FBXO31 Cure Roadmap
The first step toward a cure is a treatment, and the first step toward a treatment is a research roadmap.
Prepared for Laura Avery and the FBXO31 Foundation
Authors:
Mathuravani Thevandavakkam, PhD, Cure Guide and Director of Drug Repurposing, Perlara PBC
Uche Medoh, PhD. Cure Guide, Perlara PBC & Principal Investigator at the Arc Institute
Ethan O. Perlstein, PhD. CEO of Perlara PBC & Maggie’s Pearl, LLC & Curetopia
Executive Summary
The goal of the FBXO31 Cure Roadmap is to accelerate the identification of a treatment modality for patients with FBXO31 D344N mutation whose parent-led foundation commissioned the development of this Roadmap. This work is expected to enhance our understanding of FBXO31 function and its impairments leading to new treatment options for FBXO31-related neurological disorders and potentially even for neurodevelopmental disorders associated with cerebral palsy.
Our key recommendations for the next 12-24 months are:
Initiate a drug repurposing screen on patient iPSC-derived neural progenitor cells to identify clinically actionable repurposable hits. This effort driven by Perlara is expected to yield repurposable hits that can be translated into so-called “1-to-N” trials (coordinated, parallel single-patient studies expanded to broader patient communities).
Build a drug repurposing consortium that would bring together a broader coalition of clinicians and researchers supported by the FBXO31 Foundation. The goal of this consortium is twofold: (a) enable the sharing of patient information and biosamples, and (b) delineate the role of repurposed drugs in alleviating FBXO31 pathology at a cellular level. It’s expected that these studies would be invaluable for accelerated drug development.
Additionally explore alternative drug development opportunities for FBXO31 D344N variant, including ASO (antisense oligonucleotides), PROTAC (proteolysis-targeting chimeras), and other gene therapy approaches.
Increase awareness of FBXO31-related neurological disorder, particularly via centers for neurological diseases or specializing in cerebral palsy. It’s expected that awareness leading to more sequencing efforts could further enhance the patient pool with FBXO31 variants.
Introduction
Disease Overview: Basic science
FBXO31 and Cell cycle: The cell cycle is a precisely timed series of events fundamental to normal cellular function and organismal health1. Progression through the different phases of the cell cycle is tightly regulated by specific checkpoints and controlled by regulatory proteins including cyclins and cyclin-dependent-kinases (CDKs)2. Aberrations in cell cycle mechanisms lead to an array of diseases, from cancers to neurodegenerative disorders.
The ubiquitin-proteasome system (UPS) plays a critical role in regulating the cell cycle by targeting specific proteins for degradation. The UPS consists of three subunits: E1 (ubiquitin-activating), E2 (ubiquitin-conjugating) and E3 (ubiquitin ligase), that activate ubiquitin and subsequently transfer it to the protein destined for degradation3. Specifically, UPS regulates transitions between the cell cycle phases via cyclin degradation, consequently tightly regulating the levels of cyclins and associated cyclin dependent kinases2. This system ensures that cellular function and genomic integrity are maintained3.
FBXO31 (F-box only protein 31) was discovered as a tumor suppressor factor in 20054. The F-box proteins (FBPs) are a diverse group of proteins characterized by the presence of a conserved F-box motif of ~40 amino acids5. This motif allows the protein to interact with SKP1 (S-phase kinase-associated protein 1) and constitutes the SCF (SKP1-Cullin-F-box protein) E3 ubiquitin ligase complex. The SCF complex is instrumental in targeting proteins for ubiquitin-mediated degradation, thereby regulating various cellular processes such as cellular proliferation, cell cycle progression, and apoptosis. To date, there are over 600 E3 ligases that have been identified, of which the SCF or Cullin-RING E3 ligase (CRL) family is the largest, with CRL1 being the most characterized5–7.
FBXO31, belonging to the FBXO subfamily of F proteins, is expressed across tissue types with particularly high expression in the brain and is highly expressed in the G2→G1 phase of the cell cycle4. It has a well-defined role in DNA damage response and tumorigenesis. Acting as a tumor suppressor (alterations causing loss-of-function effects)8, FBXO31 has been implicated in breast cancer4,9, melanoma10,11, hepatocellular carcinoma12, gastric cancer13,14, and glioma15. Conversely, it functions as an oncogene (alterations causing gain-of-function effects) in esophageal squamous cell carcinoma16 and lung cancer17.
