CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis.

BACKGROUND: Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR. METHODS: After conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study. RESULTS: Preclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram. CONCLUSIONS: In a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).

Trouble at the junction: When myopathy and myasthenia overlap.

Although myopathies and neuromuscular junction disorders are typically distinct, their coexistence has been reported in several inherited and acquired conditions. Affected individuals have variable clinical phenotypes but typically display both a decrement on repetitive nerve stimulation and myopathic findings on muscle biopsy. Inherited causes include myopathies related to mutations in BIN1, DES, DNM2, GMPPB, MTM1, or PLEC and congenital myasthenic syndromes due to mutations in ALG2, ALG14, COL13A1, DOK7, DPAGT1, or GFPT1. Additionally, a decrement due to muscle fiber inexcitability is observed in certain myotonic disorders. The identification of a defect of neuromuscular transmission in an inherited myopathy may assist in establishing a molecular diagnosis and in selecting patients who would benefit from pharmacological correction of this defect. Acquired cases meanwhile stem from the co-occurrence of myasthenia gravis or Lambert-Eaton myasthenic syndrome with an immune-mediated myopathy, which may be due to paraneoplastic disorders or exposure to immune checkpoint inhibitors.

Congenital myasthenic syndromes in adult neurology clinic: A long road to diagnosis and therapy.

OBJECTIVE: To investigate the diagnostic challenges of congenital myasthenic syndromes (CMS) in adult neuromuscular practice. METHODS: We searched the Mayo Clinic database for patients with CMS diagnosed in adulthood in the neuromuscular clinic between 2000 and 2016. Clinical, laboratory, and electrodiagnostic data were reviewed. RESULTS: We identified 34 patients with CMS, 30 of whom had a molecular diagnosis (14 DOK7, 6 RAPSN, 2 LRP4, 2 COLQ, 2 slow-channel syndrome, 1 primary acetylcholine receptor deficiency, 1 AGRN, 1 GFPT1, and 1 SCN4A). Ophthalmoparesis was often mild and present in 13 patients. Predominant limb-girdle weakness occurred in 19 patients. Two patients had only ptosis. Age at onset ranged from birth to 39 years (median 5 years). The median time from onset to diagnosis was 26 years (range 4-56 years). Thirteen patients had affected family members. Fatigable weakness was present when examined. Creatine kinase was elevated in 4 of 23 patients (range 1.2-4.2 times the upper limit of normal). Repetitive nerve stimulation revealed a decrement in 30 patients. Thirty-two patients were previously misdiagnosed with seronegative myasthenia gravis (n = 16), muscle diseases (n = 15), weakness of undetermined cause (n = 8), and others (n = 4). Fifteen patients received immunotherapy or thymectomy without benefits. Fourteen of the 25 patients receiving pyridostigmine did not improve or worsen. CONCLUSION: Misdiagnosis occurred in 94% of the adult patients with CMS and causes a median diagnostic delay of nearly 3 decades from symptom onset. Seronegative myasthenia gravis and muscle diseases were the 2 most common misdiagnoses, which led to treatment delay and unnecessary exposure to immunotherapy, thymectomy, or muscle biopsy.

Neuromuscular Complications of Programmed Cell Death-1 (PD-1) Inhibitors.

PURPOSE OF REVIEW: In recent years, immune checkpoint inhibitors have been increasingly used in patients with metastatic cancers with favorable oncological outcomes; however, there have also been increasing number of cancer survivors who have developed immune-related adverse events. Little is known about PD-1 inhibitor-associated neuromuscular complications. RECENT FINDINGS: Neuromuscular disorders are the most common neurological complication reported in PD-1 inhibitor-treated patients. Myasthenia gravis, immune-mediated myopathies, and Guillain-Barre syndrome are among commonly reported immune-related neuromuscular complications. HyperCKemia occurs frequently in patients with PD-1 inhibitor-associated myasthenia gravis, indicating coexisting myopathies or myocarditis. Oculobulbar weakness is a unique and common presentation of PD-1 inhibitor-associated immune-mediated myopathies with or without concomitant myasthenia gravis. High-dose steroid monotherapy may be associated with clinical deterioration in some patients with PD-1 inhibitor-associated myasthenia gravis, immune-mediated myopathies, or Guillain-Barre syndrome. PD-1 inhibitor-associated neuromuscular complications have some characteristic features compared to their idiopathic counterparts. Although steroid monotherapy is commonly used in non-neuromuscular autoimmune disorders triggered by anti-PD-1 therapy, this may lead to unfavorable outcomes in some patients with PD-1 inhibitor-associated neuromuscular complications.

A novel ACTA1 mutation causing progressive facioscapuloperoneal myopathy in an adult.

We report a 58-year-old woman with slowly progressive facio-scapulo-peroneal muscle weakness due to congenital nemaline myopathy (NM) caused by a novel ACTA1 mutation (c.118A>G, p.Met271Val). In adult patients, congenital NM should be distinguished from sporadic late-onset nemaline myopathy (SLONM), which is a treatable acquired muscle disease often associated with monoclonal gammopathy or HIV infection. Both congenital NM and SLONM are characterized by the presence of nemaline rods in muscle. The patient's clinical history of difficulty running since childhood and weakness in other family members favored a congenital NM. The type 1 fiber atrophy and clusters of rods in normal size muscle fibers supported the diagnosis of congenital NM and prompted genetic molecular testing, which led to discovery of the novel ACTA1 variant causative of the myopathy.

