the grooming and cognitive inflexibility. If you wait 10 weeks to start treatment, you cannot rescue any of these deficits. These types of sensitive periods have been now demonstrated in a number of animal models of neurodevelopmental disorders. As mentioned earlier, PHC patients can be diagnosed very early in life with these cardiac tumors. We've actually formed a consortium to look at any biomarkers that might be associated
with development of autism and epilepsy in babies born with tuberous sclerosis, this is a five site consortia that's been funded by the NIH. And I'll show you the findings. We did prospective EEG of babies born with tuberous sclerosis and we looked at the onset of abnormalities on EEG and onset of clinical seizures. To summarize this work, the onset of epileptic discharges on the EEG was around 4 months. The onset of clinical seizures were around 6 and a half to seven months. There's a roughly two to three month period where
the EEG is abnormal and the clinical seizures have not started. Based on that, we have initiated a prevention trial. It's called Preventing Epilepsy Using Vigabatrin In Infants with Tuberous Scherosis Complex (PREVeNT trial). Vigabatrin is an
antiseizure medication that works on the GABA pathway and we're looking at the developmental impact of early versus delayed treatment and the results should be obtained within about 12 months. Now I want to briefly mention that TSC is not the only disorder where mechanistic have been tested. Fragile X is another cause of intellectual disability in autism. The metabotropic glutamate receptor (mGluR) theory puts inhibition of metabotropic glutamate receptors should reduce these symptoms and in fact this works very well in animal models. There's been a couple of studies in the clinic and they have not shown a significant efficacy. This important
paper highlight that the clinical trials in the future should consider initiating treatment in a younger population if longer treatment duration, longer placebo run ins and identifying new markers to better assess behavioral and cognitive benefits. So I've talked about fragile X and tuberous sclerosis. There's another large number of genes that are associated with neurodevelopmental disorders. How can we start to address those?
One of the potentials is identifying these convergent mechanisms and one mechanism we focused on is the mTOR pathway activation. We formed a consortium to study three gene related disorders in the clinic. With funding from the rare diseases clinical research network, funded by these four NIH Institutes. This is a multi center study where we’re performing detailed neurotological and behavioral assessment of these patients longitudinally in the natural history setting. We also have a pilot project looking at the effect of [away from mic] in Phelan-McDermid Syndrome patients. I want to summarize what I talked about. Pre clinical studies are leading to clinical trials
in rare genetic variants associated with neurodevelopmental disorders. Trials are currently taking place looking at symptoms. Prevention trials are being launched and I think are going to play an important role going forward. And biomarkers improve outcome measures and in depth natural history studies can accelerate these trials. I want to highlight that timing will be critical. The distribution of the therapy will be crucial, especially for cell autonomous treatments and the dosage of gene expression may be essentially for these disorders in which the range is narrow. I want to acknowledge in addition to the many of the collaborators we have in this field and acknowledge our funding sources. Thank you very much.
Geetha Senthil: Thank you, Mustafa. Do you want to introduce the next speaker? Mustafa Sahin: Thank you. Our next speaker is Dr. PJ Brooks and he's going to give DNA targeted therapeutic platforms for rare genetic diseases. PJ Brooks: Great. Thanks. So thank you for the opportunity to talk to you today about the different NIH programs. Very much looking forward to doing that. I'll actually go through three of them. Platform vector gene therapies, bespoke gene therapy consortium, and the NIH
common fund somatic cell genome editing consortium. Then I'll hit a couple key questions I think that are relevant for further discussion about genetic therapy development. This is kind of the slide that most of have already showed you. We're developing and identifying many disorders with a known molecular basis but the rate at which we're developing therapies is far too slow, particularly when we're talking about disease where we know the molecular basis. We ought to be able to do better. One way we think about this is to stop thinking
about one disease at a time and thinking about platform approaches to multiple diseases and certainly genetic therapies are a great example of that. So thinking first about AAV, which we heard about from Guangping Gao, there's really good news. There's excellent safety record, recent clinical success stories, two approved products in the U.S., and really a lot of pre clinical success stories. We're really great at treating
and curing diseases in mice and animal models. The real problem is getting to gene therapy clinical trials, which again is typically done one disease at a time, which is slow and inefficient and doesn't really take into account the potential for learning and the platform capacity of AAV. And also to the extent there's a commercial aspect to this, there's an obvious bias towards the most common rare diseases. But it kind of makes sense.
