Multidisciplinary approaches to combat emerging viruses: diagnostics, therapeutic gene and vaccine delivery, and nanotherapeutics.

Emerging viruses, such as filoviruses (Ebola, Marburg), SARS and MERS coronaviruses, and Zika, pose significant threats to global public health, particularly for individuals with co-morbidities. To address these challenges, this review article explores multidisciplinary strategies for combatting emerging viruses. We emphasize the importance of developing accurate diagnostics, innovative therapeutic gene and vaccine delivery systems, and long-acting nanotherapeutics. These approaches are designed to enhance the safety and efficacy of treatments against these deadly pathogens. We discuss the collaborative efforts of virologists, geneticists, formulation scientists, clinicians, immunologists, and medicinal chemists in advancing these therapeutic modalities.

A Comprehensive Review of Monoclonal Antibodies in Modern Medicine: Tracing the Evolution of a Revolutionary Therapeutic Approach.

Monoclonal antibodies (mAbs) have emerged as potent therapeutic agents, revolutionizing the landscape of modern medicine. This comprehensive review traces the evolution of mAbs from their inception to their current prominence, highlighting key milestones in their development and exploring their diverse therapeutic applications. Beginning with an overview of their molecular structure and mechanisms of action, we delve into the production and engineering of mAbs, including hybridoma technology and recombinant DNA techniques. Therapeutic applications across various medical disciplines, including cancer treatment, autoimmune diseases, and infectious diseases, are examined in detail, showcasing the significant clinical successes of mAbs. Furthermore, this review discusses the challenges and opportunities in manufacturing scalability, cost-effectiveness, and access to therapies. Looking ahead, the implications of mAbs in future research and clinical practice are explored, emphasizing the potential for next-generation mAbs, personalized medicine, and integration with emerging modalities such as immunotherapy and gene therapy. In conclusion, the evolution of monoclonal antibodies underscores their transformative impact on healthcare and their continued promise to advance the frontiers of medicine.

A Risk-Difference Meta-Analysis for the Prophylactic Treatments of Chronic Migraine.

Chronic migraine (CM) imposes significant personal, societal, and financial burdens, historically lacking specific prophylactic treatments. Monoclonal antibodies (mAbs) targeting calcitonin gene-related peptide (CGRP) represent a novel, mechanism-based, and migraine-specific prophylactic approach. Four mAbs, namely, erenumab, fremanezumab, galcanezumab, and eptinezumab, have been marketed, although head-to-head trials with standard anti-migraine treatments are absent. This study aimed to compare the efficacy and safety of anti-CGRP mAbs with standard anti-migraine treatments using a cross-trial indirect model of the absolute risk difference (ARD) of a 50% responder rate, in order to express the final results in terms of the number needed to treat (NNT) and number needed to harm (NNH). Phase 3 and 2b randomized controlled trials (RCTs) for CM prophylaxis were searched in the MEDLINE and CENTRAL databases with specific inclusion and exclusion criteria. The ARD between groups for the percentage of trial participants who reported a 50% reduction in monthly migraine days and the differences in the number of adverse events (AEs), serious adverse events (SAEs), and participants who withdrew from each RCT were calculated, and subsequently, the NNT and NNH were calculated for each one of the outcome measures. In total, eight RCTs were considered eligible. A similar efficacy and safety have been demonstrated among CGRP mAbs and all standard CM treatments. The results of the ARD for the total number of studies concerning efficacy, total adverse events, serious adverse events, and dropout from the RCTs ranged from -0.688 (95% confidence interval (CI): -0.821-(-0.513)) to -0.018 (95% CI: -0.044-(0.007)), from 0.032 (95% CI: -0.041, 0.104) to -0.380 (95% CI: -0.589, -0.126), from -0.025 (95% CI: -0.046, -0.006) to 0.014 (95% CI: -0.015, 0.42), from 0.048 (95% CI: -0.112, 0.014) to 0.232 (95% CI: -0.016, 0.458) correspondingly. All anti-CGRP mAbs showed a roughly equal statistically significant ARD and similar NNTs, ranging from 5 to 8, while the ARD of onbotulinum toxin A (oBTA) was not significant with an NNT 56. The two studies of topiramate showed contradictory results, the one significant while the other not, with NNTs 2 and 22, respectively. All four anti-CGRP mAbs showed an invariably high efficacy among their studies, in terms of the ARD and its derivative measure of NNT, in contrast to oBTA, while in topiramate, the results are contradictory between the two studies.

