Lysine-Derived Charge-Altering Releasable Transporters: Targeted Delivery of mRNA and siRNA to the Lungs.

Targeted delivery of nucleic acid therapeutics to the lungs could transform treatment options for pulmonary disease. We have previously developed oligomeric charge-altering releasable transporters (CARTs) for in vivo mRNA transfection and demonstrated their efficacy for use in mRNA-based cancer vaccination and local immunomodulatory therapies against murine tumors. While our previously reported glycine-based CART-mRNA complexes (G-CARTs/mRNA) show selective protein expression in the spleen (mouse, >99%), here, we report a new lysine-derived CART-mRNA complex (K-CART/mRNA) that, without additives or targeting ligands, shows selective protein expression in the lungs (mouse, >90%) following systemic IV administration. We further show that by delivering siRNA using the K-CART, we can significantly decrease expression of a lung-localized reporter protein. Blood chemistry and organ pathology studies demonstrate that K-CARTs are safe and well-tolerated. We report on the new step economical, organocatalytic synthesis (two steps) of functionalized polyesters and oligo-carbonate-co-α-aminoester K-CARTs from simple amino acid and lipid-based monomers. The ability to direct protein expression selectively in the spleen or lungs by simple, modular changes to the CART structure opens fundamentally new opportunities in research and gene therapy.

Oligo(serine ester) Charge-Altering Releasable Transporters: Organocatalytic Ring-Opening Polymerization and their Use for in Vitro and in Vivo mRNA Delivery.

RNA technology is transforming life science research and medicine, but many applications are limited by the accessibility, cost, efficacy, and tolerability of delivery systems. Here we report the first members of a new class of dynamic RNA delivery vectors, oligo(serine ester)-based charge-altering releasable transporters (Ser-CARTs). Composed of lipid-containing oligocarbonates and cationic oligo(serine esters), Ser-CARTs are readily prepared (one flask) by a mild ring-opening polymerization using thiourea anions and, upon simple mixing with mRNA, readily form complexes that degrade to neutral serine-based products, efficiently releasing their mRNA cargo. mRNA/Ser-CART transfection efficiencies of >95% are achieved in vitro. Intramuscular or intravenous (iv) injections of mRNA/Ser-CARTs into living mice result in in vivo expression of a luciferase reporter protein, with spleen localization observed after iv injection.

Variants in the transcriptional corepressor BCORL1 are associated with an X-linked disorder of intellectual disability, dysmorphic features, and behavioral abnormalities.

BCORL1, a transcriptional corepressor, is involved in negative gene regulation through associations with several protein complexes including Class II histone deacetylases (HDACs). Acquired somatic mutations in BCORL1 have been implicated in the pathogenesis of several malignancies, but germline mutations of BCORL1 have not been associated with a specific genetic syndrome. We report five individuals from three pedigrees with phenotypes including intellectual disability, behavioral difficulties, and dysmorphic features who were found via whole exome sequencing to have variants in BCORL1. In silico analysis of these variants strongly suggests pathogenicity. We propose that hemizygous pathogenic variants in BCORL1 underlie a newly identified X-linked epigenetic syndrome.

Natural history and genotype-phenotype correlations in 72 individuals with SATB2-associated syndrome.

SATB2-associated syndrome (SAS) is an autosomal dominant disorder characterized by significant neurodevelopmental disabilities with limited to absent speech, behavioral issues, and craniofacial anomalies. Previous studies have largely been restricted to case reports and small series without in-depth phenotypic characterization or genotype-phenotype correlations. Seventy two study participants were identified as part of the SAS clinical registry. Individuals with a molecularly confirmed diagnosis of SAS were referred after clinical diagnostic testing. In this series we present the most comprehensive phenotypic and genotypic characterization of SAS to date, including prevalence of each clinical feature, neurodevelopmental milestones, and when available, patient management. We confirm that the most distinctive features are neurodevelopmental delay with invariably severely limited speech, abnormalities of the palate (cleft or high-arched), dental anomalies (crowding, macrodontia, abnormal shape), and behavioral issues with or without bone or brain anomalies. This comprehensive clinical characterization will help clinicians with the diagnosis, counseling and management of SAS and help provide families with anticipatory guidance.

