Protein Replacement Research Articles

Rescue of the First Mitochondrial Membrane Carrier, the mPiC, by TAT-Mediated Protein Replacement Treatment

The mitochondrial phosphate carrier (mPiC), encoded by the nuclear gene SLC25A3, is synthesized with an N-terminus mitochondrial targeting sequence (MTS), enabling its import into the mitochondria. mPiC imports inorganic phosphate (Pi) into the mitochondrial matrix for ATP production and other matrix phosphorylation reactions, as well as regulates mitochondrial Ca2+ uptake and buffering of matrix Ca2+. PiC also imports copper (Cu), crucial to COX subunit holoenzyme assembly. Variants in SLC25A3 exist and lead to mPiC deficiency (MPCD), cause a rare autosomal recessive disease with no current cure; patients with MPCD usually die within the first year of life. We have developed a novel therapeutic approach using TAT-mPiC fusion protein for cellular delivery since the TAT peptide enables delivery of proteins across biological membranes. We designed, produced, and purified the TAT-mPiC fusion protein. The fusion protein is delivered into the mitochondria and localizes within the mIM, its natural cellular location, as a processed protein. Treatment of mPiC-knockdown cells with TAT-mPiC fusion protein increased cell growth and improved bioenergetic capabilities, as measured by oxygen consumption rate (OCR), ATP production, and reduction in lactate secretion. Most importantly, TAT-mPiC restored Pi and Cu delivery into the mitochondrial matrix. TAT-mPiC fusion protein also restored the mitochondrial activity of cells harboring various mitochondrial defects. This study presents the first successful delivery of a mitochondrial transmembrane carrier using the TAT-fusion system, offering a potential early treatment strategy for newborns with mPiC deficiency.

SDF-1α mRNA therapy in peripheral artery disease

Stromal cell-derived factor-1 alpha (SDF-1α) is a promising target for therapeutic angiogenesis in ischemic diseases such as peripheral artery disease (PAD). However, the clinical application of SDF-1α protein or plasmid-based gene therapy is unsuccessful. mRNA therapy has shown great promise in protein replacement. Here, we developed DOPE-lipid nanoparticles (LNPs) encapsulating SDF-1α mRNA (LNP@SDF-1α) for efficient gene delivery. In vitro, LNP@SDF-1α treatment of human umbilical vein endothelial cells (HUVECs) significantly enhanced endothelial migration, tube formation, and increased monocyte adhesion, demonstrating robust pro-angiogenic activity. In vivo, LNP@SDF-1α transfected HUVECs showed enhanced angiogenic capacity in a murine Matrigel plug model. Furthermore, in a mouse hindlimb ischemia model, intramuscular injection of LNP@SDF-1α into ischemic limbs accelerated blood flow recovery, as assessed by laser speckle contrast imaging. Immunofluorescence staining revealed a marked increase in capillary and arteriole densities in treated tissues. Angiogenic protein profiling demonstrated an upregulation of pro-angiogenic factors, including VEGF and Ang-1, and a downregulation of anti-angiogenic factors. No significant toxicity was observed in major organs, indicating the safety of this approach. Our study demonstrates that SDF-1α mRNA therapy, delivered via DOPE-LNPs, significantly promotes vascular regeneration in ischemic tissues by enhancing angiogenesis and arteriogenesis, thereby restoring blood perfusion. This approach presents a promising therapeutic option for PAD and suggests broader applications of mRNA-based therapies for ischemic diseases.

Messenger RNA Nanomedicine: Innovations and Future Directions.