Development of a colloidal gold immunochromatographic strip for the simultaneous detection of porcine epidemic diarrhea virus and transmissible gastroenteritis virus.

In recent years, porcine diarrhea-associated viruses have caused significant economic losses globally. These viruses present similar clinical symptoms, such as watery diarrhea, dehydration, and vomiting. Co-infections with porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are common. For the rapid and on-site preliminary diagnosis on the pig farms, this study aimed to develop a colloidal gold immunochromatography assay (GICA) strip for the detection of PEDV and TGEV simultaneously. The GICA kit showed that there was no cross-reactivity with the other five common porcine viruses. With visual observation, the lower limits were approximately 104 TCID50/mL and 104 TCID50/mL for PEDV and TGEV, respectively. The GICA strip could be stored at 4°C or 25°C for 12 months without affecting its efficacy. To validate the GICA strip, 121 clinical samples were tested. The positive rates of PEDV and TGEV were 42.9 and 9.9%, respectively, and the co-infection rate of the two viruses was 5.8% based on the duplex GICA strip. Thus, the established GICA strip is a rapid, specific, and stable tool for on-site preliminary diagnosis of PEDV- and TGEV-associated diarrhea.

New and emerging drug therapies for Alzheimer disease.

Established drug therapies for Alzheimer disease (cholinesterase inhibitors and memantine) do not modify the disease course and provide only modest clinical benefit. Biomarker measures of amyloid, tau and neurodegeneration have been integral to Alzheimer disease clinical trials for biologic drugs, for patient selection and efficacy monitoring. At the time of writing, two monoclonal antibodies targeting the amyloid-beta protein (aducanumab and lecanemab) have been approved in the USA, and two agents (lecanemab and donanemab) are under evaluation by the Therapeutic Goods Administration in Australia. Clinical trials have demonstrated that monoclonal antibodies are effective at removing amyloid from the brain in people with early Alzheimer disease. Cognitive benefits are statistically significant, but do not achieve the minimal clinically important difference. Amyloid-related imaging abnormalities of vasogenic oedema and microhaemorrhages occur more frequently on treatment; although these are usually asymptomatic or transient, in some people they are serious or fatal. Targeting amyloid as a unimodal strategy is unlikely to be sufficient and future therapies may need to be multimodal, targeting multiple pathogenic pathways. The burden of dementia is greatest in the older population where mixed dementia pathology dominates; the relationship between biomarkers, clinical phenotype and pathology attenuates; and frailty and comorbidity impact cognition. This creates challenges in identifying effective therapies for the group where dementia is most prevalent.

Classification of anticancer drugs: an update with FDA- and EMA-approved drugs.

Anticancer systemic therapy comprises a complex and growing group of drugs. Some of the new agents with novel mechanisms of action that have appeared are difficult to fit in the groups of classical chemotherapy, hormones, tyrosine-kinase inhibitors, and monoclonal antibodies. We propose a classification based on two levels of information: the site of action and the mechanism of action. Regarding the former, drugs can exert their action in the tumor cell, the tumor vasculature, the immune system, or the endocrine system. The mechanism of action refers to the molecular target.

The efficacy of antivirals, corticosteroids, and mAbs as acute COVID treatments in reducing the incidence of long COVID: a Systematic Review and meta-analysis.

