Beyond Gene Silencing: Harnessing Regulatory RNAs To Increase Gene Expression In Haploinsufficient Disease

By Dan Tardiff, Ph.D., Chief Scientific Officer, CAMP4 Therapeutics Corp.

A myriad of monogenic human diseases are caused by inadequate protein levels and function. Among these include haploinsufficient disorders, which result from expression of only half the amount of functional protein due to nonsense, frameshift, deletion, or severe loss-of-function (LoF) missense mutations in one allele. These disorders present a significant therapeutic challenge and unmet medical need across various indications.

Restoring protein levels presents a logical approach to mitigate disease pathology and improve clinical symptoms. However, this is no easy task given inherent biological complexity and challenges associated with modalities capable of increasing protein expression. While adeno-associated virus-mediated gene delivery has the potential to restore functional genes, hurdles remain with tolerability, biodistribution, and the risks of inadequate effect in a  one-and-done approach. As an alternative, antisense oligonucleotides (ASOs) offer a titratable modality that can be programmed to act on RNA biology in several ways to change gene expression.   

Splice-switching oligonucleotides (SSOs), for example, can redirect splicing to increase the amount of mRNA that encodes functional protein. ASOs, such as nusinersen for spinal muscular atrophy (Biogen/Ionis Pharmaceuticals; approved) and zorevunersen for Dravet syndrome (Stoke Therapeutics; Phase 3), validate this approach in diseases of the central nervous system. Other approaches, such as targeting microRNA bindings sites in the 3’ untranslated region of mRNA to improve stability or upstream open reading frames to suppress translation, offer potential mechanisms to modulate RNA function to increase protein output. A challenge, however, is that each gene is unique and may lack the requisite features that make them amenable to these different approaches.  

An emerging class of RNA, called regulatory RNAs (regRNAs), has been shown to play a fundamental role in modulating transcription of most genes. We have demonstrated for multiple genes that regRNAs can be targeted with ASOs to increase transcription to a potentially biologically meaningful extent. CMP-002, our lead ASO candidate, is advancing toward the clinic for a developmental epileptic encephalopathy (DEE) called SYNGAP1-related disorder (SRD).

Regulatory RNAs As A Lever To Increase Gene Transcription

RegRNAs are a subset of long noncoding RNAs expressed from promoters and enhancers. While promoters drive expression of mRNA in the sense direction, they initiate transcription in the antisense direction to generate noncoding promoter-associated RNAs (paRNAs). Active enhancers are bidirectionally transcribed to generate enhancer RNAs (eRNAs). These RNAs act locally where they interact with transcription factors to modulate gene expression. We established our RAP Platform to generate genome-wide regulatory maps across diverse cell types and tissues, characterize regRNAs expressed from regulatory sequences, and discover ASOs that drive the specific upregulation of transcription.

To date, we have presented data demonstrating upregulation of multiple disease genes in the liver and CNS, specifically, OTC, CPS1, PGRN, LDLR, and SYNGAP1. A consistent observation has been that regRNA-targeting ASOs operate within a physiological range. This ability to precisely target an RNA to increase gene expression to a relatively modest extent lends itself to therapeutic applications where achievable increases in transcription may have a meaningful clinical benefit. This approach is ideal for haploinsufficiencies where even partial restoration of gene expression can have a significant impact on disease.

SYNGAP1-Related Disorder Is A Haploinsufficiency Of Significant Unmet Medical Need

SRD is a DEE caused by SYNGAP1 haploinsufficiency. SRD patients exhibit a constellation of symptoms, ranging from intellectual disability, general developmental delays, behavioral challenges, limited communication, disrupted sleep, motor dysfunction, and epilepsy. There are no approved disease-modifying therapies, and no clinical trials have been performed in SRD patients with a precision therapeutic approach.

The broad impact of SYNGAP1 mutations on symptomology arises from the core function that SynGAP (the protein) plays in postsynaptic nerve terminals. As a GTPase-activating protein, SynGAP negatively regulates Ras, which ultimately controls AMPA receptor (AMPAR) concentrations in the postsynaptic membrane. Reductions in SynGAP lead to disinhibition of Ras, increased AMPAR, and enhanced synaptic strength and hyperexcitability. Restoring SynGAP at the synapse may improve synaptic activity by normalizing AMPAR at the postsynaptic membrane.