FBXO31's role in the DNA damage response includes halting cell cycle progression to allow for damage assessment and repair, particularly through pathways involving Ataxia telangiectasia mutated kinase (ATM)18, DNA-dependent protein kinase (DNA-PK), and Ataxia telangiectasia and Rad3-related protein (ATR) that maintain genomic stability by detecting DNA double-strand breaks by homologous recombination and non-homologous end joining, and in response to replication stress and single-strand breaks respectively19. In response to genotoxic stress, FBXO31 facilitates cyclin D1 degradation20.
Substrate specificity and neuromodulation of FBXO31: Cyclin D1, a substrate of SCFFBXO31, plays a pivotal role in initiating and advancing the cell cycle through the G1 phase in response to external stimuli20,21. Considered a proto-oncogene22, changes in the ubiquitination process and the subsequent stability of cyclin D1 have been identified as cancer biomarkers 23, indicative of the progression of the disease.
An unconventional function for cyclin D1 in neuronal stem cells is its effect on cell fate influencing whether cells proliferate or differentiate by regulating the G1 phase24. In pathological contexts, nuclear cyclin D1 can prompt apoptosis in post-mitotic neurons25. Interestingly, cyclin D1's presence in the cytoplasm of differentiating neurons and neuroblastoma cells suggests roles in preventing apoptosis or promoting cell cycle withdrawal26. Additionally, cytoplasmic cyclin D1 in the cytoplasm supports neuritogenesis in response to nerve growth factor (NGF) and contributes to neuronal plasticity during hippocampal development27,28. More recently, cyclin D1 was identified to modulate the activity of Gamma-aminobutyric acid (GABA), a crucial neurotransmitter in the brain, and thereby neuronal inhibition29. Deficits in GABA receptor function are implicated in various neurological disorders, from anxiety to epilepsy, with a significant role in synaptic inhibition and neurodevelopment in the hippocampus and cortex30,31.
An emerging role for FBXO31 points towards it being a regulator of neuronal morphogenesis and polarity32. These two distinct processes create the information flow necessary for brain functions such as memory, learning, and emotion. In addition to Parkinson’s disease protein Parkin I, FBXO31 is one of few E3 ubiquitin ligases that localizes to the centrosome, the microtubule organizing center, that regulates neuronal polarity, spindle formation motility and adhesion crucial to cell division32.Malfunctioning centrosomes potentiate cortical defects during development (the region of the brain associated with sensory, and motor development)33,34. The ligase activity of FBXO31 drives neuronal morphogenesis and is attributed to axon and dendritic growth, and axon specification. FBXO31 also targets centrosome polarity protein Par6c for degradation35. Reduction in Par6 degradation by another E3 ligase (Smurf1) drives the formation of a Par3/Par6/aPKC complex36,37 and thereby axon specification and polarity. Conversely, FBXO31 drives the proteasomal degradation of Par6c (the most abundant Par6 protein) regulating axonal growth and neuronal polarity32.
These examples underscore the significance of FBXO31 in maintaining cellular function, supporting normal development, and potentially influencing the pathogenesis of neurodegenerative diseases. Other key FBXO31 substrates include MCM7, MDM2, CDT1, FOXM1, Slug, Histone H2AX, TRF1, FAT10 perturbations of which can impact neurogenesis, neuronal survival, synaptic plasticity, and in general brain development38.
It has been proposed that the conserved binding pocket of FBXO31 enables the selective recognition of C-terminal peptides with an amide group (CTAPs), thereby serving as a distinct marker for proteins targeted for degradation by FBXO31, including hormones and neuropeptides39. Mutations affecting the binding domain of FBXO31 disrupt this specificity, leading to the degradation of newly identified proteins lacking amidation, while proteins originally marked with CTAPs accumulate within the cell. This toxic gain-of-function mutation in FBXO31 affects the function of downstream substrates but also results in the accumulation of protein degradation products, thereby disrupting the cell cycle. Dysregulation of this process in tissues where FBXO31 is highly expressed such as the brain 4 and skeletal muscles40, can significantly impact the development of neuro and skeleton-muscular disorders.