BACKGROUND: Whether treatment during acute COVID results in protective efficacy against long COVID incidence remains unclear. OBJECTIVES: To assess the relationship between acute COVID treatments of antivirals, corticosteroids, and monoclonal antibodies (mAbs) and long COVID incidence, and their effects in different populations and individual symptoms. DATA SOURCES: Searches were conducted up to Jan 29, 2024 in PubMed, Medline, Web of Science, and Embase. STUDY ELIGIBILITY CRITERIA: Articles that reported long COVID incidence post-acute COVID with a follow-up of at least 30 days with no language restrictions. PARTICIPANTS: Patients with a COVID-19 diagnosis history. INTERVENTIONS: Patients treated with antivirals, corticosteroids or mAbs. ASSESSMENT OF RISK OF BIAS: Quality assessment was based on Newcastle-Ottawa scale, ROBINS-I and Cochrane risk of bias tool. METHODS OF DATA SYNTHESIS: Basic characteristics were documented for each study. Random forest model and meta-regression was used to evaluate correlation between treatments and long COVID. RESULTS: Our search identified 2363 records, 32 of which were included in the qualitative synthesis and 25 included into the meta-analysis. Effect size from 14 papers investigating acute COVID antiviral treatment concluded its protective efficacy against long COVID (OR 0.61, 95% CI: 0.48-0.79, p = 0.0002); however, corticosteroid (OR 1.57, 95% CI: 0.80-3.09, p = 0.1913) and mAbs treatments (OR 0.94, 95% CI: 0.56-1.56, p = 0.8012) did not generate such effect. Subsequent subgroup analysis revealed that antivirals provided stronger protection in the aged, male, unvaccinated and non-diabetic populations. Furthermore, antivirals effectively reduced eight out of the twenty-two analyzed long COVID symptoms. DISCUSSION: Our meta-analysis determined that antivirals reduced long covid incidence across populations and should thus be recommended for acute COVID treatment. There was no relationship between mAbs treatment and long COVID, but studies should be conducted to clarify acute COVID corticosteroids' potential harmful effects on the post-acute phase of COVID.

Production and evaluation of anti-BP26 monoclonal antibodies for the serological detection of animal brucellosis.

Brucella BP26 proves to be a highly immunogenic antigen with excellent specificity in brucellosis detection. In China, the authorized use of the Bp26-deleted vaccine M5ΔBP26 for preventing small ruminant brucellosis highlights the importance of developing accurate detection methods targeting BP26, particularly for the diagnosis of differentiation between infected and vaccinated animals (DIVA). Using the traditional mouse hybridoma technique, we successfully obtained 12 monoclonal antibodies (mAbs) targeting BP26. The efficacy of these mAbs in detecting various animal brucellosis cases using the competitive ELISA method was evaluated. Among them, only the E10 mAb exhibited significant efficiency, being inhibited by 100, 97.62, and 100% of brucellosis-positive sera from cattle, small ruminants, and canines, respectively. The E10-based competitive enzyme-linked immunosorbent assay (cELISA) outperformed the BP26-based indirect enzyme-linked immunosorbent assay (iELISA) in accuracy, particularly for cattle and small ruminant brucellosis, with cELISA sensitivity reaching 97.62% compared to 64.29% for iELISA for small ruminants. Although cELISA showed slightly lower specificity than iELISA, it still maintained high accuracy in canine brucellosis detection. The epitope of mAb E10 was identified in the amino acid sequence QPIYVYPDDKNNLKEPTITGY, suggesting its potential as a diagnostic antigen for brucellosis. In conclusion, the E10-based cELISA presents an effective means of detecting animal brucellosis, particularly significant for DIVA diagnosis in China, where the BP26-mutant vaccine is widely used.

Patient-Reported Outcomes in COVID-19 Treatment with Monoclonal Antibodies Reveal Benefits in Return to Usual Activities.

INTRODUCTION: This study aimed to assess the effects of a monoclonal antibody (mAb) combination on symptoms, daily function, and overall health-related quality of life. METHODS: We analyzed patient-reported outcomes data from symptomatic outpatients in a phase 1/2/3 trial. Patients with confirmed SARS-CoV-2 infection and ≥ 1 risk factor for severe COVID-19 received mAb treatment (casirivimab plus imdevimab 1200 mg) or placebo. Prespecified exploratory assessments included time to sustained symptoms resolution, usual health, and return to usual activities (assessed daily for 29 days). The trial was conducted from September 2020 to February 2021, prior to widespread COVID-19 vaccination programs and Omicron-lineage variants against which casirivimab + imdevimab is not active. RESULTS: In this analysis 736 outpatients received mAb and 1341 received placebo. Median time to sustained symptoms resolution was consistently shorter with mAb versus placebo (≥ 2 consecutive days: 14 vs 17 days, [nominal p = 0.0017]; ≥ 3 consecutive days: 17 vs 21 days, [nominal p = 0.0046]). Median time to sustained return to usual health and usual activities were both consistently shorter with mAb versus placebo (≥ 2 consecutive days: 12 vs 15 days [nominal p = 0.0001] and 9 vs 11 days [nominal p = 0.0001], respectively; ≥ 3 consecutive days: 14 vs 18 days [nominal p = 0.0003] and 10 vs 13 days [nominal p = 0.0041], respectively). CONCLUSIONS: mAb treatment against susceptible SARS-CoV-2 strains improved how patients feel and function, as evidenced by shortened time to sustained symptoms resolution and return to usual health and activities. Future studies are warranted to assess the patient experience with next generation mAbs. CLINICALTRIALS: GOV: Registration number, NCT04425629; Submission date June 11, 2020.