Customizable Lipid Nanoparticle Materials for the Delivery of siRNAs and mRNAs.

RNAs are a promising class of therapeutics given their ability to regulate protein concentrations at the cellular level. Developing safe and effective strategies to deliver RNAs remains important for realizing their full clinical potential. Here, we develop lipid nanoparticle formulations that can deliver short interfering RNAs (for gene silencing) or messenger RNAs (for gene upregulation). Specifically, we study how the tail length, tail geometry, and linker spacing in diketopiperazine lipid materials influences LNP potency with siRNAs and mRNAs. Eight lipid materials are synthesized, and 16 total formulations are screened for activity in vitro; the lead material is evaluated with mRNA for in vivo use and demonstrates luciferase protein expression in the spleen. In undertaking this approach, not only do we develop synthetic routes to delivery materials, but we also reveal structural criteria that could be useful for developing next-generation delivery materials for RNA therapeutics.

New targets for monitoring and therapy in Barth syndrome.

PURPOSE: Barth syndrome (BTHS), an X-linked disorder caused by defects in TAZ, is the only known single-gene disorder of cardiolipin remodeling. We hypothesized that through analysis of affected individuals, we would gain a better understanding of the range of clinical features and identify targets for monitoring and therapy. METHODS: We conducted a multidisciplinary investigation involving 42 patients with BTHS, including echocardiograms, muscle strength testing, functional exercise capacity testing, physical activity assessments, cardiolipin analysis, 3-methylglutaconic acid analysis, and review of genotype data. We analyzed data points to provide a quantitative spectrum of disease characteristics and to identify relationships among phenotype, genotype, and relevant metabolites. RESULTS: Echocardiography revealed considerable variability in cardiac features. By contrast, almost all patients had significantly reduced functional exercise capacity. Multivariate analysis revealed significant relationships between cardiolipin ratio and left ventricular mass and between cardiolipin ratio and functional exercise capacity. We additionally identified genotypes associated with a less severe metabolic and clinical profile. CONCLUSION: We defined previously unrecognized metabolite/phenotype/genotype relationships, established targets for therapeutic monitoring, and validated avenues for clinical assessment. In addition to providing insight into BTHS, these studies also provide insight into the myriad of multifactorial disorders that converge on the cardiolipin pathway.Genet Med 18 10, 1001-1010.

Clinical whole exome sequencing in child neurology practice.

OBJECTIVE: Whole exome sequencing (WES) represents a significant breakthrough in clinical genetics as a powerful tool for etiological discovery in neurodevelopmental disorders. To better characterize the genetic landscape of neurodevelopmental disorders, we analyzed patients in our pediatric neurogenetics clinic who underwent WES. METHODS: We performed a retrospective cohort study on 78 patients with various neurodevelopmental disabilities and unrevealing workup prior to WES. We characterized their molecular diagnoses, clinical features, and whether their previous treatment plan changed due to WES results. RESULTS: The overall presumptive diagnostic rate for our cohort was 41% (n = 32 of 78 patients). Nineteen patients had a single autosomal dominant (AD) disorder, 11 had a single autosomal recessive (AR) disorder, 1 had an X-linked dominant disorder, and 1 had both an AD and an AR disorder. The 32 patients with pathogenic or likely pathogenic variants exhibited various neurobehavioral and neuroimaging abnormalities, including intellectual disability/developmental delay (n = 28), cerebral palsy-like encephalopathy (n = 11), autism spectrum disorder (n = 5), delayed/hypomyelination (n = 7), and cerebellar abnormalities (n = 9). The results of WES affected management for all patients with a presumptive diagnosis, triggering reproductive planning (n = 27), disease monitoring initiation (n = 4), investigation of systemic involvement of the disorder(s) (n = 6), alteration of presumed disease inheritance pattern (n = 7), changing of prognosis (n = 10), medication discontinuation (n = 5) or initiation (n = 2), and clinical trial education (n = 3). INTERPRETATION: The high diagnostic yield of WES supports its use in pediatric neurology practices. It may also lead to earlier diagnosis, impacting medical management, prognostication, and family planning. WES therefore serves as a critical tool for the child neurologist.