Disease Overview: Clinical Features of FBXO31-related cerebral palsy
To date, there have been six reported cases of a neurodegenerative disorder related to cerebral palsy caused by mutations in FBXO3141. All affected patients have a de novo D334N (p. Asp334Asn) mutation. This mutation is located in the cyclin D1 binding region of FBXO31 and alters the electrostatic charge in the binding pocket, leading to reduced levels of cyclin D1 in these patients compared to controls42. Conflicting studies suggest that FBXO31 amelioration increases levels of cyclin D139. Modulation via CTAP and switching in electrostatic charge in the binding domain likely together alter cyclin D1 binding and induce substrate-related toxicity.
The characteristic effect of mutation is a form of brain injury categorized as cerebral palsy typified by poor coordination, stiff muscles, weak muscles, and tremors. Infants, heterozygous for mutation, are born full-term and are not diagnosed with abnormalities prenatally. Hypotonia, low muscle tone, is identified in neonates followed by a diagnosis of cerebral palsy. MRI scans reveal a reduction in white matter distribution and content in certain patients. Gross motor milestones such as rolling over, sitting, crawling or walking are delayed in infants. Speech impairments (primarily receptive language) and cognitive deficits including attention deficit hyperactivity disorder are present. Central and peripheral motor pathways are affected leading to spasticity and dystonia. Children present with global developmental delay and varying levels of intellectual disability. However, underlying motor or neurodevelopmental issues do not worsen over time. Literature reports one patient with intestinal malrotation in whom bowel resection was required (see Figure 1 on clinical presentation below)43 (includes observations by FBXO31 Foundation).
Recessive mutations in FBXO31 offer insights into a potential model for understanding the cerebral palsy phenotype associated with the D334N mutation. A 5-base pair deletion in FBXO31 results in a frameshift mutation that introduces a premature stop codon p.(Cys283asnfs*81). This mutation is characterized by dysmorphia and intellectual disability but does not manifest as cerebral palsy44. The truncation of the protein leads to its degradation via nonsense-mediated decay resulting in reduced levels of FBXO31. Consequently, this reduction prevents any potential toxic gain-of-function effects. Although decreased FBXO31 levels likely disrupt normal neuronal development, compensatory mechanisms mitigate the extent of damage44,45. This model posits that any intervention that corrects the gain-of-function toxicity will result in a significant correction of the pathophysiology. As such, there are many therapeutic approaches with the potential to positively impact the progression of FBXO31-related cerebral palsy (see Therapeutic Readiness).
Disease Models: Cellular Models
A crucial step in developing cures is establishing disease models to investigate disease mechanisms and test potential therapies. Reliable disease models provide essential evidence of safety and efficacy before clinical trials. The development of models here is essential for understanding how aberrant FBXO31 activity or lack thereof influences proteostasis and neurological dysfunction. Cellular models, the first to be developed, are relatively simple to generate and handle in vitro. These models are vital for large-scale therapeutic screenings and for exploring the activity and mechanisms of new treatments.
Developing a model system for FBXO31 is complicated by several factors:
Substrate Interaction: FBXO31, like other F-box proteins, has multiple binding partners, making it difficult to delineate their interactions and downstream effects. Functional redundancy among F-box proteins46 can also obscure the specific effects of FBXO31 mutations.
Cell-Type Specificity: FBXO31's function varies across different cell types and tissues, making it challenging to develop models that accurately reflect its role in specific contexts, such as neuronal cells versus other cell types; developmental stages (during neuronal development vs post development).
Model Organism Limitations: Common model organisms may not fully replicate human FBXO31-related disease phenotypes due to differences in gene expression, protein function, and developmental processes, limiting the model's relevance.
This often requires the establishment of different types of cellular models to answer questions iterating FBOX31 dysfunction and therapeutics. Thanks to efforts in academic discovery science, however, several options exist to overcome these challenges.
Patient iPSCs are currently in development and will serve as the foundational research asset in drug repurposing screens, and beyond.
Disease Models: Whole-Organism Models
At present, there is a lack of published whole-organism models for FBXO31. Owing to their genetic similarity to humans, mouse models are indispensable for studying the pathological mechanism of human diseases, as well as for identifying new treatments. The FBXO31 mouse and Zebrafish model are currently under development and/or validation.
Therapeutic Readiness:
Several therapeutic approaches have potential applicability to address the understood mechanisms of FBXO31-related cerebral palsy, offering potential benefits to patients. These modalities vary significantly in development strategy, precedence, timeline, and likelihood of success. In this section, we delineate these modalities within the context of an overarching treatment strategy for patients affected by FBXO31-related cerebral palsy.
