Early life seizures and epileptic spasms in STXBP1-related disorders.

OBJECTIVE: Individuals with disease-causing variants in STXBP1 frequently have epilepsy onset in the first year of life with a variety of seizure types, including epileptic spasms. However, the impact of early onset seizures and antiseizure medication (ASM) on the risk of developing epileptic spasms and impact on their trajectory are poorly understood, limiting informed and anticipatory treatment, as well as trial design. METHODS: We retrospectively reconstructed seizure and medication histories in weekly intervals for individuals with STXBP1 developmental and epileptic encephalopathy (DEE) with epilepsy onset in the first year of life and quantitatively analyzed longitudinal seizure histories and medication response. RESULTS: We included 61 individuals with early onset seizures, 29 of whom had epileptic spasms. Individuals with neonatal seizures were likely to have continued seizures after the neonatal period (25/26). The risk of developing epileptic spasms was not increased in individuals with neonatal seizures or early infantile seizures (21/41 vs. 8/16, odds ratio [OR] = 1, 95% confidence interval [CI] = .3-3.9, p = 1). We did not find any ASM associated with the development of epileptic spasms following prior seizures. Individuals with prior seizures (n = 16/21, 76%) had a higher risk of developing refractory epileptic spasms (n = 5/8, 63%, OR = 1.9, 95% CI = .2-14.6, p = .6). Individuals with refractory epileptic spasms had a later onset of epileptic spasms (n = 20, median = 20 weeks) compared to individuals with nonrefractory epileptic spasms (n = 8, median = 13 weeks, p = .08). SIGNIFICANCE: We provide a comprehensive assessment of early onset seizures in STXBP1-DEE and show that the risk of epileptic spasms is not increased following a prior history of early life seizures, nor by certain ASMs. Our study provides baseline information for targeted treatment and prognostication in early life seizures in STXBP1-DEE.

Delineating clinical and developmental outcomes in STXBP1-related disorders.

STXBP1-related disorders are among the most common genetic epilepsies and neurodevelopmental disorders. However, the longitudinal epilepsy course and developmental end points, have not yet been described in detail, which is a critical prerequisite for clinical trial readiness. Here, we assessed 1281 cumulative patient-years of seizure and developmental histories in 162 individuals with STXBP1-related disorders and established a natural history framework. STXBP1-related disorders are characterized by a dynamic pattern of seizures in the first year of life and high variability in neurodevelopmental trajectories in early childhood. Epilepsy onset differed across seizure types, with 90% cumulative onset for infantile spasms by 6 months and focal-onset seizures by 27 months of life. Epilepsy histories diverged between variant subgroups in the first 2 years of life, when individuals with protein-truncating variants and deletions in STXBP1 (n = 39) were more likely to have infantile spasms between 5 and 6 months followed by seizure remission, while individuals with missense variants (n = 30) had an increased risk for focal seizures and ongoing seizures after the first year. Developmental outcomes were mapped using milestone acquisition data in addition to standardized assessments including the Gross Motor Function Measure-66 Item Set and the Grasping and Visual-Motor Integration subsets of the Peabody Developmental Motor Scales. Quantification of end points revealed high variability during the first 5 years of life, with emerging stratification between clinical subgroups. An earlier epilepsy onset was associated with lower developmental abilities, most prominently when assessing gross motor development and expressive communication. We found that individuals with neonatal seizures or early infantile seizures followed by seizure offset by 12 months of life had more predictable seizure trajectories in early to late childhood compared to individuals with more severe seizure presentations, including individuals with refractory epilepsy throughout the first year. Characterization of anti-seizure medication response revealed age-dependent response over time, with phenobarbital, levetiracetam, topiramate and adrenocorticotropic hormone effective in reducing seizures in the first year of life, while clobazam and the ketogenic diet were effective in long-term seizure management. Virtual clinical trials using seizure frequency as the primary outcome resulted in wide range of trial success probabilities across the age span, with the highest probability in early childhood between 1 year and 3.5 years. In summary, we delineated epilepsy and developmental trajectories in STXBP1-related disorders using standardized measures, providing a foundation to interpret future therapeutic strategies and inform rational trial design.

Opioid Exposure Differentially Impacts Esophageal Body Contraction Over the Lower Esophageal Sphincter.