PD-1 Inhibitor-associated Myopathies: Emerging Immune-mediated Myopathies.

Programmed death-1 (PD-1) inhibitors are increasingly used in cancer immunotherapy. Various immune-related adverse events are reported, including infrequent individual case reports of myositis or rhabdomyolysis. The frequency and diagnostic spectrum of immune-related adverse events affecting skeletal muscle in PD-1 inhibitor-treated patients are unknown. We searched the Mayo Clinic Pharmacy database (2014-2016) to identify patients who developed myopathies during or after PD-1 inhibitor therapy. Among 654 cancer patients received PD-1 inhibitors (pembrolizumab=389; nivolumab=264; both=1), we identified 5 patients (pembrolizumab=5) with biopsy-proven myopathies (2 necrotizing myopathy, 1 early dermatomyositis, and 2 nonspecific myopathy). Four patients developed concomitant autoimmune disorders. Weakness occurred after a median of 2 treatments (range, 1-4). All patients had proximal or axial weakness. Four patients had either bulbar or extraocular weakness, but only 1 patient had acetylcholine receptor antibodies. Creatine kinase levels were elevated in 3 patients (necrotizing myopathy=2; nonspecific myopathy=1). Brain magnetic resonance imaging revealed abnormal T2 signal and enhancement of extraocular muscles in 1 patient with ophthalmoparesis. Pembrolizumab was discontinued in all patients. All patients received immunosuppressive therapy, with fatal outcome in 2 necrotizing myopathy patients and favorable outcome in others. We conclude that myopathy is a rare, but unique complication of PD-1 inhibitors with frequent involvement of extraocular or bulbar muscles, mimicking myasthenia gravis. Muscle biopsy is an important test for PD-1 inhibitor-treated patients who develop oculobulbar weakness and hyperCKemia, to distinguish patients with necrotizing myopathy from myasthenia gravis. Necrotizing myopathy patients may require more aggressive immunotherapy due to their grave prognosis.

Neurological Complications Associated With Anti-Programmed Death 1 (PD-1) Antibodies.

Importance: Neurological complications are an increasingly recognized consequence of the use of anti-programmed death 1 (PD-1) antibodies in the treatment of solid-organ tumors, with an estimated frequency of 4.2%. To date, the clinical spectrum and optimum treatment approach are not established. Objective: To investigate the frequency, clinical spectrum, and optimum treatment approach to neurological complications associated with anti-PD-1 therapy. Design, Setting, and Participants: This single-center, retrospective cohort study was conducted from either September or December 2014 (the approval dates of the study drugs by the US Food and Drug Administration) to May 19, 2016. All patients receiving anti-PD-1 monoclonal antibodies were identified using the Mayo Cancer Pharmacy Database. Patients with development of neurological symptoms within 12 months of anti-PD-1 therapy were included. Patients with neurological complications directly attributable to metastatic disease or other concurrent cancer-related treatments were excluded. Main Outcomes and Measures: Clinical and pathological characteristics, time to development of neurological symptoms, and modified Rankin Scale (mRS) score. Results: Among 347 patients treated with anti-PD1 monoclonal antibodies (pembrolizumab or nivolumab), 10 (2.9%) developed subacute onset of neurological complications. Seven patients were receiving pembrolizumab, and 3 patients were receiving nivolumab. The patients included 8 men and 2 women. Their median age was 71 years (age range, 31-78 years). Neurological complications occurred after a median of 5.5 (range, 1-20) cycles of anti-PD-1 inhibitors. Complications included myopathy (n = 2), varied neuropathies (n = 4), cerebellar ataxia (n = 1), autoimmune retinopathy (n = 1), bilateral internuclear ophthalmoplegia (n = 1), and headache (n = 1). Peripheral neuropathies included axonal and demyelinating polyradiculoneuropathies (n = 2), length-dependent neuropathies (n = 1), and asymmetric vasculitic neuropathy (n = 1). The time to maximum symptom severity varied from 1 day to more than 3 months. The median mRS score was 2.5 (range, 1-5), indicating mild to moderate disability. Five patients experienced other systemic immune-mediated complications, including hypothyroidism (n = 3), colitis (n = 2), and hepatitis (n = 1). Treatment with anti-PD-1 antibodies was discontinued in 7 patients. Treatment included corticosteroids (n = 7), intravenous immunoglobulin (n = 3), and plasma exchange (n = 1). Nine patients improved, with a median mRS score of 2 (range, 0-6). One patient with severe necrotizing myopathy died. Conclusions and Relevance: Neurological adverse events associated with anti-PD-1 therapy have a diverse phenotype, with more frequent neuromuscular complications. Although rare, they will likely be encountered with increasing frequency as anti-PD-1 therapy expands to other cancers. The time of onset is unpredictable, and evolution may be rapid and life-threatening. Prompt recognition and discontinuation of anti-PD-1 therapy is recommended. In some cases, immune rescue treatment may be required.