Underlying the loss of SynGAP are mutations in SYNGAP1 that result in partial or complete loss of SynGAP expressed from the affected allele. Most patients (~70%–80%) have nonsense or frameshift mutations that likely result in mRNA degradation by nonsense-mediated decay, resulting in protein levels that are approximately 50% of normal. Missense mutations make up much of the remaining mutations and are generally considered to be LoF, either by reducing activity or destabilizing the protein.

Interestingly, there are rare mutations in exons 1–4 that, due to the presence of multiple transcriptional start sites and isoforms, spare shorter isoforms from the effects of mutation. The result is presumed to be intermediate SynGAP levels. While still impacted, these patients present with milder symptoms and provide human genetic evidence that gene dosage tracks with clinical severity.

Given that modest increases in SynGAP may achieve meaningful clinical benefit, we believed that SRD is an ideal indication for our approach. Thus, we explored the potential of targeting a SYNGAP1 regRNA with ASOs to increase gene expression as a potential therapeutic strategy.

CMP-002 Increases SYNGAP1 In Cells, Humanized Mice Model, And Monkeys

We identified multiple regulatory regions for SYNGAP1 and characterized their associated regRNAs. The SYNGAP1 regRNA we selected for targeting was expressed in human induced pluripotent stem cell (iPSCs)-derived neurons and human brain tissue and also conserved in monkeys.

A comprehensive discovery campaign led to identification of CMP-002, our development candidate that is advancing to the clinic. CMP-002 increased SYNGAP1 levels in immortalized cells and restored SYNGAP1 to wild-type levels in neurons derived from patient iPSCs. The elevation in SYNGAP1 was the direct effect of CMP-002 on transcription, as evidenced by both chromatin immunoprecipitation for RNA polymerase II and nascent RNA labeling. CMP-002 hybridization to the regRNA disrupted regRNA secondary structure, which we believe results in altered interactions with transcription factors to increase SYNGAP1 transcription.

Although the regRNA was expressed in vivo, we needed to confirm that ASO activity — and, by extension, regRNA function — remained operative in a more complex biological context. We therefore evaluated the in vivo pharmacology of CMP-002 in a humanized SYNGAP1 mouse model. In this model, the full human SYNGAP1 gene, including its key regulatory regions, replaced the mouse gene and was bred as either wild-type (two copies) or heterozygous/haploinsufficient (one copy). Consistent with the in vitro data, a single intracerebroventricular (ICV) dose of CMP-002 increased expression, restoring SynGAP to near wild-type levels.

While SynGAP levels were increased, the key question was whether this effect would translate into improvements in disease-relevant phenotypes. SRD is a disorder characterized by an array of neurodevelopmental and seizure phenotypes, so it is critical to establish that CMP-002 intervention retains its impact across a broad range of phenotypes.

CMP-002 was administered to neonatal mice. In this model, learning and memory, motor function, and open field activity were assessed approximately two weeks later. The expected deficits were observed in haploinsufficient mice and were rescued upon treatment with CMP-002. We extended our evaluation to assess seizures using a chemical-induction assay, given haploinsufficient mice do not exhibit countable spontaneous motor seizures. A GABA antagonist previously shown to increase seizure burden and severity in SYNGAP1 haploinsufficient mice was administered one month after ASO administration. We replicated the enhanced sensitivity of the haploinsufficient mice and, importantly, demonstrated that CMP-002 reduced seizure burden and severity.

Taken together, these data demonstrated that CMP-002 administration and the resulting increases in SynGAP levels were associated with improvements in both neurodevelopmental deficits and seizures. This demonstration of broad improvement across multiple phenotypes in animal models bolsters confidence in our approach as we advance CMP-002 as a potential disease-modifying therapy.