BACKGROUND & AIMS: Studies with limited sample sizes have investigated association of chronic opioid use with motility disorders of esophagogastric junction and esophageal body peristalsis. Our aims were to use a large cohort of patients to assess (1) the impact of opioid exposure on clinical and manometric characteristics, and (2) the association of opioid exposure with higher long-term symptom burden. METHODS: Patients recruited from a tertiary medical center who underwent high-resolution manometry (HRM) between 2007 and 2018 were included. Demographics, opiate exposure, clinical symptoms, and HRM parameters were compared. Patient-Reported Outcomes Measurement Information System-Gastrointestinal swallowing domain (PROMIS-GI swallowing domain) and Eckardt score were administered via phone interviews in patients with hypercontractile esophagus (HE) or distal esophageal spasm (DES) to determine long-term symptom burden between opioid and nonopioid users. RESULTS: Our cohort included 4075 patients (869 with opiate exposure with median morphine milligram equivalent [interquartile range] of 30 [10-45]). Patients in the opioid group were significantly more likely to have dysphagia (65% vs 51%, P < .01) and diagnosis of DES (11% vs 5%, P < .01) and HE (9% vs 3%, P < .01). Partial opioid agonists were not associated with motility abnormalities. Patients on opioids had significantly higher symptom burden on median (interquartile range) follow-up of 8.9 years (5.8-10.4) post manometric diagnosis with median PROMIS-GI swallowing domain score of 21.5 (17-25) compared with the nonopioid group at 15 (9.8-21, P = .03). CONCLUSIONS: Nearly 2 of 3 patients with opioid exposure undergoing HRM have dysphagia and more than 25% of them with dysphagia as the primary symptom have a diagnosis of either DES or HE. Opioid users with spastic disorders have higher symptom burden long-term compared with nonopioid users.

STXBP1: fast-forward to a brighter future - a patient organization perspective.

Syntaxin-binding protein 1 related disorder (STXBP1-RD) is a rare neurologic disorder associated with global neurodevelopmental delay, intellectual disability, early-onset epilepsy, motor abnormalities, and autism. The underlying pathophysiology stems from a de novo mutation in the STXBP1 gene, which codes for the STXBP1 protein. The STXBP1 protein is involved in synaptic vesicle fusion and neurotransmitter release. Pathogenic variants in the STXBP1 gene generally result in haploinsufficiency, an impairment in neurotransmitter release, and subsequent dysfunction in neuronal communication. The STXBP1 Foundation was founded in 2017 to support families of children with STXBP1-RD and accelerate the development of effective therapies and, ultimately, a cure for the disorder. The Foundation initially supported research aimed at better understanding the complex phenotypic presentation of the disease as well as the development of animal and cellular models usable by the research community to more fully characterize STXBP1 function and disease pathogenicity. In 2023, the Foundation embarked on its STXBP1 Fast Forward Strategic Plan, which includes a prospective natural history study and substantive biomarker work to drive forward the development of new precision therapies for STXBP1-RD.

STXBP1: fast-forward to a brighter future STXBP1-related disorder (STXBP1-RD) is a rare and severe brain condition. It causes delays in development, learning problems, seizures starting at an early age, movement challenges, and sometimes autism. The main problem comes from a new mutation in the STXBP1 gene, which makes a protein needed for brain cells to communicate properly. When this gene doesn't work right, there's not enough of the protein, leading to trouble with brain cell communication. To help families dealing with this disorder and speed up the search for effective therapies, the STXBP1 Foundation started in 2017. At first, they funded studies to understand the disease better and create models for testing treatments. Then, in 2023, they launched the STXBP1 Fast Forward Strategic Plan. This plan includes studying how the disorder progresses naturally and researching markers that could help develop precise treatments for STXBP1-RD.

Herpes simplex virus vector mediated gene therapy of tumor necrosis factor-α blockade for bladder overactivity and nociception in rats.