Mutations in FARS2 and non-fatal mitochondrial dysfunction in two siblings.

Recently, mutations in FARS2, which encodes for mitochondrial phenylalanyl-tRNA synthetase, have been implicated in autosomal recessive combined oxidative phosphorylation deficiency 14. Associated clinical features in three previously reported patients with confirmed FARS2 mutations include infantile onset epilepsy, and a fatal Alpers-like encephalopathy. Herein, we report on two siblings with global developmental delay, dysarthria and tremor and compound heterozygous FARS2 abnormalities. They have a heterozygous missense mutation, c.1255C>T which predicts p.Arg419Cys in exon 7 of FARS2, inherited from their father and uncovered on exome sequencing, and an interstitial deletion of chromosome 6p25.1 inherited from their mother and uncovered on SNP array. This interstitial deletion includes all of exon 6 and parts of introns 5 and 6 of FARS2. Biochemical studies were also consistent with a mitochondrial disorder. While these siblings had considerable developmental difficulties, they are making consistent developmental progress and appear to be considerably less severely affected than the other patients reported in the literature with FARS2 associated mitochondrial disease. Thus, this study expands the phenotypic spectrum of FARS2 related disease and emphasizes intragenic deletion in the list of causative mutations.

Clinical laboratory studies in Barth Syndrome.

Barth Syndrome is a rare X-linked disorder characterized principally by dilated cardiomyopathy, skeletal myopathy and neutropenia and caused by defects in tafazzin, an enzyme responsible for modifying the acyl chain moieties of cardiolipin. While several comprehensive clinical studies of Barth Syndrome have been published detailing cardiac and hematologic features, descriptions of its biochemical characteristics are limited. To gain a better understanding of the clinical biochemistry of this rare disease, we measured hematologic and biochemical values in a cohort of Barth Syndrome patients. We characterized multiple biochemical parameters, including plasma amino acids, plasma 3-methylglutaconic acid, cholesterol, cholesterol synthetic intermediates, and red blood cell membrane fatty acid profiles in 28 individuals with Barth Syndrome from ages 10 months to 30 years. We describe a unique biochemical profile for these patients, including decreased plasma arginine levels. We further studied the plasma amino acid profiles, cholesterol, cholesterol synthetic intermediates, and plasma 3-methylglutaconic acid levels in 8 female carriers and showed that they do not share any of the distinct, Barth Syndrome-specific biochemical laboratory abnormalities. Our studies augment and expand the biochemical profiles of individuals with Barth Syndrome, describe a unique biochemical profile for these patients, and provide insight into the possible underlying biochemical pathology in this disorder.

Unraveling Complexities in Genetically Elusive Long QT Syndrome.

Genetic testing has become standard of care for patients with long QT syndrome (LQTS), providing diagnostic, prognostic, and therapeutic information for both probands and their family members. However, up to a quarter of patients with LQTS do not have identifiable Mendelian pathogenic variants in the currently known LQTS-associated genes. This absence of genetic confirmation, intriguingly, does not lessen the severity of LQTS, with the prognosis in these gene-elusive patients with unequivocal LQTS mirroring genotype-positive patients in the limited data available. Such a conundrum instigates an exploration into the causes of corrected QT interval (QTc) prolongation in these cases, unveiling a broad spectrum of potential scenarios and mechanisms. These include multiple environmental influences on QTc prolongation, exercise-induced repolarization abnormalities, and the profound implications of the constantly evolving nature of genetic testing and variant interpretation. In addition, the rapid advances in genetics have the potential to uncover new causal genes, and polygenic risk factors may aid in the diagnosis of high-risk patients. Navigating this multifaceted landscape requires a systematic approach and expert knowledge, integrating the dynamic nature of genetics and patient-specific influences for accurate diagnosis, management, and counseling of patients. The role of a subspecialized expert cardiogenetic clinic is paramount in evaluation to navigate this complexity. Amid these intricate aspects, this review outlines potential causes of gene-elusive LQTS. It also provides an outline for the evaluation of patients with negative and inconclusive genetic test results and underscores the need for ongoing adaptation and reassessment in our understanding of LQTS, as the complexities of gene-elusive LQTS are increasingly deciphered.