One of the things we're thinking about is if we design clinical trials for multiple diseases at a time, really taking advantage of the platform capacity of AAV vectors, we can increase the efficiency and reduce the time of clinical trial startup. At a very rough level, this is kind of the representation of AAV. There's a Capsid with a viral DNA replaced by the therapeutic human DNA and depending on the therapeutic human DNA you put in there, you can generate therapies for different diseases simply by swapping it out. You can almost think of AAV as a delivery vehicle like a delivery box for example, in
fact one that sort of pre addressed if you will to certain specific organs and cell types. And given this obvious platform capacity, you do wonder if we do multiple diseases, are there not steps that could be taken to increase the efficiency. Or to say it another way, do you really have to repeat all of the steps in the pre clinical development every time, even if you're just swapping out one therapeutic gene? So the goal of PaVe-GT is to put that question to the test. We see this as an experimental
pilot product. It's really a translational science experiment. And the idea is, we think of it as a sort of public AAV gene therapy development approach, where we’re going to be going forward for clinical trials for rare diseases, four different rare diseases under study by intramural investigators at the NIH clinical center. These diseases, at least at the time we began, were of no known commercial interest. And all four diseases we'll be using the same viral vector, in this case AAV9, the same route of administration. Same production and purification methods. As they say at the FDA, the process is the product. And we are going to keep the whole process the same and simply change out the
therapeutic constructs. And then I think the different thing about this as we go forward through the process and to the FDA, we will be making all of that information public, including the methods, the protocols, all the regulating documents, including our discussion through the FDA, and ultimately improved INDs and make them available to the website so other people can use them and hopefully use them in a cut and paste approach. So as another illustration of this, at the top you can think of what you might consider to be the null hypothesis which is we have got four different diseases here. On the left we have got two organic acidemias, PCCA deficiency and MMAB deficiency to on the right to neuromuscular diseases, DOK7. And the null hypothesis is that for each one of these, we have to do everything in parallel, all of these steps, proof of concept, CMC, biodistribution, toxicology, etc. But the question we’re are going to ask in PaVe-GT is since we are using the same platform, maybe we don't have to do all these things. Maybe we can reduce the number of
biodistribution studies or toxicology studies and also find ways to increase the efficiency and streamline the CMC process. These are the kinds of questions we'll be asking as we go along to the FDA. We'll get the answers back and make those answers publicly available to make the process transparent and hopefully help everybody understand how the clinical trials are developed and make them more efficient with the goal of benefiting all stakeholders with the particular focus on those focused on diseases of no commercial interest. So the project is ongoing. This is the team. It's a great team effort at the NIH. And everybody
involved is at the NIH, I particularly wanted to highlight my colleagues Donald Lo and Elizabeth Ottinger, and Anne Pariser, the investigators involved, Chuck Venditti and Carsten Bonnemann from NINDS. There's the websites you can follow on the progress and we also have a recent publication going into PaVe-GT in more detail. Moving on to the next project, which is the bespoke gene therapy consortium, which is a public/private partnership, again focused on making AAV gene therapy a reality for genetic diseases affecting populations too small to be viable from the current commercial perspective. This is a public/private partnership organized by the foundation for NIH, which is not part of the NIH, by the way, not part of the Federal Government. The FNIH, the FDA Center for biologics, NCATS taken kind of a leadership role but also involving many other NIH colleagues as I'll highlight later.
So the BGTC really has two different components to it. One component is focused on the basic biology of AAV as it relates to translation and the production of recombinant AAV vectors for gene therapy. So optimizing and better understanding of the basic biology of how we make vectors in manufacturing facilities and then also can we enhance therapeutic gene expression once these vectors go into patients.
And then the larger component of the BGT is actually supporting some clinical trials. And in the process, streamlining the manufacturing and analytics for the vectors involved in the trials and the clinical development process. So in terms of the clinical component, we envision here a pilot project involving perhaps five to six diseases still to be determined what those diseases are. And by doing this in a concerted way, to streamline the process for going through the idea of gene therapy all the way out to the clinical trial and standardizing things like the vectors available, the process in toxicology and testing and limited number of delivery methods and doing this in a consortium so all the things we learn from the process are reported back to the consortium to support iterative learning. And ultimately all of the work that comes out of this, we plan to put into the public domain as well so it can benefit the whole community and all stakeholders. Where we are here, this has been a fairly long development process beginning with the concept evaluation, well over about nine months ago, initial concept was approved. We're now
in the process of getting and finalizing the support from different stakeholders, including the government, private companies and nonprofits and are working on the finalized detailed research plan and again, want to thank my colleagues at NIH, Patina from NIH and Chris working with this on this effort along with the FDA Center for Biologics, private sector partners and others, it is very much a collaborative effort. Once we get all the funding agreements in place, we anticipate launching the program hopefully in the first quarter of 2021, like many other efforts, this one has also been slowed early on by the COVID pandemic. Then the final program I want to talk about is the NIH somatic cell genome editing consortium (SCGE). This is an effort supported by the NIH common fund to the Office of the NIH director and involves participation, again, many different NIH Institutes and centers. NCATS is taking
a lead role but it's very much a collaborative effort as well. The focus of the SCGE is to lower the barriers for new genome editing therapies by a variety of initiatives including better animal models and animal systems to test genome editing reagents and delivery systems. These are animals that have reported genes in them so we'll be able to assess editing in all cells and tissues in these animals, including rodents, pigs and non human primates. Another component is focused on assessing unintended biological effects of genome editors.