PURPOSE: We examined the effects of tumor necrosis factor-α blockade on bladder overactivity and nociception using replication defective HSV vectors expressing tumor necrosis factor-α soluble receptor. MATERIALS AND METHODS: HSV vectors expressing tumor necrosis factor-α soluble receptor or ß-galactosidase/green fluorescent protein as the control were injected into the bladder wall of female Sprague-Dawley® rats. Green fluorescent protein was observed with fluorescent microscopy in the bladder and L6 dorsal root ganglia. mRNA and protein expression of tumor necrosis factor-α, and interleukin-1ß and 6 as well as myeloperoxidase activity in the bladder were determined by quantitative reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay 4 hours after intravesical resiniferatoxin administration. c-Fos positive neurons were counted in the L6 spinal dorsal horn. Cystometry and behavioral analyses were also performed. RESULTS: Green fluorescent protein expression was confirmed in the bladder and L6 dorsal root ganglia. Resiniferatoxin administration significantly increased tumor necrosis factor-α mRNA and protein levels in the bladder in controls. Tumor necrosis factor-α mRNA was also increased in the tumor necrosis factor-α soluble receptor group, although tumor necrosis factor-α protein up-regulation was suppressed. The up-regulation of interleukin-1ß and 6 mRNA and protein levels, and the myeloperoxidase activity seen in controls were suppressed in the tumor necrosis factor-α soluble receptor group. c-Fos positive cells in the L6 spinal dorsal horn were less prominent in the tumor necrosis factor-α soluble receptor group than in controls. On cystometry the significant decrease in intercontraction intervals after resiniferatoxin infusion detected in controls was not seen in the tumor necrosis factor-α soluble receptor group. On behavioral analyses freezing behavior was significantly decreased in the tumor necrosis factor-α soluble receptor group without affecting licking behavior. CONCLUSIONS: HSV vector mediated tumor necrosis factor-α blockade gene therapy in the bladder and bladder afferent pathways decreases the bladder pain and overactivity induced by nociceptive bladder stimuli.

Early life seizures and epileptic spasms in STXBP1-related disorders.

Background and Objectives: Individuals with disease-causing variants in STXBP1 frequently have epilepsy onset in the first year of life with a variety of seizure types, including epileptic spasms. However, the impact of early-onset seizures and anti-seizure medication (ASM) on the risk of developing epileptic spasms and impact on their trajectory is poorly understood, limiting informed and anticipatory treatment, as well as trial design. Methods: We retrospectively reconstructed seizure and medication histories in weekly intervals for individuals with STXBP1-related disorders with epilepsy onset in the first year of life and quantitatively analyzed longitudinal seizure histories and medication response. Results: We included 61 individuals with early onset seizures, 29 of whom had epileptic spasms. Individuals with neonatal seizures were likely to have continued seizures after the neonatal period (25/26). The risk of developing epileptic spasms was not increased in individuals with neonatal seizures or early infantile seizures (21/41 vs. 8/16; OR 1, 95% CI 0.3-3.9, p = 1). We did not find any ASM associated with the development of epileptic spasms following prior seizures. Individuals with prior seizures (n = 16/21, 76%) had a higher risk to develop refractory epileptic spasms (n = 5/8, 63%, OR =1.9, 95% CI 0.2-14.6, p = 0.6). Individuals with refractory epileptic spasms had a later onset of epileptic spasms (n = 20, median 20 weeks) compared to individuals with non-refractory epileptic spasms (n = 8, median 13 weeks; p = 0.08). When assessing treatment response, we found that clonazepam (n = 3, OR 12.6, 95% CI 2.2-509.4; p < 0.01), clobazam (n=7, OR 3, 95% CI 1.6-6.2; p < 0.01), topiramate (n=9, OR 2.3, 95% CI 1.4-3.9; p < 0.01), and levetiracetam (n=16, OR 1.7, 95% CI 1.2-2.4; p < 0.01) were more likely to reduce seizure frequency and/or to maintain seizure freedom with regards to epileptic spasms than other medications. Discussion: We provide a comprehensive assessment of early-onset seizures in STXBP1-related disorders and show that the risk of epileptic spasms is not increased following a prior history of early-life seizures, nor by certain ASM. Our study provides baseline information for targeted treatment and prognostication in early-life seizures in STXBP1-related disorders.

A disease concept model for STXBP1-related disorders.

