Duration of SARS-CoV-2 mRNA vaccine persistence and factors associated with cardiac involvement in recently vaccinated patients.

At the start of the COVID-19 pandemic, the BNT162b2 (BioNTech-Pfizer) and mRNA-1273 (Moderna) mRNA vaccines were expediently designed and mass produced. Both vaccines produce the full-length SARS-CoV-2 spike protein for gain of immunity and have greatly reduced mortality and morbidity from SARS-CoV-2 infection. The distribution and duration of SARS-CoV-2 mRNA vaccine persistence in human tissues is unclear. Here, we developed specific RT-qPCR-based assays to detect each mRNA vaccine and screened lymph nodes, liver, spleen, and myocardium from recently vaccinated deceased patients. Vaccine was detected in the axillary lymph nodes in the majority of patients dying within 30 days of vaccination, but not in patients dying more than 30 days from vaccination. Vaccine was not detected in the mediastinal lymph nodes, spleen, or liver. Vaccine was detected in the myocardium in a subset of patients vaccinated within 30 days of death. Cardiac ventricles in which vaccine was detected had healing myocardial injury at the time of vaccination and had more myocardial macrophages than the cardiac ventricles in which vaccine was not detected. These results suggest that SARS-CoV-2 mRNA vaccines routinely persist up to 30 days from vaccination and can be detected in the heart.

Development of an oral treatment that rescues gait ataxia and retinal degeneration in a phenotypic mouse model of familial dysautonomia.

Familial dysautonomia (FD) is a rare neurodegenerative disease caused by a splicing mutation in elongator acetyltransferase complex subunit 1 (ELP1). This mutation leads to the skipping of exon 20 and a tissue-specific reduction of ELP1, mainly in the central and peripheral nervous systems. FD is a complex neurological disorder accompanied by severe gait ataxia and retinal degeneration. There is currently no effective treatment to restore ELP1 production in individuals with FD, and the disease is ultimately fatal. After identifying kinetin as a small molecule able to correct the ELP1 splicing defect, we worked on its optimization to generate novel splicing modulator compounds (SMCs) that can be used in individuals with FD. Here, we optimize the potency, efficacy, and bio-distribution of second-generation kinetin derivatives to develop an oral treatment for FD that can efficiently pass the blood-brain barrier and correct the ELP1 splicing defect in the nervous system. We demonstrate that the novel compound PTC258 efficiently restores correct ELP1 splicing in mouse tissues, including brain, and most importantly, prevents the progressive neuronal degeneration that is characteristic of FD. Postnatal oral administration of PTC258 to the phenotypic mouse model TgFD9;Elp1Δ20/flox increases full-length ELP1 transcript in a dose-dependent manner and leads to a 2-fold increase in functional ELP1 in the brain. Remarkably, PTC258 treatment improves survival, gait ataxia, and retinal degeneration in the phenotypic FD mice. Our findings highlight the great therapeutic potential of this novel class of small molecules as an oral treatment for FD.

Selective retinal ganglion cell loss and optic neuropathy in a humanized mouse model of familial dysautonomia.

Familial dysautonomia (FD) is an autosomal recessive neurodegenerative disease caused by a splicing mutation in the gene encoding Elongator complex protein 1 (ELP1, also known as IKBKAP). This mutation results in tissue-specific skipping of exon 20 with a corresponding reduction of ELP1 protein, predominantly in the central and peripheral nervous system. Although FD patients have a complex neurological phenotype caused by continuous depletion of sensory and autonomic neurons, progressive visual decline leading to blindness is one of the most problematic aspects of the disease, as it severely affects their quality of life. To better understand the disease mechanism as well as to test the in vivo efficacy of targeted therapies for FD, we have recently generated a novel phenotypic mouse model, TgFD9; IkbkapΔ20/flox. This mouse exhibits most of the clinical features of the disease and accurately recapitulates the tissue-specific splicing defect observed in FD patients. Driven by the dire need to develop therapies targeting retinal degeneration in FD, herein, we comprehensively characterized the progression of the retinal phenotype in this mouse, and we demonstrated that it is possible to correct ELP1 splicing defect in the retina using the splicing modulator compound (SMC) BPN-15477.