The first approach involves drug screening, which Perlara is currently undertaking in collaboration with industry partners (refer to Biotechnology Readiness). The second modality, antisense oligonucleotide therapy, will be clinically developed by a biotechnology company and would be accessible through compassionate use pathways. Additionally, we discuss two emerging modalities that are currently in development. While these approaches have longer development timelines, they represent promising avenues for expanding treatment options over the next decade.
Modalities: Drug Repurposing Screen
The fastest route in developing a clinical treatment modality that could significantly help patients with FBXO31-related cerebral palsy is to screen drugs that are already approved for activities that might be beneficial therapeutically. Since we don't fully understand the specific reasons why the disease happens or how drugs work in these cases, unbiased drug repurposing screens can often give us valuable and promising results.
Lastly, a key challenge for this approach is the question of generalizability. While it is understood that FBXO31-related cerebral palsy shares common underlying molecular mechanisms, it is not guaranteed that the most promising hits with one patient’s cells will also apply to other patients due to the small patient pool. Validating the generalizability of a screening hit will require access to a broader array of patient-derived cell lines (see Biobanking).
Modalities: RNase H-Dependent ASO
A Gapmer ASO is a specific type of antisense oligonucleotide (ASO) designed to induce the degradation of target RNA through the recruitment of RNase H, an enzyme that specifically degrades the RNA strand of an RNA/DNA hybrid. This allele-specific ASO specifically targets the mutated RNA and forms a complementary RNA/DNA hybrid with the target RNA, which is recognized and cleaved by RNase H. This degradation of the target RNA reduces the levels of the corresponding mRNA (specifically the mutant FBXO31), leading to decreased production of the target protein. Gapmer ASOs are FDA-approved, Mipomersen and Inotersen are examples of FDA-approved ASO gapmers, used for treating familial hypercholesterolemia and hereditary transthyretin amyloidosis, respectively.
The FBXO31 D334N mutation is amenable to this intervention on two counts: (1) FBXO31 is haplosufficient, meaning one functional copy is enough to maintain normal function, and (2) this method specifically eliminates the toxic gain-of-function of the mutant allele. Recessive mutations in FBXO31 support this, as the severity of the phenotype is significantly reduced when the truncated FBXO31 is targeted for non-mediated decay, leaving only one functional FBXO31 copy.
Modalities: PROTACs
PROTACs (Proteolysis Targeting Chimeras) are a novel class of therapeutic molecules designed to target specific proteins for degradation within cells and are part of a larger class of molecules known as heterobifunctional molecules. These molecules contain two binding domains and function by recruiting their respective targets into proximity with each other They leverage the ubiquitin-proteasome system to achieve selective degradation of pathogenic proteins, offering a potential approach to treat diseases caused by aberrant protein function. They typically consist of two parts: one that binds to the target protein and another that recruits an E3 ubiquitin ligase7, which tags the protein for degradation by the cell's proteasome machinery. PROTACs have been described as “targeted protein degraders”.
In gain-of-function mutations where the FBXO31 D334N ubiquitinates and degrades target protein excessively (evinced by low cyclin D1 levels), PROTAC could be used to selectively degrade the mutant E3 ligase reducing toxic activity. Conversely, PROTACS can also be designed to recruit other functional E3 ligases to degrade substrates the mutant FBXO31 can no longer process.
The caveat with this approach is that it requires a clear biomarker to phenotype connection.
Modalities: Symptomatic Treatments
Common symptomatic treatments include pharmacological medications to manage symptoms of various neuromotor and psychiatric symptoms. Patients currently work with their physicians to develop the best management route. It’s expected that drug repurposing hits from the screen will broaden the scope of immediately accessible drugs that could improve the quality of life.
Deep Brain Stimulation (DBS) is an invasive treatment option aimed at managing symptoms. It involves using a neurostimulator to produce electrical pulses that regulate abnormal neural activity in precise brain regions through implanted electrodes. DBS effectively alters neural circuit activity by modifying neuronal firing patterns and potentially influencing neurotransmitter release48. This therapy has demonstrated efficacy in treating movement disorders such as Parkinson’s disease and dystonia, as well as certain psychiatric conditions like treatment-resistant depression and obsessive-compulsive disorder49.