OBJECTIVE: STXBP1-related disorders are rare genetic epilepsies and neurodevelopmental disorders, but the impact of symptoms across clinical domains is poorly understood. Disease concept models are formal frameworks to assess the lived experience of individuals and their families and provide a basis for generating outcome measures. METHODS: We conducted semistructured, qualitative interviews with 19 caregivers of 16 individuals with STXBP1-related disorders and 7 healthcare professionals. We systematically coded themes using NVivo software and grouped concepts into the domains of symptoms, symptom impact, and caregiver impact. We quantified the frequency of concepts throughout the lifespan and across clinical subgroups stratified by seizure history and developmental trajectories. RESULTS: Over 25 hours of interviews, we coded a total of 3626 references to 38 distinct concepts. In addition to well-recognized clinical features such as developmental delay (n = 240 references), behavior (n = 201), and seizures (n = 147), we identified previously underrepresented symptoms including gastrointestinal (n = 68) and respiratory symptoms (n = 24) and pain (n = 30). The most frequently referenced symptom impacts were autonomy (n = 96), socialization (n = 64), and schooling (n = 61). Emotional impact (n = 354), support (n = 200), and daily life & activities (n = 108) were highly cited caregiver impacts. We found that seizures were more commonly referenced in infancy than in other age groups, while behavior and socialization were more likely to be referred to in childhood. We found that caregivers of individuals with ongoing seizures were less likely to reference developmental delay, possibly due to the relatively high impact of seizures. SIGNIFICANCE: STXBP1-related disorders are complex conditions affecting a wide range of clinical and social domains. We comprehensively mapped symptoms and their impact on families to generate a comprehensive disease model as a foundation for clinical endpoints in future trials.

Delineating clinical and developmental outcomes in STXBP1-related disorders.

STXBP1-related disorders are among the most common genetic epilepsies and neurodevelopmental disorders. However, the longitudinal epilepsy course and developmental endpoints have not yet been described in detail, which is a critical prerequisite for clinical trial readiness. Here, we assessed 1,281 cumulative patient-years of seizure and developmental histories in 162 individuals with STXBP1-related disorders and established a natural history framework. STXBP1-related disorders are characterized by a dynamic pattern of seizures in the first year of life and high variability in neurodevelopmental trajectories in early childhood. Epilepsy onset differed across seizure types, with 90% cumulative onset for infantile spasms by 6 months and focal-onset seizures by 27 months of life. Epilepsy histories diverged between variant subgroups in the first 2 years of life, when individuals with protein-truncating variants and deletions in STXBP1 (n=39) were more likely to have infantile spasms between 5 and 6 months followed by seizure remission, while individuals with missense variants (n=30) had an increased risk for focal seizures and ongoing seizures after the first year. Developmental outcomes were mapped using milestone acquisition data in addition to standardized assessments including the Gross Motor Function Measure-66 Item Set and the Grasping and Visual-Motor Integration subsets of the Peabody Developmental Motor Scales. Quantification of endpoints revealed high variability during the first five years of life, with emerging stratification between clinical subgroups, most prominently between individuals with and without infantile spasms. We found that individuals with neonatal seizures or early infantile seizures followed by seizure offset by 12 months of life had more predictable seizure trajectories in early to late childhood than compared to individuals with more severe seizure presentations, including individuals with refractory epilepsy throughout the first year. Characterization of anti-seizure medication response revealed age-dependent response over time, with phenobarbital, levetiracetam, topiramate, and adrenocorticotropic hormone effective in reducing seizures in the first year of life, while clobazam and the ketogenic diet were effective in long-term seizure management. Virtual clinical trials using seizure frequency as the primary outcome resulted in wide range of trial success probabilities across the age span, with the highest probability in early childhood between 1 year and 3.5 years. In summary, we delineated epilepsy and developmental trajectories in STXBP1-related disorders using standardized measures, providing a foundation to interpret future therapeutic strategies and inform rational trial design.

Gene therapy for pain: results of a phase I clinical trial.