Broadening anticancer spectrum by preprocessing and treatment of T- lymphocytes expressed FcγRI and monoclonal antibodies for refractory cancers.

Background: Chimeric antigen receptor T (CAR-T) cell therapies have achieved remarkable success in the treatment of hematological tumors. However, given the distinct features of solid tumors, particularly heterogeneity, metabolic aggressiveness, and fewer immune cells in tumor microenvironment (TME), the practical utility of CAR-T cells for solid tumors remains as a challenging issue. Meanwhile, although anti-PD-1 monoclonal antibody (mAb) has shown clinical efficacy, most mAbs also show limited clinical benefits for solid tumors due mainly to the issues associated with the lack of immune cells in TME. Thus, the infiltration of targeted immunological active cells into TME could generate synergistic efficacy for mAbs. Methods: We present a combinational strategy for solid tumor treatment, which combines armored-T cells to express Fc-gamma receptor I (FcγRI) fragment on the surfaces for targeting various tumors with therapeutically useful mAbs. Choosing CD20 and HER-2 as the targets, we characterized the in vitro and in vivo efficacy and latent mechanism of the combination drug by using flow cytometry, ELISA and other methods. Results: The combination and preprocessing of armored T-cells with corresponding antibody of Rituximab and Pertuzumab exerted profound anti-tumor effects, which is demonstrated to be mediated by synergistically produced antibody-dependent cellular cytotoxicity (ADCC) effects. Meanwhile, mAb was able to carry armored-T cell by preprocessing for the infiltration to TME in cell derived xenograft (CDX) model. Conclusions: This combination strategy showed a significant increase of safety profiles from the reduction of antibody doses. More importantly, the present strategy could be a versatile tool for a broad spectrum of cancer treatment, with a simple pairing of engineered T cells and a conventional antibody.

Molecular pathways and targeted therapies in head and neck cancers pathogenesis.

The substantial heterogeneity exhibited by head and neck cancer (HNC), encompassing diverse cellular origins, anatomical locations, and etiological contributors, combined with the prevalent late-stage diagnosis, poses significant challenges for clinical management. Genomic sequencing endeavors have revealed extensive alterations in key signaling pathways that regulate cellular proliferation and survival. Initiatives to engineer therapies targeting these dysregulated pathways are underway, with several candidate molecules progressing to clinical evaluation phases, including FDA approval for agents like the EGFR-targeting monoclonal antibody cetuximab for K-RAS wild-type, EGFR-mutant HNSCC treatment. Non-coding RNAs (ncRNAs), owing to their enhanced stability in biological fluids and their important roles in intracellular and intercellular signaling within HNC contexts, are now recognized as potent biomarkers for disease management, catalyzing further refined diagnostic and therapeutic strategies, edging closer to the personalized medicine desideratum. Enhanced comprehension of the genomic and immunological landscapes characteristic of HNC is anticipated to facilitate a more rigorous assessment of targeted therapies benefits and limitations, optimize their clinical deployment, and foster innovative advancements in treatment approaches. This review presents an update on the molecular mechanisms and mutational spectrum of HNC driving the oncogenesis of head and neck malignancies and explores their implications for advancing diagnostic methodologies and precision therapeutics.

Harnessing natural killer cells for refractory/relapsed non-Hodgkin lymphoma: biological roles, clinical trials, and future prospective.

Non-Hodgkin lymphomas (NHLs) are heterogeneous and are among the most common hematological malignancies worldwide. Despite the advances in the treatment of patients with NHLs, relapse or resistance to treatment is anticipated in several patients. Therefore, novel therapeutic approaches are needed. Recently, natural killer (NK) cell-based immunotherapy alone or in combination with monoclonal antibodies, chimeric antigen receptors, or bispecific killer engagers have been applied in many investigations for NHL treatment. The functional defects of NK cells and the ability of cancerous cells to escape NK cell-mediated cytotoxicity within the tumor microenvironment of NHLs, as well as the beneficial results from previous studies in the context of NK cell-based immunotherapy in NHLs, direct our attention to this therapeutic strategy. This review aims to summarize clinical studies focusing on the applications of NK cells in the immunotherapy of patients with NHL.