Not just looking at off target sequencing or off target genetic effects but going more forward and asking what are the biological consequences of some of the editing effects. And all these studies are carried out in human cell systems with a goal of ultimately reducing some of the animal usage in the regulatory process. That's an aspirational goal of the SCGE long term.
There's also a component involved looking at better ways to be able to monitor genome edited cells in vivo. The biggest component of the program is focused on delivery of genome editing machinery, given that's one of the big needs in the field. We have a smaller component on expanding the human genome editing repertoire, including developing of new editors. We didn't want to put a lot of effort or funding into that because it's a very hot area to begin with, but we felt it was important to have some of that in our consortium. We'll also have a coordinating center, a dissemination coordinator center which will be primarily responsible for generating a toolkit that will be publicly available and allow interested stakeholders to take a look at what has been produced by the SCGE and see the data upon which the results are based.
And this slide kind of shows where we see the SCGE in terms of the developmental process of new therapies. We are not supporting any direct clinical trials with the SCGE. That's beyond the scope. But the way we see it is some of the tools we're developing would be filling gaps in the process of getting do an IND. So you might imagine that for certain diseases there would be a need for the ability to deliver a genome editor to some specific cell type which doesn't exist at present and through the efforts of the SCGE investigators, we could provide some of the delivery methods that could then be used by patient advocate groups or small business entities, et cetera, to develop an IND. That's the way we see it is being an IND enabling effort.
We would like to think as an aspirational goal that some of the biological systems we're developing here might fill some gaps in the regulatory process and streamline that as well, getting around the need for testing in animals, at least where that would be possible. Because that's obviously a great cost and expensive aspect of the regulatory pathway. So this is the different give you a sense of different components. You can see on the right when people say it's all about delivery, we took that very seriously. We have a total
of 20 different grants focused on different approaches to deliver genome editors including modified AAVs. And one of those investigators is Guagnping Gao. We have got several working on nanoparticles, modified viruses, basically synthetic viral particles, and also one on adenovirus. Quite a diverse group of awards. I believe six or seven of them are focused on delivery to the nervous system, at least to some degree. And below is the website so you can see more about the program. Listening to Mustafa talk about the idea of platforms that would be appropriate for use in multiple diseases and also Guangping talking about that with the delivery of modified tRNAs, I think one of the things that really gets me most excited about genome editing is the idea that you could have ultimately a single biologic that would be applicable to multiple diseases and that would be a single editor. And one of particular interest was developed
in part in support by the SCGE, the prime editor developed by David Liu's lab. You can see based on the way it's designed, it is anticipated that this single editor could in principle at least be applicable to almost 90% of all genetic diseases. And if this works as anticipated and ultimately gets into clinical trials, you could imagine then that applying this to different diseases would simply be a matter of changing the sequence of the guide RNA to direct it to different locations within the genome. I think these kind of ideas have really huge implications for getting past this one disease at a time approach and getting to a lot of diseases in an efficient manner.
Then I thought at the end to kind of, because we at NCATS think a lot about generalized approaches rather than specific diseases, we get a lot of questions, people come to us saying they're thinking about developing a gene therapy for the disease. I find myself always thinking about the same questions as I have those conversations. I thought I would raise some of them here for the sake of discussion. Assuming one is interested in genetic therapy and you can develop a genetic therapy for a disease, some of the key questions you ultimately would have to answer or at least address are which cell type or cell types and organs and cell types are you going to need to target to have a therapeutic effect. And amongst those cell types, how many of those cells do you need to correct to have a therapeutic effect? Do you know the answer to these questions? I think for many cases it's going to be very important to know the answers to the questions.