OBJECTIVE: Preclinical evidence indicates that gene transfer to the dorsal root ganglion using replication-defective herpes simplex virus (HSV)-based vectors can reduce pain-related behavior in animal models of pain. This clinical trial was carried out to assess the safety and explore the potential efficacy of this approach in humans. METHODS: We conducted a multicenter, dose-escalation, phase I clinical trial of NP2, a replication-defective HSV-based vector expressing human preproenkephalin (PENK) in subjects with intractable focal pain caused by cancer. NP2 was injected intradermally into the dermatome(s) corresponding to the radicular distribution of pain. The primary outcome was safety. As secondary measures, efficacy of pain relief was assessed using a numeric rating scale (NRS), the Short Form McGill Pain Questionnaire (SF-MPQ), and concurrent opiate usage. RESULTS: Ten subjects with moderate to severe intractable pain despite treatment with >200mg/day of morphine (or equivalent) were enrolled into the study. Treatment was well tolerated with no study agent-related serious adverse events observed at any point in the study. Subjects receiving the low dose of NP2 reported no substantive change in pain. Subjects in the middle- and high-dose cohorts reported pain relief as assessed by NRS and SF-MPQ. INTERPRETATION: Treatment of intractable pain with NP2 was well tolerated. There were no placebo controls in this relatively small study, but the dose-responsive analgesic effects suggest that NP2 may be effective in reducing pain and warrants further clinical investigation.

HSV delivery of a ligand-regulated endogenous ion channel gene to sensory neurons results in pain control following channel activation.

Persistent pain remains a tremendous health problem due to both its prevalence and dearth of effective therapeutic interventions. To maximize pain relief while minimizing side effects, current gene therapy-based approaches have mostly exploited the expression of pain inhibitory products or interfered with pronociceptive ion channels. These methods do not enable control over the timing or duration of analgesia, nor titration to analgesic efficacy. Here, we describe a gene therapy strategy that potentially overcomes these limitations by providing exquisite control over therapy with efficacy in clinically relevant models of inflammatory pain. We utilize a herpes simplex viral (HSV) vector (vHGlyRα1) to express a ligand-regulated chloride ion channel, the glycine receptor (GlyR) in targeted sensory afferents; the subsequent exogenous addition of glycine provides the means for temporal and spatial control of afferent activity, and therefore pain. Use of an endogenous inhibitory receptor not normally present on sensory neurons both minimizes immunogenicity and maximizes therapeutic selectivity.

Centralizing neurofibromatosis experimental tool knowledge with the NF Research Tools Database.

Experimental tools and resources, such as animal models, cell lines, antibodies, genetic reagents and biobanks, are key ingredients in biomedical research. Investigators face multiple challenges when trying to understand the availability, applicability and accessibility of these tools. A major challenge is keeping up with current information about the numerous tools available for a particular research problem. A variety of disease-agnostic projects such as the Mouse Genome Informatics database and the Resource Identification Initiative curate a number of types of research tools. Here, we describe our efforts to build upon these resources to develop a disease-specific research tool resource for the neurofibromatosis (NF) research community. This resource, the NF Research Tools Database, is an open-access database that enables the exploration and discovery of information about NF type 1-relevant animal models, cell lines, antibodies, genetic reagents and biobanks. Users can search and explore tools, obtain detailed information about each tool as well as read and contribute their observations about the performance, reliability and characteristics of tools in the database. NF researchers will be able to use the NF Research Tools Database to promote, discover, share, reuse and characterize research tools, with the goal of advancing NF research. Database URL: https://tools.nf.synapse.org/.

Suppression of detrusor-sphincter dyssynergia by herpes simplex virus vector mediated gene delivery of glutamic acid decarboxylase in spinal cord injured rats.

PURPOSE: We investigated whether replication defective herpes simplex virus vectors encoding genes of glutamic acid decarboxylase, the gamma-aminobutyric acid synthesis enzyme, could suppress detrusor-sphincter dyssynergia in rats with spinal cord injury. MATERIALS AND METHODS: One week after spinalization herpes simplex virus vectors expressing glutamic acid decarboxylase and green fluorescent protein were injected into the bladder wall. Spinal cord injured rats without herpes simplex virus injection (sham treated) and those injected with LacZ encoding herpes simplex virus vectors served as controls. Three weeks after viral injection we simultaneously recorded urethral and intravesical pressure in awake rats. RESULTS: In the glutamic acid decarboxylase group the urethral pressure increase during bladder contraction was significantly decreased by 77% to 79% compared with that in the sham treated and LacZ groups. Bladder activity and urethral baseline pressure did not differ among the 3 groups. Intrathecal application of the gamma-aminobutyric acid-A receptor antagonist bicuculline almost completely reversed the decrease in the urethral pressure increase during bladder contractions while intrathecal saclofen (Tocris Cookson, Ellisville, Missouri), a gamma-aminobutyric acid-B receptor antagonist, partially reversed it. In the glutamic acid decarboxylase group the mRNA of glutamic acid decarboxylase 67 was significantly increased in L6-S1 dorsal root ganglia, which is where bladder afferents originate, compared with that in the LacZ group. CONCLUSIONS: Herpes simplex virus based glutamic acid decarboxylase gene transfer to bladder afferent pathway may represent a novel approach to detrusor-sphincter dyssynergia in cases of spinal cord injury.