Clinical Research and Development Readiness:
Developing new therapeutics, particularly in rare diseases, requires hand-in-glove coordination between clinicians and researchers, especially at the earliest stages. Having reviewed the scientific landscape, laboratory model systems, and promising technologies that define the space of possible therapeutic candidates, here we explore the readiness of the clinical healthcare system to support the development of these candidates as well as their evaluation in human patients - the final, costliest, and riskiest stage of the drug development process.
Clinical Research: Patient Identification and Community Engagement
Engagement of the patient community in the drug development process is essential. The recruitment of an adequate number of patients for clinical trials remains a significant bottleneck in the path to FDA approval, often making the clinical development phase the most time-consuming aspect of the entire drug development pipeline. Consequently, early initiatives that diagnose, identify, and register FBXO31 patients can yield substantial benefits, both in terms of cost-effectively accelerating drug development and improving health outcomes for the patient community.
Patients with FBXO31-related cerebral palsy are identified primarily through genetic testing, particularly in individuals presenting with atypical cerebral palsy features, such as non-spastic motor defects, cognitive deficits, and behavioral issues. These features, while characteristic, often require a multidisciplinary diagnostic approach, involving neurologists, developmental pediatricians, and psychiatrists, to prompt referral for genetic screening. Despite these diagnostic avenues, many patients face a prolonged diagnostic journey.
The challenge of accurately diagnosing FBXO31-related disorders is compounded by the need for broader awareness among clinicians regarding the condition and its genetic basis. To this end, whole exome sequencing is expected to emerge as a powerful diagnostic tool in the identification of FBXO31 mutations among cerebral palsy patients. WES allows for the comprehensive analysis of all coding regions in the genome, making it particularly useful in uncovering rare genetic variants like those associated with FBXO3150. This approach is especially valuable when standard diagnostic methods fail to provide a clear diagnosis. This is also expected to uncover more patients with FBXO31-related cerebral palsy who may be mistaken for other neurodevelopmental disorders, leading to delays in appropriate identification. Establishing a patient registry could facilitate better tracking and management of these cases.
Community engagement is critical for advancing the understanding and treatment of FBXO31-related disorders. The FBXO31 Foundation would lead efforts in engaging patients and connecting them with resources, a coordinated effort involving awareness campaigns, support groups, and partnerships with advocacy organizations that are essential. These initiatives are expected to aid in patient identification and ensure that patients remain informed and engaged in research and clinical trials, ultimately accelerating the development of effective therapies.
Clinical Research: Natural History
A detailed understanding of the natural history of a disease is fundamentally important to a successful drug development campaign, particularly in the case of a rare disease. There are two major reasons for this: first, the natural progression of a disease offers clues as to how a drug that corrects it might work; second, a robust natural history study has significant regulatory implications. As part of a modernization of its handling of the regulation of new medicines for rare diseases, FDA now accepts natural history as a control group on a case-by-case basis. Much as a strong patient network can accelerate a clinical development timeline by facilitating enrollment, a natural history control group accelerates timelines by reducing the total number of patients required to produce a signal of efficacy that FDA requires for a drug to be marketed. This also has the advantage of reducing trial costs and addresses ethical concerns associated with withholding a potentially effective drug from a patient, often a child, with severe disease to recapitulate a natural history that we already understand (see Regulatory Landscape and Strategy).
Unfortunately, dedicated longitudinal natural history studies for FBXO31-related conditions are currently unavailable. The absence of such studies presents a significant gap in our understanding of FBXO31-related cerebral palsy and its progression. Natural history studies are essential for identifying clear prognostic indicators, such as the age of onset and genetic markers, and for establishing key clinical indicators of disease severity. The insights gained from such studies could play a critical role in the development of inclusion criteria and clinical endpoints for future therapeutic interventions.
Currently, natural history studies for FBXO31 are also complicated by the small patient pool. To this end, whole exome sequencing of cerebral palsy patients is expected to increase the FBXO31 patient pool. A larger pool enhances the statistical power of these studies and improves the identification of crucial surrogate endpoints, such as biomarkers. These endpoints are essential for monitoring disease progression, assessing therapy efficacy, and have significant implications for accelerating the drug development process through regulatory strategies.
Future investigations should focus on applying modern laboratory techniques, such as single-cell genetic analysis, proteomics, and metabolomics, to observe longitudinal changes associated with disease severity or progression in FBXO31-related conditions. Moreover, while large-scale genome-wide association studies may not yet be feasible due to the limited number of FBXO31 patients, exploring the role of nuclear genetic background in these conditions could yield valuable insights and potential biomarkers for disease progression.