C-reactive Protein Elevation Is Associated With QTc Interval Prolongation in Patients Hospitalized With COVID-19.

Background: The relationship between inflammation and corrected QT (QTc) interval prolongation is currently not well defined in patients with COVID-19. Objective: This study aimed to assess the effect of marked interval changes in the inflammatory marker C-reactive protein (CRP) on QTc interval in patients hospitalized with COVID-19. Methods: In this retrospective cohort study of hospitalized adult patients admitted with COVID-19 infection, we identified 85 patients who had markedly elevated CRP levels and serial measurements of an ECG and CRP during the same admission. We compared mean QTc interval duration, and other clinical and ECG characteristics between times when CRP values were high and low. We performed mixed-effects linear regression analysis to identify associations between CRP levels and QTc interval in univariable and adjusted models. Results: Mean age was 58 ± 16 years, of which 39% were women, 41% were Black, and 25% were White. On average, the QTc interval calculated via the Bazett formula was 15 ms higher when the CRP values were "high" vs. "low" [447 ms (IQR 427-472 ms) and 432 ms (IQR 412-452 ms), respectively]. A 100 mg/L increase in CRP was associated with a 1.5 ms increase in QTc interval [ß coefficient 0.15, 95% CI (0.06-0.24). In a fully adjusted model for sociodemographic, ECG, and clinical factors, the association remained significant (ß coefficient 0.14, 95% CI 0.05-0.23). Conclusion: An interval QTc interval prolongation is observed with a marked elevation in CRP levels in patients with COVID-19.

Acquired Long QT and Ventricular Arrhythmias in the Setting of Acute Inflammation: A Case Series.

We report a case series of 4 patients with transient marked QTc prolongation and ventricular arrhythmias in the setting of inflammation with very high ferritin levels. Three patients were positive for coronavirus disease-2019. In the setting of an acute rise in inflammatory markers, electrocardiography screening for QTc prolongation is warranted. (Level of Difficulty: Beginner.).

Anxiety and depression in inherited channelopathy patients with implantable cardioverter-defibrillators.

BACKGROUND: Implantable cardioverter-defibrillators (ICDs) are an effective treatment in some patients with inherited heart disease, including inherited channelopathies, yet they have also been shown to impact patients' psychological health. OBJECTIVE: We sought to improve understanding of the level of anxiety and depression as well as device acceptance among inherited channelopathy patients with an ICD. METHODS: Eligible patients seen at Johns Hopkins Hospital were sent a survey, which included the Hospital Anxiety and Depression Scale (HADS), Cardiac Anxiety Questionnaire (CAQ), and the Florida Patient Acceptance Survey (FPAS). Student t tests and χ2 tests were used to identify associations with abnormal anxiety and depression scores. RESULTS: Among eligible patients (n = 65), 32 individuals (49%) completed the survey. The rate of device-related complications was 34%, and 41% of patients experienced 1 or more ICD shocks. Twelve patients (38%) had an abnormal HADS anxiety subscore and 5 patients (16%) had an abnormal HADS depression subscore (score ≥ 8). Secondary-prevention ICDs were associated with an abnormal HADS anxiety subscore (P = .03). Experiencing ICD shock(s), device complications, age, sex, and family history of sudden cardiac death were not statistically associated with anxiety or depression. Overall, respondents demonstrated high device acceptance by FPAS (79.9 ± 2.9, maximum total score 100) and moderately high cardiac-specific anxiety by CAQ total score (1.53 ± 0.12). CONCLUSION: A high prevalence of generalized anxiety was identified among inherited channelopathy patients with ICDs. High device acceptance and lack of association with ICD shocks or complications indicate that further research is necessary to understand this increased incidence.

Incorporation of genetic testing significantly increases the number of individuals diagnosed with familial hypercholesterolemia.