The Cardiac Arrest Support Tier: a service evaluation.

AIM: This service evaluation seeks to determine whether the pre-hospital Cardiac Arrest Support Tier (CAST), implemented by a Hazardous Area Response Team (HART), was clinically effective, feasible and acceptable during its pilot year. METHODS: Chest compression feedback, provision of Return of Spontaneous Circulation (ROSC) care and CAST paramedic exposure to Out-of-Hospital Cardiac Arrest (OHCA) were audited. The number of incidents that CAST responded to and the number of staff it committed were also assessed. An online questionnaire was used to gauge acceptability of the project among frontline Ambulance Service Trust staff. RESULTS: CAST attended 178 OHCAs and committed a median of three (IQR 2-3) paramedics to each incident. In comparison to data from both South Western Ambulance Service Foundation Trust (SWASFT) and the National Ambulance Service in England, CAST delivered the full complement of post-ROSC care more frequently during the same period (CAST = 80% vs SWASFT = 68.5% vs England = 77.46%). CAST paramedics had a median exposure to 15.5 (IQR 12-19) OHCAs during the pilot year. Unfortunately, chest compression feedback was unavailable due to ongoing equipment inaccuracies and failure.Additionally 64.6% (n = 42/65) of SWASFT respondents believed CAST to be beneficial to resuscitation attempts, 63.1% (n = 41/65) would like CAST to continue to support resuscitation attempts in the future and 55.6% (n = 35/63) felt supported by CAST staff on scene. CONCLUSION: CAST is logistically feasible, is acceptable to the majority of SWASFT staff and demonstrated the successful delivery of evidence-based practice (EBP) to OHCA incidents.

Protein kinase C epsilon contributes to basal and sensitizing responses of TRPV1 to capsaicin in rat dorsal root ganglion neurons.

Phosphorylation of the vanilloid receptor (TRPV1) by protein kinase C epsilon (PKCepsilon) plays an important role in the development of chronic pain. Here, we employ a highly defective herpes simplex virus vector (vHDNP) that expresses dominant negative PKCepsilon (DNPKCepsilon) as a strategy to demonstrate that PKCepsilon is essential for: (i) maintenance of basal phosphorylation and normal TRPV1 responses to capsaicin (CAPS), a TRPV1 agonist and (ii) enhancement of TRPV1 responses by phorbol esters. Phorbol esters induced translocation of endogenous PKCepsilon to the plasma membrane and thereby enhanced CAPS currents. These results were extended to an in-vivo pain model in which vHDNP delivery to dorsal root ganglion neurons caused analgesia in CAPS-treated, acutely inflamed rat hind paws. These findings support the conclusion that in addition to receptor sensitization, PKCepsilon is essential for normal TRPV1 responses in vitro and in vivo.

The therapeutic potential of gene transfer for the treatment of peripheral neuropathies.

Peripheral neuropathy is a common medical problem with numerous aetiologies. Unfortunately, for the majority of cases there is no available medical solution for the underlying cause, and the only option is to try to treat the resulting symptoms. Treatment options exist when neuropathy results in positive symptoms such as pain, but there is a significant lack of treatments for negative symptoms such as numbness and weakness. Systemic application of growth factor peptides has shown promise in protecting nerves from neuropathic insults in preclinical animal studies, but translation into human trials has been problematic and disappointing. Significant advancements have been made in the past few years in utilising gene therapy approaches to treat peripheral neuropathy by expressing neuroprotective gene products either systemically or in specific nervous tissues. For example, plasmids expressing vascular endothelial growth factor injected into muscle, or herpes-simplex-virus-based vectors expressing neurotrophin gene products delivered to dorsal root ganglion neurons, have been used to protect peripheral nerve function in animal models of diabetes-associated peripheral neuropathy. Many published studies support the feasibility of this approach, although several questions still need to be addressed as gene therapy to treat peripheral neuropathy moves out of the laboratory and into the clinic.