Clinical Research: Biobanking
Reliable patient samples are essential for advancing research and therapeutic development for FBXO31-related disorders. The foundation of patient sample collection, cataloging, and distribution lies in the biobank or biorepository. Currently, there is no dedicated biobank for FBXO31, highlighting a critical gap in resources for researchers and drug developers in this space.
To support the creation of effective cellular models for research and drug screening, the most valuable samples for biobanking would include blood, skin, and muscle biopsies. While blood samples are easier to collect, skin and muscle biopsies offer cells that are more representative of the tissues most affected by FBXO31-related cerebral palsy. Fibroblasts from skin biopsies and myocytes from muscle biopsies, although more challenging to obtain and culture, are vital for creating robust and physiologically relevant models. These models are crucial for accurately replicating the disease environment in vitro, thereby enhancing the potential for developing effective therapies.
In addition to supporting research, biobanks play a key role in natural history studies by providing the high-quality, longitudinal samples needed to track disease progression. As more patients with FBXO31-related disorders are identified through methods such as whole exome sequencing, biobanking will become increasingly important for collecting and preserving these samples. This will not only aid in the discovery of biomarkers and therapeutic targets but will also strengthen the overall drug development pipeline by offering a centralized, accessible resource for researchers working to combat FBXO31-related conditions.
Cognizant of the above, the FBXO31 Foundation is leading efforts toward establishing an easily accessible biobank for FBXO31 and making any patient-derived cellular model(s) accessible to the wider research community that would significantly bolster efforts to understand and treat this rare disorder.
Clinical Development: Regulatory Landscape and Strategy
The FDA serves a critical function in the healthcare system by acting as the gatekeeper to the pharmaceutical market. Any new therapeutic must demonstrate both safety and efficacy in human clinical trials to gain FDA approval. Additionally, the FDA is responsible for the ethical evaluation and approval of these clinical trials, balancing the risks and potential benefits to patients. As such, a well-crafted regulatory strategy is essential for successful drug development.
Historically, the FDA has maintained a degree of detachment from drug developers to preserve its neutrality. However, the increasing need for innovative regulatory approaches, particularly for rare diseases, has led to the FDA becoming more involved early in the development process, often acting as a partner and resource. The FDA now offers several regulatory designations to aid the development of drugs in areas that are typically underserved by the biopharmaceutical industry. For therapeutic candidates targeting FBXO31-related cerebral palsy, several of these designations are likely applicable, each with significant implications for the development process.
Clinical Development: Biomarkers and Candidate Surrogate Endpoints
As we explore the clinical development pathway for FBXO31-related therapies, identifying and validating biomarkers remains a critical step in establishing robust surrogate endpoints. Biomarkers serve as key indicators that can provide early insights into disease progression, therapeutic efficacy, and overall patient outcomes. In the context of cerebral palsy, several biomarkers have emerged as potential candidates for further investigation, however, there are no approved biomarkers specific to FBXO31-related cerebral palsy.
This is a significant bottleneck to clinical readiness for an FBXO31 therapeutic. FDA will require significant evidence that any surrogate biomarker serving as a primary endpoint in a clinical trial is highly representative of the desired clinical outcome. As such, research toward the identification and validation of potential surrogate biomarker candidates and their specific relevance to FBXO31-related cerebral palsy disease progression should be prioritized and supported.
Neuroimaging biomarkers, including findings from MRI and Cranial ultrasound, provide critical information on structural and metabolic changes in the brain. These imaging modalities could serve as early indicators of disease severity and treatment response.
Existing research into biomarkers for cerebral palsy yields some interesting starting points. Biochemical biomarkers, such as metabolites in cerebrospinal fluid (CSF) or blood-based inflammatory markers, will offer another layer of insight into the underlying pathophysiology of FBXO31-related cerebral palsy51. At present, no biochemical biomarker consistently identifies patients with cerebral palsy, this could be attributed to clinical heterogeneity of the disease. Given the specific aberration in cyclin D1 function and levels in FBXO31 D334N, cyclin D1 could be developed as a potential biomarker specific to FBXO31-related cerebral palsy.