Developmental regulation of neuronal gene expression by Elongator complex protein 1 dosage.

Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by a mutation in the Elongator complex protein 1 (ELP1) gene that leads to a tissue-specific reduction of ELP1 protein. Our work to generate a phenotypic mouse model for FD headed to the discovery that homozygous deletion of the mouse Elp1 gene leads to embryonic lethality prior to mid-gestation. Given that FD is caused by a reduction, not loss, of ELP1, we generated two new mouse models by introducing different copy numbers of the human FD ELP1 transgene into the Elp1 knockout mouse (Elp1-/-) and observed that human ELP1 expression rescues embryonic development in a dose-dependent manner. We then conducted a comprehensive transcriptome analysis in mouse embryos to identify genes and pathways whose expression correlates with the amount of ELP1. We found that ELP1 is essential for the expression of genes responsible for nervous system development. Further, gene length analysis of the differentially expressed genes showed that the loss of Elp1 mainly impacts the expression of long genes and that by gradually restoring Elongator, their expression is progressively rescued. Finally, through evaluation of co-expression modules, we identified gene sets with unique expression patterns that depended on ELP1 expression.

A deep learning approach to identify gene targets of a therapeutic for human splicing disorders.

Pre-mRNA splicing is a key controller of human gene expression. Disturbances in splicing due to mutation lead to dysregulated protein expression and contribute to a substantial fraction of human disease. Several classes of splicing modulator compounds (SMCs) have been recently identified and establish that pre-mRNA splicing represents a target for therapy. We describe herein the identification of BPN-15477, a SMC that restores correct splicing of ELP1 exon 20. Using transcriptome sequencing from treated fibroblast cells and a machine learning approach, we identify BPN-15477 responsive sequence signatures. We then leverage this model to discover 155 human disease genes harboring ClinVar mutations predicted to alter pre-mRNA splicing as targets for BPN-15477. Splicing assays confirm successful correction of splicing defects caused by mutations in CFTR, LIPA, MLH1 and MAPT. Subsequent validations in two disease-relevant cellular models demonstrate that BPN-15477 increases functional protein, confirming the clinical potential of our predictions.

Factors associated with myocardial SARS-CoV-2 infection, myocarditis, and cardiac inflammation in patients with COVID-19.

COVID-19 has been associated with cardiac injury and dysfunction. While both myocardial inflammatory cell infiltration and myocarditis with myocyte injury have been reported in patients with fatal COVID-19, clinical-pathologic correlations remain limited. The objective was to determine the relationships between cardiac pathological changes in patients dying from COVID-19 and cardiac infection by SARS-CoV-2, laboratory measurements, clinical features, and treatments. In a retrospective study, 41 consecutive autopsies of patients with fatal COVID-19 were analyzed for the associations between cardiac inflammation, myocarditis, cardiac infection by SARS-CoV-2, clinical features, laboratory measurements, and treatments. Cardiac infection was assessed by in situ hybridization and NanoString transcriptomic profiling. Cardiac infection by SARS-CoV-2 was present in 30/41 cases: virus+ with myocarditis (n = 4), virus+ without myocarditis (n = 26), and virus- without myocarditis (n = 11). In the cases with cardiac infection, SARS-CoV-2+ cells in the myocardium were rare, with a median density of 1 cell/cm2. Virus+ cases showed higher densities of myocardial CD68+ macrophages and CD3+ lymphocytes, as well as more electrocardiographic changes (23/27 vs 4/10; P = 0.01). Myocarditis was more prevalent with IL-6 blockade than with nonbiologic immunosuppression, primarily glucocorticoids (2/3 vs 0/14; P = 0.02). Overall, SARS-CoV-2 cardiac infection was less prevalent in patients treated with nonbiologic immunosuppression (7/14 vs 21/24; P = 0.02). Myocardial macrophage and lymphocyte densities overall were positively correlated with the duration of symptoms but not with underlying comorbidities. In summary, cardiac infection with SARS-CoV-2 is common among patients dying from COVID-19 but often with only rare infected cells. Cardiac infection by SARS-CoV-2 is associated with more cardiac inflammation and electrocardiographic changes. Nonbiologic immunosuppression is associated with lower incidences of myocarditis and cardiac infection by SARS-CoV-2.