Dopamine Gene Profiling to Predict Impulse Control and Effects of Dopamine Agonist Ropinirole.

Dopamine agonists can impair inhibitory control and cause impulse control disorders for those with Parkinson disease (PD), although mechanistically this is not well understood. In this study, we hypothesized that the extent of such drug effects on impulse control is related to specific dopamine gene polymorphisms. This double-blind, placebo-controlled study aimed to examine the effect of single doses of 0.5 and 1.0 mg of the dopamine agonist ropinirole on impulse control in healthy adults of typical age for PD onset. Impulse control was measured by stop signal RT on a response inhibition task and by an index of impulsive decision-making on the Balloon Analogue Risk Task. A dopamine genetic risk score quantified basal dopamine neurotransmission from the influence of five genes: catechol-O-methyltransferase, dopamine transporter, and those encoding receptors D1, D2, and D3. With placebo, impulse control was better for the high versus low genetic risk score groups. Ropinirole modulated impulse control in a manner dependent on genetic risk score. For the lower score group, both doses improved response inhibition (decreased stop signal RT) whereas the lower dose reduced impulsiveness in decision-making. Conversely, the higher score group showed a trend for worsened response inhibition on the lower dose whereas both doses increased impulsiveness in decision-making. The implications of the present findings are that genotyping can be used to predict impulse control and whether it will improve or worsen with the administration of dopamine agonists.

Reversible cerebral vasoconstriction in Guillain-Barré syndrome.

We present a 51-year-old woman with clinical and neurophysiological evidence of Guillain-Barré syndrome (GBS) who developed a generalised headache and autonomic dysfunction with sinus tachycardia, hypertension, gastrointestinal motility symptoms and urinary retention. MRI/MRA demonstrated cerebral vasoconstriction and a small convexity subarachnoid haemorrhage which resolved after 3 months. Reversible cerebral vasoconstriction syndrome (RCVS) is characterised by headache, focal neurological deficits or seizures, and reversible cerebral vasoconstriction. To our knowledge, this is the first reported case of RCVS complicating autonomic dysfunction in GBS. This case depicts a rare complication of a common condition and also sheds light on the potential mechanism of RCVS. Neurologists should be aware that autonomic dysfunction can lead to RCVS in GBS.

Rupture of thin films with resonant substrate patterning.

We study the stability and rupture of thin liquid films on patterned substrates. It is shown that striped patterning on a length scale comparable to that of the spinodal instability leads to a resonance effect and an imperfect bifurcation of equilibrium film shapes. Weakly nonlinear analysis gives predictions for film shapes, stability, growth rates, and rupture times, which are confirmed by numerical solution of the thin-film equation. Film behavior is qualitatively different in the resonant patterning regime, but with sufficiently large domains rupture occurs on a spinodal length scale regardless of patterning. Instabilities transverse to the patterning are examined and shown to behave similarly as disturbances to films on uniform substrates. We explain some previously reported effects in terms of the imperfect bifurcation.

Epinephrine delivery during neonatal resuscitation: comparison of direct endotracheal tube vs catheter inserted into endotracheal tube administration.

OBJECTIVES: The optimal method for epinephrine administration during neonatal resuscitation is not known. We hypothesized that epinephrine will be delivered more efficiently when administered via a feeding catheter inserted into the endotracheal tube (C-ETT) vs when administered directly into the ETT (D-ETT). Our objectives were to (1) compare the delivery of epinephrine to the distal end of the ETT when administered via D-ETT vs C-ETT; (2) measure the retention of epinephrine within the ETT vs the feeding catheter used for the drug delivery; and (3) compare the delivery of the drug with and without an air flush after administration via C-ETT. METHODS: All experiments were performed in vitro, simulating epinephrine administration during neonatal resuscitation, according to the standard guidelines. Radiolabeled epinephrine, diluted to 1 microCi/ml, was used and experiments were repeated at least 4 times. Epinephrine administration via D-ETT was followed by one manual breath via a self-inflating bag attached to the ETT. Epinephrine delivery via C-ETT was followed by 1 ml saline flush, and in some experiments, this was also followed by a 1 cm(3) air flush. Epinephrine delivery and retention were assessed by measuring the radioactive content of the effluent fluid and that of the ETT or of the feeding catheter used for drug delivery. RESULTS: Significantly higher dosage of the drug was delivered when administered via D-ETT vs C-ETT, if air flush following C-ETT method was not used. However, with an air flush following the saline flush after the drug instillation, there was no difference in the amount of epinephrine delivered between the two methods. Retention in the ETT wall or the catheter was <7.5% of the administered dose with either method. CONCLUSIONS: Without an air flush following C-ETT method of epinephrine delivery, higher dosage of the drug is delivered via D-ETT vs C-ETT method. An air flush following the saline flush during C-ETT method improves drug delivery. Given that the C-ETT method is more cumbersome and time consuming, and does not improve drug delivery, D-ETT administration should be the method of choice for epinephrine delivery during neonatal resuscitation.