BACKGROUND: It is estimated that less than 10% of cases of familial hypercholesterolemia (FH) in the United States have been diagnosed. Low rates of diagnosis may in part be attributable to affected patients not meeting the clinical diagnostic criteria of the Dutch Lipid Clinic Network (DLCN), Simon Broome, or US MEDPED diagnostic criteria. OBJECTIVE: The objective of this study was to assess the utility of incorporating genetic testing into a patient's evaluation for FH. METHODS: We retrospectively reviewed patients seen in the Advanced Lipids Disorders Clinic at Johns Hopkins Hospital between January 2015 and May 2018. Between June 2018 and December 2018, patients were consented to a prospective registry. DLCN, Simon Broome, and MEDPED criteria were applied to each patient, before and after genetic testing. Genetic testing included sequencing and deletion duplication analysis of four genes (LDLR, PCSK9, APOB, and LDLRAP1). RESULTS: The retrospective review and prospective study identified 135 adult probands who were seen in our clinic for evaluation of heterozygous FH. Twenty-nine individuals (21%) were heterozygous for a pathogenic or likely pathogenic monogenic variant. Before genetic testing, using the DLCN criteria, 35 (26%) individuals met criteria for a definite diagnosis of FH. Thirty patients (22%) met criteria using Simon Broome, and 29 (21%) patients met criteria using US MEDPED before genetic analysis. Depending on the criteria, incorporating genetic testing identified 11-14 additional patients with FH. CONCLUSIONS: Incorporating genetic testing diagnosed almost 50% more patients with definite FH in comparison to classification solely on clinical grounds.

Functional exercise capacity, strength, balance and motion reaction time in Barth syndrome.

BACKGROUND: Barth syndrome (BTHS) is an X-linked disorder caused by defects in TAZ with key clinical features including cardiomyopathy, neutropenia and skeletal myopathy. In order to gain a better understanding of the range of clinical features, identify targets for monitoring, and increase knowledge of natural history of the disease, we conducted muscle strength testing, functional exercise capacity testing, physical activity assessment, balance assessment and motion reaction time testing in 33 affected individuals and 14 controls. We analyzed data points to provide a cross-sectional quantitative spectrum of disease characteristics. We also compared these data points to the matched data points collected two years prior to provide insight into effects of BTHS over time. RESULTS: In comparison to controls, pediatric subjects with BTHS present with significantly impaired balance and motion reaction time while adult subjects with BTHS present with significantly impaired motion reaction time. In comparison to controls, subjects with BTHS presented with decreased functional exercise capacity (assessed via 6 MWT), knee extensor strength (both assessed via handheld dynamometry and five times sit-to-stand (5 TSTS)), and self-reported physical activity. Comparison of functional exercise capacity, knee extensor strength and self-reported physical activity from identical cohorts in 2014 and 2016 BTHS showed that the deficits in 6 MWT do not change significantly over the 2 year time span. CONCLUSION: In this comprehensive assessment of musculoskeletal parameters in a cross-section of individuals with BTHS, we uncovered deficits in motion reaction time and balance, which were previously not known to be abnormal in in BTHS. We also confirmed results of our previous study showing that pediatric and adult subjects with BTHS have decreased functional exercise capacity, knee extensor strength, and physical activity in comparison to controls, r, verifying the importance of including these measures as part of the regular clinical assessment in individuals with BTHS, as well as introducing 5 TSTS as an additional testing parameter. Perhaps most importantly, we demonstrated that 6 MWT results do not significantly vary in pediatric and adult cohorts with BTHS over a 2-year period, supporting this as a reliable quantitative measure of therapeutic outcomes in clinical studies and for clinical monitoring.

Ubiquity of polystyrene digestion and biodegradation within yellow mealworms, larvae of Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae).