Additionally, abnormalities in neurotransmitter systems, such as altered levels of dopamine, serotonin, and GABA, have been associated with motor and cognitive impairments in FBXO31-related cerebral palsy. These neurotransmitters could serve as biomarkers to monitor the effectiveness of therapeutic interventions. Lastly, neuroinflammatory biomarkers such as elevated levels of cytokines such as IL-6, IL-8 could be instrumental in monitoring neuroinflammation and metabolic disturbances associated with the disorder51.
These biomarkers can be evaluated using either a targeted ELISA panel for protein quantification or a qPCR approach for assessing gene expression levels.
Functional biomarkers, assessed through tools like the Gross Motor Function Measure (GMFM) and neurodevelopmental scales, provide a direct link to patient outcomes, capturing the impact of disease on motor and cognitive functions43. These assessments could be crucial in defining meaningful clinical endpoints, particularly in the context of long-term therapeutic efficacy.
Lastly, imaging-based functional biomarkers, such as those derived from functional MRI (fMRI) and electroencephalography (EEG), have the potential to reveal alterations in brain activity and connectivity patterns that correlate with clinical symptoms. As the development of FBXO31-targeted therapies progresses, the identification and validation of these biomarkers will be pivotal in establishing surrogate endpoints that can accelerate the drug approval process.
While challenges remain, particularly in the variability and validation of these biomarkers, their integration into the clinical development strategy for FBXO31-related cerebral palsy could significantly enhance our ability to track disease progression, evaluate therapeutic interventions, and ultimately improve patient outcomes.
Clinical Development: Clinical Endpoints
In nearly all cases, the primary relevant outcome to FDA (and, generally, to patients) is whether the drug produces some kind of clinical benefit that is meaningful. Even if surrogate endpoints are used to approve a drug, ongoing investigation to demonstrate such clinical benefit will continue, and if there is no evidence of clinical benefit the drug can be removed from the marketplace. As such, even though defining strong surrogate endpoints is key at this stage, it is important to consider clinical endpoints as well.
Clinical endpoints are generally more challenging, expensive, and time-consuming to measure than biomarker endpoints. Clinical endpoints are more subjective, measured over longer periods, and capture more inherent biological variability than biomarker endpoints do. Thus, reaching a clinical endpoint is a high bar that most therapeutic candidates do not achieve - but to offer a patient anything less than this high standard would be unacceptable.
Several clinical endpoints have been defined for cerebral palsy in general and their use can be cited in clinical trials for FBXO31-related cerebral palsy. These endpoints in both children and adults focus on evaluating the impact of the condition on functional abilities, quality of life, and overall health43. For children, key endpoints include assessments of motor function through tools like the Gross Motor Function Measure (GMFM) and fine motor skill evaluations, functional independence using the Functional Independence Measure (FIM) and Pediatric Evaluation of Disability Inventory (PEDI), and cognitive and communication skills through relevant developmental tests. Quality of life is measured using instruments like the Pediatric Quality of Life Inventory (PedsQL), and muscle tone and spasticity are evaluated with the Ashworth Scale and Modified Tardieu Scale. In adults, similar endpoints are utilized, with additional focus on mobility and movement through tests like the Timed Up and Go (TUG) and Six-Minute Walk Test (6MWT), pain assessment with the Visual Analog Scale (VAS), and quality of life measures adapted for adult populations. Both age groups also benefit from assessments of psychosocial factors and secondary conditions to provide a comprehensive understanding of the disease’s impact and guide effective management strategies. Given the clinical heterogeneity of cerebral palsy, the above endpoints will be suitably refined to define standards for FBXO31-related cerebral palsy.
Biotechnology Ecosystem Readiness
Having explored the scientific landscape and considerations for the clinical development of therapeutic candidates, we now turn our attention to the biotechnology ecosystem crucial for advancing therapies for FBXO31-related cerebral palsy. This ecosystem encompasses academic institutions, industrial biotechnology, and both public and private funding sources. The successful development of new therapies for FBXO31 nearly always requires the engagement of multiple players within this ecosystem.
In the course of preparing this Roadmap, we’ve identified potential academic partners with knowledge and expertise that will enable accelerating the discovery of potential treatments and cures. At present, patient-derived neuronal cell lines are under generation and an in silico RNA sequencing drug repurposing screening approach employing multiple patients is being considered to identify potential drug repurposing candidates and lead-like molecules for FBXO31 therapeutics.
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