Because the next one is assuming you know those cells, the question would be do you have a way to deliver your genetic therapy to those cells. And of course that depends very much on what the therapy is. We've heard about AAV has a tremendous capacity to develop therapeutic genes to certain cell types but as yet we don't have one that can go to basically all cell types in an organ and in some cases they're very much cell type specific. Whereas some other approaches such as antisense oligonucleotides could potentially go to a much greater number of cell types in the brain. If you have got drugs like that that can get to essentially all the different cell types in an organ, you may in fact not need to know the cell types you have to target because you'll be targeting them anyway. Then of course there are constraints based on the time of the course
of the disease. As was mentioned, this can be a particular issue in the neurodevelopmental disorders and also neurodegenerative disorders, where if you're looking at delivering therapies to a cell population that's dying off, you have to consider how many cells are still there by the time you develop the therapy. Geetha Senthil: Two minutes. PJ Brooks: Yep, got it. Finally, is clinical trial endpoints. If you're going to do a clinical
trial you have to have something to measure that's acceptable to the FDA and optimally this would be based on Natural History data such as may come from Natural History studies like the one that Mustafa mentioned as well. These are some general thoughts that might come up later in the meeting. Just want to thank my other colleagues at the Office of Rare diseases research. We have got a lot of other programs I didn't have time to talk about. I'll stop there. Thank you.
Guangping Gao: Thank you, PJ. Our next speaker, it's our fearless leader in the field, president elect of ASGCT Beverly Davidson. Please, Beverly. Beverly Davidson: Can you see my slides? Guangping Gao: Yes. Beverly Davidson: Okay. Fantastic. First of all, I also want to thank NIMH and Guangping and Mustafa and Sitra [video cut out] ... There was enough presented that we thought we would
probably span five days and to get it into just a couple days is really impressive. I'm going to bring up something that I think the field has been looking for, for some time. This was also brought up in the chatbox after Guangping's presentation and that is how do we think about really refining control of gene expression for a number of applications? In my laboratory, these are my disclosures, in my laboratory, we work on repeat expansion disease. This is just a cartoon showing you an example of where many of these repeats can occur within the genome. And my lab focuses on spinocerebellar ataxia … [video cut out] Huntington's disease, due to a polyglutamine repeat expansion in exon 1, the HTT gene.
If we think about targeting dominant alleles, you've heard both from PJ and from Guangping that there's a number of approaches for these disorders and folks are looking at going after the protein using various approaches. Of course, there are antisense nucleotide approaches to reduce the RNA that's encoding the mutant Huntington, those are in the clinic and moving along in clinical trials and very exciting. There are also efforts to induce splicing changes and also AAV delivery of RNA interference expressing vectors for therapy for Huntington's. Some of these can be allele specific. We've also heard about approaches targeting reduction of both mutant allele as well as the wild type allele. Of course we’ve also heard about approaches to target DNA and that was brought up in one of PJ's last slides, and that was to look at editing machinery. That's
really where I want to focus on here. And the work I want to present today was largely spear headed by Alex Mas Monteys in the lab, who is an assistant professor at the University of Pennsylvania. For Huntington's disease we have to think critically about what part of the gene to target. Our RNA interference approaches and ASO-based approaches are really targeted to any part of the gene. And the impact is a reduction of both alleles to some degree but not … [video cut out] a little bit more careful because we are essentially going to reduce the expression of the gene in that cell to nothing. There's some recent papers that have come out of Joe Bates and David Hausman's labs, shows if you do target downstream with exon one, you’re still going to get aberrant splicing of exon one, which is where that CAG expansion occurs. So you may not be doing any good by editing downstream of
exon one. More recently also Laura Ranum's group has shown that there may be transcription off the CAG repeat, both strands, with subsequent translation of those repeat consequences leading to additional forms of toxicity. Our idea was let's get rid of exon one and do it in an allele specific way.