Academics researchers and "citizen scientists" from 22 countries confirmed that yellow mealworms, the larvae of Tenebrio molitor Linnaeus, can survive by eating polystyrene (PS) foam. More detailed assessments of this capability for mealworms were carried out by12 sources: five from the USA, six from China, and one from Northern Ireland. All of these mealworms digested PS foam. PS mass decreased and depolymerization was observed, with appearance of lower molecular weight residuals and functional groups indicative of oxidative transformations in extracts from the frass (insect excrement). An addition of gentamycin (30 mg g-1), a bactericidal antibiotic, inhibited depolymerization, implicating the gut microbiome in the biodegradation process. Microbial community analyses demonstrated significant taxonomic shifts for mealworms fed diets of PS plus bran and PS alone. The results indicate that mealworms from diverse locations eat and metabolize PS and support the hypothesis that this capacity is independent of the geographic origin of the mealworms, and is likely ubiquitous to members of this species.

Synthesis and Biological Evaluation of Ionizable Lipid Materials for the In Vivo Delivery of Messenger RNA to B Lymphocytes.

B lymphocytes regulate several aspects of immunity including antibody production, cytokine secretion, and T-cell activation; moreover, B cell misregulation is implicated in autoimmune disorders and cancers such as multiple sclerosis and non-Hodgkin's lymphomas. The delivery of messenger RNA (mRNA) into B cells can be used to modulate and study these biological functions by means of inducing functional protein expression in a dose-dependent and time-controlled manner. However, current in vivo mRNA delivery systems fail to transfect B lymphocytes and instead primarily target hepatocytes and dendritic cells. Here, the design, synthesis, and biological evaluation of a lipid nanoparticle (LNP) system that can encapsulate mRNA, navigate to the spleen, transfect B lymphocytes, and induce more than 60 pg of protein expression per million B cells within the spleen is described. Importantly, this LNP induces more than 85% of total protein production in the spleen, despite LNPs being observed transiently in the liver and other organs. These results demonstrate that LNP composition alone can be used to modulate the site of protein induction in vivo, highlighting the critical importance of designing and synthesizing new nanomaterials for nucleic acid delivery.

DNM1 encephalopathy: A new disease of vesicle fission.

OBJECTIVE: To evaluate the phenotypic spectrum caused by mutations in dynamin 1 (DNM1), encoding the presynaptic protein DNM1, and to investigate possible genotype-phenotype correlations and predicted functional consequences based on structural modeling. METHODS: We reviewed phenotypic data of 21 patients (7 previously published) with DNM1 mutations. We compared mutation data to known functional data and undertook biomolecular modeling to assess the effect of the mutations on protein function. RESULTS: We identified 19 patients with de novo mutations in DNM1 and a sibling pair who had an inherited mutation from a mosaic parent. Seven patients (33.3%) carried the recurrent p.Arg237Trp mutation. A common phenotype emerged that included severe to profound intellectual disability and muscular hypotonia in all patients and an epilepsy characterized by infantile spasms in 16 of 21 patients, frequently evolving into Lennox-Gastaut syndrome. Two patients had profound global developmental delay without seizures. In addition, we describe a single patient with normal development before the onset of a catastrophic epilepsy, consistent with febrile infection-related epilepsy syndrome at 4 years. All mutations cluster within the GTPase or middle domains, and structural modeling and existing functional data suggest a dominant-negative effect on DMN1 function. CONCLUSIONS: The phenotypic spectrum of DNM1-related encephalopathy is relatively homogeneous, in contrast to many other genetic epilepsies. Up to one-third of patients carry the recurrent p.Arg237Trp variant, which is now one of the most common recurrent variants in epileptic encephalopathies identified to date. Given the predicted dominant-negative mechanism of this mutation, this variant presents a prime target for therapeutic intervention.

Bioinspired Alkenyl Amino Alcohol Ionizable Lipid Materials for Highly Potent In Vivo mRNA Delivery.

Thousands of human diseases could be treated by selectively controlling the expression of specific proteins in vivo. A new series of alkenyl amino alcohol (AAA) ionizable lipid nanoparticles (LNPs) capable of delivering human mRNA with unprecedented levels of in vivo efficacy is demonstrated. This study highlights the importance of utilizing synthesis tools in tandem with biological inspiration to understand and improve nucleic acid delivery in vivo.