The new frontier in CHO cell line development: From random to targeted transgene integration technologies.

Cell line development represents a crucial step in the development process of a therapeutic glycoprotein. Chinese hamster ovary (CHO) cells are the most frequently employed mammalian host cell system for the industrial manufacturing of biologics. The predominant application of CHO cells for heterologous recombinant protein expression lies in the relative simplicity of stably introducing ectopic DNA into the CHO host cell genome. Since CHO cells were first used as expression host for the industrial production of biologics in the late 1980s, stable genomic transgene integration has been achieved almost exclusively by random integration. Since then, random transgene integration had become the gold standard for generating stable CHO production cell lines due to a lack of viable alternatives. However, it was eventually demonstrated that this approach poses significant challenges on the cell line development process such as an increased risk of inducing cell line instability. In recent years, significant discoveries of new and highly potent (semi)-targeted transgene integration systems have paved the way for a technological revolution in the cell line development sector. These advanced methodologies comprise the application of transposase-, recombinase- or Cas9 nuclease-mediated site-specific genomic integration techniques, which enable a scarless transfer of the transgene expression cassette into transcriptionally active loci within the host cell genome. This review summarizes recent advancements in the field of transgene integration technologies for CHO cell line development and compare them to the established random integration approach. Moreover, advantages and limitations of (semi)-targeted integration techniques are discussed, and benefits and opportunities for the biopharmaceutical industry are outlined.

Monoclonal antibodies targeting interleukins for systemic lupus erythematosus: updates in early clinical drug development.

INTRODUCTION: The advent of biological therapies has already revolutionized treatment strategies and disease course of several rheumatologic conditions, and monoclonal antibodies (mAbs) targeting cytokines and interleukins represent a considerable portion of this family of drugs. In systemic lupus erythematosus (SLE) dysregulation of different cytokine and interleukin-related pathways have been linked to disease development and perpetration, offering palatable therapeutic targets addressable via such mAbs. AREAS COVERED: In this review, we provide an overview of the different biological therapies under development targeting cytokines and interleukins, with a focus on mAbs, while providing the rationale behind their choice as therapeutic targets and analyzing the scientific evidence linking them to SLE pathogenesis. EXPERT OPINION: An unprecedented number of clinical trials on biological drugs targeting different immunological pathways are ongoing in SLE. Their success might allow us to tackle present challenges of SLE management, including the overuse of glucocorticoids in daily clinical practice, as well as SLE heterogenicity in treatment response among different individuals, hopefully paving the way toward precision medicine.

Belimumab concentration measurements using a homologous anti-idiotype immunoassay.

Monitoring belimumab concentrations in patients can be a valuable tool for assessing treatment response and for personalizing drug doses. Various assay formats may be used to measure concentrations of therapeutic monoclonal antibodies. A particularly useful format involves the use of anti-idiotype monoclonal antibodies, selected to be highly specific to the antibody of interest. Here, we describe the development of a specific, high-affinity anti-idiotype antibody to belimumab, and the application of this antibody in a homologous sandwich ELISA to measure belimumab concentrations.

The Effect of Calcitonin Gene-Related Peptide Monoclonal Antibodies on Quality of Life among Migraine Patients: Pilot Study at the Hospital of Lithuanian University of Health Sciences Kaunas Clinics.