… [video cut out] polymorphisms that insist with the mutation on the mutant allele. For a fully editable Cas9 approach we would take advantage of a snip present on the mutant allele along with a common neutronic slip to allow for a deletion of exon one and the approach would essentially leave the non mutated allele intact. When we tested this initially in cells that express the snips on both alleles, you could see that we could effectively get very robust knock down looking at Huntington expression here at the RNA level and here at the protein level. And you can see that these snips effectively removed exon one, the most effective … [video cut out] we next moved into Huntington cells. We worked
in both cells that had a PAM on the normal allele as well as those with PAMs on the mutant allele. In this data I'll show you a little bit of work from this. You can see that we have knock down with the guide sequences targeting upstream of exon one with common neutronic sequence, we get about 50% reduction of exon one. Can you all still see my slides? [Male speaker]: Yes, we can see the slides. We just turned off the video to improve the sound. Beverly Davidson: Okay. Thank you. You can see we can reduce the expression of the mutant Huntington. This is a Western blot of a relatively small compared to what we're used to in the
mouse tissues. You can see a relative reduction of the protein levels here that correspond again to about the same levels of knock down at the RNA level. We next moved this into in vivo using a BACHD mouse model that expresses the full length human Huntington's transgene and also has this common snip in the 5'UTR that allowed us to test for the effectiveness of this approach. We developed AAVs that expressed Cas9 and
the guides and introduced those into one side of the rodent brain. When we looked BIO-PCR assay for cleavage between the injected and not injected side, you can see robust editing and the tail here was a control, this was a direct injection of parenchyma with no systemic deliver. … [sound cut out] And this is all well and good. We can reduce the levels of Huntington and leaves the normal allele intact. We need to think of moving
this forward and improving the safety of this. The nuclease are foreign entities and elicit immune responses in cells so how can we make this safer. Guangping mentioned various ways for AAV targeting so that you’re only going to deliver the vector to the right cell, you can use microRNA targeting sequences in the three prime UTR. The other ways to improve the safety for AAV delivery is to regulate the expression for a short burst that's sufficient for editing. I think this is going to be critical as we move these foreign entities into cells. So I'm going to side step and talk about how we reintroduce for you the gene that when mutated causes spinal muscular atrophy. You'll hear very beautiful talk later from Jerry
Mendell's group. Just to remind you, in healthy individuals we have a normal SMN1 gene and various copies of SMN2 pseudogene. And this SMN2 pseudogene, sometimes exon 7 is skipped and sometimes exon 7 is included. The disease severity of SMN2 has to do with how many copies of this pseudogene you have and how much functioning full length SMN2 can complement the mutated SMN1 in SMA patients. And fortunately there are two drugs that can induce or enhance that
exon skipping. One is … [sound cut out] the other is approved for use in Europe. What both of these small molecules do is essentially promote exon 7 inclusion. We reasoned this is great for SMN2 but can we take advantage of the splicing phenomena and these orally bioavailable molecules for regulation of gene expression. The idea is really quite simple.
You take this cassette from SMN2E6, E7, E8, and infuse gene of interest, in general you get very low expression of gene of interest and with a drug you would get protein synthesis. So we took this and engineered a report of vectors initially and then we also altered the exon splicing junctions here. We could make it constitutively active or … [sound cut out] shown by the splicing assay here. Here’s the wild type construct and you can see it very much mirrors what we see in patients, about a 10% exon 7 spliced in. If we modulate these sequences here we can get it down to less than 1% and this is the constitutively active.
Does this work for gene expression? Indeed it is. This is an example of luciferase. When we use this system with a drug, we get about a 10 fold induction of gene expression, which may be fine for some cases, but possibly not enough for others. With that low level of induction we decided to go back and treat cells with very low dose drug, a couple of orders of magnitude lower, and screen … [sound cut out] responded low dose drug. This was work done by Paul Ranum, post doc in the lab together with Alex Mas Monteys. And when they looked at cells that were treated at low dosage or human cells, this is an example of one of the genes in which in the presence of drug you get this novel splicing event. We evaluated these five candidates here for their inducibility in the system that I just showed. So we tested
all these non SMN2 mini gene candidates. In the absence of drug, they're essentially off. In the presence of drug, now you're seeing we're getting roughly with the first gene candidate a 200-fold induction. This is much more sensitive than our previous SMN2 mini gene cassette. This gives you an idea of how on and off it is just using GFP as a reporter. These are tissue culture cells either treated with DMSO or the drug. In this case it's LMI070 this system also works with [away from mic] using other cassettes. You can see expression control.
We also tested how responsive this was with regards to promoters. This is just an example where we used RSV promoter, PGK promoter and minimal mCMV, which is a weak promoter because it's stripped of enhancer elements. Just looking at the levels of induction we could achieve with this, you can see we can control by low dose drug or by using the different promoters to get the level of gene expression that we think we need. And importantly this drug,
again I told you is orally bioavailable and brain penetrable and the beauty of this is there's a relatively rapid wash out, so you could dose an animal once or maybe once or twice a week, or you could dose it at low dose more often, depending what levels of expression you were trying to achieve. This just shows you the dose responsiveness of the splicing here using LMI070 as an example, and this is just the folded induction which we were seeing. So w