Molecular basis of inhibition of acid sensing ion channel 1A by diminazene.

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation permeable ion channels expressed primarily in neurons. Here we employed site-directed mutagenesis and electrophysiology to investigate the mechanism of inhibition of ASIC1a by diminazene. This compound inhibits mouse ASIC1a with a half-maximal inhibitory concentration (IC50) of 2.4 µM. At first, we examined whether neutralizing mutations of Glu79 and Glu416 alter diminazene block. These residues form a hexagonal array in the lower palm domain that was previously shown to contribute to pore opening in response to extracellular acidification. Significantly, single Gln substitutions at positions 79 and 416 in ASIC1a reduced diminazene apparent affinity by 6-7 fold. This result suggests that diminazene inhibits ASIC1a in part by limiting conformational rearrangement in the lower palm domain. Because diminazene is charged at physiological pHs, we assessed whether it inhibits ASIC1a by blocking the ion channel pore. Consistent with the notion that diminazene binds to a site within the membrane electric field, diminazene block showed a strong dependence with the membrane potential. Moreover, a Gly to Ala mutation at position 438, in the ion conduction pathway of ASIC1a, increased diminazene IC50 by one order of magnitude and eliminated the voltage dependence of block. Taken together, our results indicate that the inhibition of ASIC1a by diminazene involves both allosteric modulation and blocking of ion flow through the conduction pathway. Our findings provide a foundation for the development of more selective and potent ASIC pore blockers.

The Thumb Domain Mediates Acid-sensing Ion Channel Desensitization.

Acid-sensing ion channels (ASICs) are cation-selective proton-gated channels expressed in neurons that participate in diverse physiological processes, including nociception, synaptic plasticity, learning, and memory. ASIC subunits contain intracellular N and C termini, two transmembrane domains that constitute the pore, and a large extracellular loop with defined domains termed the finger, ß-ball, thumb, palm, and knuckle. Here we examined the contribution of the finger, ß-ball, and thumb domains to activation and desensitization through the analysis of chimeras and the assessment of the effect of covalent modification of introduced Cys at the domain-domain interfaces. Our studies with ASIC1a-ASIC2a chimeras showed that swapping the thumb domain between subunits results in faster channel desensitization. Likewise, the covalent modification of Cys residues at selected positions in the ß-ball-thumb interface accelerates the desensitization of the mutant channels. Studies of accessibility with thiol-reactive reagents revealed that the ß-ball and thumb domains reside apart in the resting state but that they become closer to each other in response to extracellular acidification. We propose that the thumb domain moves upon continuous exposure to an acidic extracellular milieu, assisting with the closing of the pore during channel desensitization.

Conformational Fine-Tuning of Pore-Forming Peptide Potency and Selectivity.

To better understand the sequence-structure-function relationships that control the activity and selectivity of membrane-permeabilizing peptides, we screened a peptide library, based on the archetypal pore-former melittin, for loss-of-function variants. This was accomplished by assaying library members for failure to cause leakage of entrapped contents from synthetic lipid vesicles at a peptide-to-lipid ratio of 1:20, 10-fold higher than the concentration at which melittin efficiently permeabilizes the same vesicles. Surprisingly, about one-third of the library members are inactive under these conditions. In the negative peptides, two changes of hydrophobic residues to glycine were especially abundant. We show that loss-of-function activity can be completely recapitulated by a single-residue change of the leucine at position 16 to glycine. Unlike the potently cytolytic melittin, the loss-of-function peptides, including the single-site variant, are essentially inactive against phosphatidylcholine vesicles and multiple types of eukaryotic cells. Loss of function is shown to result from a shift in the binding-folding equilibrium away from the active, bound, α-helical state toward the inactive, unbound, random-coil state. Accordingly, the addition of anionic lipids to synthetic lipid vesicles restored binding, α-helical secondary structure, and potent activity of the "negative" peptides. While nontoxic to mammalian cells, the single-site variant has potent bactericidal activity, consistent with the anionic nature of bacterial membranes. The results show that conformational fine-tuning of helical pore-forming peptides is a powerful way to modulate their activity and selectivity.