Background: Migraine has a negative impact on patients' quality of life, with the frequency of attacks being associated with greater disability and poorer health status. Frequent migraine-type headaches require prophylactic treatment, which has so far been of limited effectiveness until advent of calcitonin gene-related peptide (CGRP) monoclonal antibody. Materials and Methods: A prospective analysis was conducted of data from 41 migraine patients who experienced 4 or more monthly migraine days (MMD) longer than three months. At the beginning of the study, treatment with monoclonal antibodies against CGRP (fremanezumab 225 mg or erenumab 70 or 140 g per month) was prescribed according to the indications. The effect of the medications was evaluated after 3-month period. Results: The mean age of patients was 37.17 (±11.78) years. It was found that 17 patients (41.5%) had episodic migraine (EM) and 24 (58.5%) had chronic migraine (CM). Fremanezumab was prescribed to 26 patients (63.4%) and erenumab to 15 patients (36.6%); among the latter, 13 patients used 70 mg/month and 2 patients used 140 mg/month. Three months after treatment, CM changed to EM for 19 patients (79.2%), 27 patients (65.9%) had ≥50% reduction in the number of MMD and total migraine disability assessment (MIDAS) score was reduced by >50% in 31 patients (75.6%). Also, all areas of quality of life of patients were improved after 3 months continued treatment compared to baseline. Conclusions: For more than half the patients using fremanezumab or erenumab after 3-month period, MMD decreased by ≥50% and total MIDAS score by >50 points. All areas of quality of life were improved after prophylactic treatment of migraine.

Harnessing the potential of microalgae for the production of monoclonal antibodies and other recombinant proteins.

Monoclonal antibodies (mAbs) have become indispensable tools in various fields, from research to therapeutics, diagnostics, and industries. However, their production, primarily in mammalian cell culture systems, is cost-intensive and resource-demanding. Microalgae, diverse photosynthetic microorganisms, are gaining attention as a favorable option for manufacturing mAbs and various other recombinant proteins. This review explores the potential of microalgae as a robust expression system for biomanufacturing high-value proteins. It also highlights the diversity of microalgae species suitable for recombinant protein. Nuclear and chloroplast genomes of some microalgae have been engineered to express mAbs and other valuable proteins. Codon optimization, vector construction, and other genetic engineering techniques have significantly improved recombinant protein expression in microalgae. These accomplishments demonstrate the potential of microalgae for biopharmaceutical manufacturing. Microalgal biotechnology holds promise for revolutionizing the production of mAbs and other therapeutic proteins, offering a sustainable and cost-effective solution to address critical healthcare needs.

In-line fiber optical sensor for detection of IgG aggregates in affinity chromatography.

Therapeutic monoclonal antibodies (mAbs) are critical for treatment of a wide range of diseases. Immunoglobulin G (IgG) is the most predominant form of mAb but is prone to aggregation during production. Detection and removal of IgG aggregates are time-consuming and laborious. Chromatography is central for purification of biopharmaceuticals in general and essential in the production of mAbs. Protein purification systems are usually equipped with detectors for monitoring pH, UV absorbance, and conductivity, to facilitate optimization and control of the purification process. However, specific in-line detection of the target products and contaminating species, such as aggregates, is currently not possible using convectional techniques. Here we show a novel fiber optical in-line sensor, based on localized surface plasmon resonance (LSPR), for specific detection of IgG and IgG aggregates during affinity chromatography. A flow cell with a Protein A sensor chip was connected to the outlet of the affinity column connected to three different chromatography systems operating at lab scale to pilot scale. Samples containing various IgG concentrations and aggregate contents were analyzed in-line during purification on a Protein A column using both pH gradient and isocratic elution. Because of avidity effects, IgG aggregates showed slower dissociation kinetics than monomers after binding to the sensor chips. Possibilities to detect aggregate concentrations below 1 % and difference in aggregate content smaller than 0.3 % between samples were demonstrated. In-line detection of aggregates can circumvent time-consuming off-line analysis and facilitate automation and process intensification.

SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes for compact, ratio-tunable multi-gene expression.

Here, we present a method for expressing multiple open reading frames (ORFs) from single transcripts using the leaky scanning model of translation initiation. In this approach termed "stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes" (SEMPER), adjacent ORFs are translated from a single mRNA at tunable ratios determined by their order in the sequence and the strength of their translation initiation sites. We validate this approach by expressing up to three fluorescent proteins from one plasmid in two different cell lines. We then use it to encode a stoichiometrically tuned polycistronic construct encoding gas vesicle acoustic reporter genes that enables efficient formation of the multi-protein complex while minimizing cellular toxicity. We also demonstrate that SEMPER enables polycistronic expression of recombinant monoclonal antibodies from plasmid DNA and of two fluorescent proteins from single mRNAs made through in vitro transcription. Finally, we provide a probabilistic model to elucidate the mechanisms underlying SEMPER. A record of this paper's transparent peer review process is included in the supplemental information.