Demonstrating target engagement and preclinical activity in a relevant mouse model was a critical step in providing key proof-of-concept data that targeting the SYNGAP1 regRNA could have a meaningful impact on pathophysiology. However, these studies utilized ICV dosing, whereas administration to patients will be via intrathecal (IT) administration. We therefore turned to a monkey pharmacology study to assess biodistribution and target engagement.

CMP-002 administered by IT injection resulted in dose-dependent and linear increases in tissue concentrations throughout relevant brain regions impacted by SYNGAP1 haploinsufficiency, including multiple cortical regions and the hippocampus. Critically, the achieved tissue concentrations resulted in dose-dependent increases in SynGAP protein in the most relevant brain regions. These data provide key evidence that IT administration — the intended route of administration in the clinic — can achieve adequate tissue exposure to engage the SYNGAP1 regRNA and increase transcription and protein levels.

Preparation For A Potential CMP-002 Clinical Study In SRD Patients

CMP-002 has been explored in multiple in vitro and in vivo systems and reproducibly demonstrated increases in SYNGAP1 expression alongside demonstrated phenotypic improvement in a relevant mouse model. Based on these data, CMP-002 was advanced to GLP toxicology studies to support global regulatory submissions, which include the first regulatory submission to Australia recently announced. This is the first submission in what is intended to be a global Phase 1/2 clinical study in SYNGAP1 patients starting by the end of this year, pending regulatory clearance.

Upon approval for clinical testing, CMP-002 is planned to be administered to SRD patients in what would be the first dedicated clinical study with a potential disease-modifying therapy in this population. Based on the lack of clinical precedence, it is our focus to solicit feedback from clinicians, regulatory agencies, patient advocacy groups, and patient caregivers on the planned clinical study. In support of this trial, we have closely engaged the SYNGAP1 community, working closely with CURE SYNGAP1 and its global partners and supporting the ProMMiS natural history study sponsored by CURE SYNGAP1. As with other neurodevelopmental disorders with epilepsy, individual natural histories are critical in understanding unique patient journeys and informing potential endpoints.

The Big Picture

Precision gene upregulation presents a major challenge but also a tremendous opportunity to impact patient lives. There is building momentum in the field with development programs in Dravet syndrome (Stoke Therapeutics, Phase 3 study of SSO to increase SCN1A) and Angelman’s syndrome (Ultragenyx, Ionis Pharmaceuticals, Oak Hill Bio, multiple studies of ASOs targeting a natural antisense transcript to upregulate UBE3A) with relatively near-term clinical readouts. Despite the progress, there remains room to learn and improve the development of therapies for both this class of disease and for ASOs targeted to the CNS in general. Ongoing dialogues with patient groups, clinicians, and regulatory agencies will be key for the continued advancement of the field.

Targeting regRNAs with ASOs has the potential for broad applicability across a wide range of targets given the fundamental role regRNAs play in regulating gene transcription. Buoyed by our progress on the SYNGAP1 program, we continue to explore multiple additional targets within the DEE space and look to maximize the potential of targeting regRNAs to increase gene expression in therapeutic areas with significant unmet need.

As we say at CAMP4: Act urgently… Patients are waiting.

About The Author

Dan Tardiff, Ph.D., is chief scientific officer of CAMP4 where he oversees Platform and Discovery Teams to expand the scope of CAMP4’s discovery platform and advance the pipeline towards the clinic. Prior to joining CAMP4, Tardiff led drug discovery projects and teams in both small biotech and large pharma across multiple drug modalities. Tardiff most recently led a team in the Rare Disease Research Unit at Pfizer exploring genetic medicines for rare neurological disorders. Prior to Pfizer, he was a scientific co-founder of Yumanity Therapeutics, where he helped build a platform to discover novel therapeutics for the treatment of neurodegenerative diseases. At Yumanity, he led teams from screening through translational research to discover new therapeutic targets and drug candidates, including a clinical-stage small molecule targeting stearoyl-CoA desaturase for the potential treatment of Parkinson’s disease. Tardiff earned his bachelor’s degree in biochemistry from Stonehill College and his doctorate from Brandeis University. He completed his post-doctoral training at the Whitehead Institute for Biomedical Research with Dr. Susan Lindquist.