Toward the de novo design of antimicrobial peptides: Lack of correlation between peptide permeabilization of lipid vesicles and antimicrobial, cytolytic, or cytotoxic activity in living cells.

We previously performed a lipid vesicle-based, high-throughput screen on a 26-residue combinatorial peptide library that was designed de novo to yield membrane-permeabilizing peptides that fold into ß-sheets. The most active and soluble library members that were identified permeabilized lipid vesicles detectably, but not with high potency. Nonetheless, they were broad-spectrum, membrane-permeabilizing antibiotics with minimum sterilizing activity at low µM concentrations. In an expansion of that work, we recently performed an iterative screen in which an active consensus sequence from that first-generation library was used as a template to design a second-generation library which was then screened against lipid vesicles at very high stringency. Compared to the consensus sequence from the first library, the most active second-generation peptides are highly potent, equilibrium pore-formers in synthetic lipid vesicles. Here, we use these first- and second-generation families of peptides to test the hypothesis that a large increase in potency in bacteria-like lipid vesicles will correlate with a large improvement in antimicrobial activity. The results do not support the hypothesis. Despite a 20-fold increase in potency against bacteria-like lipid vesicles, the second-generation peptides are only slightly more active against bacteria, and at the same time, are also more toxic against mammalian cells. The results suggest that a "pipeline" strategy toward the optimization of antimicrobial peptides could begin with a vesicle-based screen for identifying families with broad-spectrum activity, but will also need to include screening or optimization steps that are done under conditions that are more directly relevant to possible therapeutic applications.

Independent contribution of extracellular proton binding sites to ASIC1a activation.

Acid-sensing ion channels (ASICs) are a group of trimeric cation permeable channels gated by extracellular protons that are mainly expressed in the nervous system. Despite the structural information available for ASIC1, there is limited understanding of the molecular mechanism that allows these channels to sense and respond to drops in extracellular pH. In this report, we employed the substituted cysteine accessibility method and site-directed mutagenesis to examine the mechanism of activation of ASIC1a by extracellular protons. We found that the modification of E238C and D345C channels by MTSET reduced proton apparent affinity for activation. Furthermore, the introduction of positively charged residues at position 345 rendered shifted biphasic proton activation curves. Likewise, channels bearing mutations at positions 79 and 416 in the palm domain of the channel showed reduced proton apparent affinity and biphasic proton activation curves. Of significance, the effect of the mutations at positions 79 and 345 on channel activation was additive. E79K-D345K required a change to a pH lower than 2 for maximal activation. In summary, this study provides direct evidence for the presence of two distinct proton coordination sites in the extracellular region of ASIC1a, which jointly facilitate pore opening in response to extracellular acidification.

Synthetic molecular evolution of pore-forming peptides by iterative combinatorial library screening.

We previously reported the de novo design of a combinatorial peptide library that was subjected to high-throughput screening to identify membrane-permeabilizing antimicrobial peptides that have ß-sheet-like secondary structure. Those peptides do not form discrete pores in membranes but instead partition into membrane interfaces and cause transient permeabilization by membrane disruption, but only when present at high concentration. In this work, we used a consensus sequence from that initial screen as a template to design an iterative, second generation library. In the 24-26-residue, 16,200-member second generation library we varied six residues. Two diad repeat motifs of alternating polar and nonpolar amino acids were preserved to maintain a propensity for non-helical secondary structure. We used a new high-throughput assay to identify members that self-assemble into equilibrium pores in synthetic lipid bilayers. This screen was done at a very stringent peptide to lipid ratio of 1:1000 where most known membrane-permeabilizing peptides, including the template peptide, are not active. In a screen of 10,000 library members we identified 16 (~0.2%) that are equilibrium pore-formers at this high stringency. These rare and highly active peptides, which share a common sequence motif, are as potent as the most active pore-forming peptides known. Furthermore, they are not α-helical, which makes them unusual, as most of the highly potent pore-forming peptides are amphipathic α-helices. Here we demonstrate that this synthetic molecular evolution-based approach, taken together with the new high-throughput tools we have developed, enables the identification, refinement, and optimization of unique membrane active peptides.

Gain-of-function analogues of the pore-forming peptide melittin selected by orthogonal high-throughput screening.

We recently developed an orthogonal, high-throughput assay to identify peptides that self-assemble into potent, equilibrium pores in synthetic lipid bilayers. Here, we use this assay as a high-throughput screen to select highly potent pore-forming peptides from a 7776-member rational combinatorial peptide library based on the sequence of the natural pore-forming peptide toxin melittin. In the library we varied ten critical residues in the melittin sequence, chosen to test specific structural hypotheses about the mechanism of pore formation. Using the new high-throughput assay, we screened the library for gain-of-function sequences at a peptide to lipid ratio of 1:1000 where native melittin is not active. More than 99% of the library sequences were also inactive under these conditions. A small number of library members (0.1%) were highly active. From these we identified 14 potent, gain-of-function, pore-forming sequences. These sequences differed from melittin in only 2-6 amino acids out of 26. Some native residues were highly conserved and others were consistently changed. The two factors that were essential for gain-of-function were the preservation of melittin's proline-dependent break in the middle of the helix and the improvement and extension the amphipathic nature of the α-helix. In particular the highly cationic carboxyl-terminal sequence of melittin, is consistently changed in the gain-of-function variants to a sequence that it is capable of participating in an extended amphipathic α-helix. The most potent variants reside in a membrane-spanning orientation, in contrast to the parent melittin, which is predominantly surface bound. This structural information, taken together with the high-throughput tools developed for this work, enable the identification, refinement and optimization of pore-forming peptides for many potential applications.

Interactions of membrane active peptides with planar supported bilayers: an impedance spectroscopy study.

Membrane active peptides exert their biological effects by interacting directly with a cell's lipid bilayer membrane. These therapeutically promising peptides have demonstrated a variety of activities including antimicrobial, cytolytic, membrane translocating, and cell penetrating activities. Here, we use electrochemical impedance spectroscopy (EIS) on polymer-cushioned supported lipid bilayers constructed on single crystal silicon to study two pairs of closely related membrane active peptides selected from rationally designed, combinatorial libraries to have different activities in lipid bilayers: translocation, permeabilization, or no activity. Using EIS, we observed that binding of a membrane translocating peptide to the lipid bilayer resulted in a small decrease in membrane resistance followed by a recovery back to the original value. The recovery may be directly attributable to peptide translocation. A nontranslocating peptide did not decrease the resistance. The other pair, two membrane permeabilizing peptides, caused an exponential decrease of membrane resistance in a concentration-dependent manner. This permeabilization of the supported bilayer occurs at peptide to lipid ratios as much as 1000-fold lower than that needed to observe effects in vesicle leakage assays and gives new insights into the fundamental peptide-bilayer interactions involved in membrane permeabilization.

Determining the mechanism of membrane permeabilizing peptides: identification of potent, equilibrium pore-formers.

To enable selection and characterization of highly potent pore-forming peptides, we developed a set of novel assays to probe 1) the potency of peptide pores at very low peptide concentration; 2) the presence or absence of pores in membranes after equilibration; 3) the interbilayer exchangeability of pore-forming peptides; and 4) the degree to which pore-forming peptides disrupt the bilayer organization at equilibrium. Here, we use these assays to characterize, in parallel, six membrane-permeabilizing peptides belonging to multiple classes. We tested the antimicrobial peptides LL37 and dermaseptin S1, the well-known natural lytic peptides melittin and alamethicin, and the very potent lentivirus lytic peptides LLP1 and LLP2 from the cytoplasmic domain of HIV GP41. The assays verified that that the antimicrobial peptides are not potent pore formers, and form only transient permeabilization pathways in bilayers which are not detectable at equilibrium. The other peptides are far more potent and form pores that are still detectable in vesicles after many hours. Among the peptides studies, alamethicin is unique in that it is very potent, readily exchanges between vesicles, and disturbs the local bilayer structure even at very low concentration. The equally potent LLP peptides do not exchange readily and do not perturb the bilayer at equilibrium. Comparison of these classes of pore forming peptides in parallel using the set of assays we developed demonstrates our ability to detect differences in their mechanism of action. Importantly, these assays will be very useful in high-throughput screening where highly potent pore-forming peptides can be selected based on their mechanism of action.