With its high potential, mRNA nanomedicine has become one of the transformative frontiers of modern therapeutic strategies for treating and preventing a wide array of diseases. This review article covers recent developments in mRNA nanomedicine and its prospects in terms of innovations in drug delivery systems, stability improvements, and targeted therapeutic applications. The versatility of mRNA means that almost any protein can potentially be encoded into it, making it a powerhouse for vaccines, gene editing, and protein replacement therapies. Recent breakthroughs in nanoparticle technology have significantly enhanced mRNA molecules' delivery efficiency and stability, surmounting previous barriers concerning rapid degradation and immune system activation. It has been developed Innovations such as LNPs, polymer-based carriers, and hybrid nanocarriers have been central to the success of targeted delivery and the sustained release of mRNA. This review further underlines the potential of mRNA nanomedicine for oncological, infectious, and genetic diseases by highlighting ongoing clinical trials, emerging therapeutic paradigms, and future directions that lay much emphasis on delivery platform optimization, mRNA stability, and broadening the scope of mRNA nanomedicine therapy. With the power of emerging technologies and solving present challenges, mRNA nanomedicine has a vast potential to revolutionize the future landscape of personalized medicine and targeted therapies.

Attitudes of female carriers of X-linked hypohidrotic ectodermal dysplasia towards prenatal treatment and their decisions during a pregnancy with a male fetus

BackgroundX-linked hypohidrotic ectodermal dysplasia (XLHED) is a severe genetic disorder that may be treatable with short-term protein replacement therapy during fetal development. This is currently being investigated in a multicenter clinical trial. Affected fetuses can be identified by the number of tooth germs during a routine ultrasound scan in mid-gestation. To understand the attitudes of female XLHED carriers towards prenatal treatment and ultrasonographic screening of the fetus, we analyzed an earlier and a very recent survey among those women and the actual decisions of potential trial participants.MethodsInitial analyses were based on a self-administered survey of 167 female XLHED carriers conducted in 2011. A similar questionnaire was completed 12 years later by 72 female XLHED carriers aged 18–45 years. Subsequently, both the path to diagnosis and further decision-making of the first 33 pregnant women screened for participation in the EDELIFE trial were investigated.ResultsMost women diagnosed with XLHED considered this disease as an obstacle to having children: About one third had decided not to have children, another third would monitor their pregnancy using invasive genetic testing. In both surveys, a small number of women stated that they would consider termination of pregnancy depending on the test result. When it came to participating in the clinical trial, 80% were likely to take part (17% moderately likely, 63% very likely). Among the first pregnant women screened for this trial, 48% underwent invasive tests, while 52% relied on non-invasive tooth germ imaging for fetal XLHED diagnosis. One pregnancy with an affected fetus was terminated, another one resulted in a miscarriage, one woman declined to participate in the trial, and 12 women (80%) decided to have the affected fetuses treated.ConclusionUltrasound-based screening and prenatal treatment of the fetus are viewed positively by the vast majority of female XLHED carriers.

Associations between national plant-based vs animal-based protein supplies and age-specific mortality in human populations

Transitions to sustainable food systems require shifts in food production and availability, particularly the replacement of animal-based protein with plant-based protein. To explore how this transition may relate to demographic patterns, we undertake an ecological analysis of global associations between age-specific mortality, total national macronutrient distributions, and protein substitution. Our dataset includes per capita daily food supply and demographic data for 101 countries from 1961–2018. After adjusting for time, population size, and economic factors, we find associations between low total protein supplies and higher mortality rates across all age groups. Early-life survivorship improves with higher animal-based protein and fat supplies, while later-life survival improves with increased plant-based protein and lower fat supplies. Here, we show that the optimal balance of protein and fat in national food supplies, which correlates with minimal mortality, varies with age, suggesting that reductions in dietary protein, especially from animal sources, may need to be managed with age-specific redistributions to balance health and environmental benefits.

Preclinical evaluation of AGT mRNA replacement therapy for primary hyperoxaluria type I disease.

Primary hyperoxaluria type 1 (PH1) is a rare inherited liver disorder caused by alanine glyoxylate aminotransferase (AGT) dysfunction, leading to accumulation of glyoxylate which is then converted into oxalate. Excessive oxalate results in kidney damage due to deposition of oxalate crystals. We have developed an mRNA-based protein replacement therapy for PH1 to restore normal glyoxylate to glycine metabolism. Sequence optimized human AGT mRNA (hAGT mRNA) was encapsulated in lipopolyplex (LPP) and produced functional AGT enzyme in peroxisomes. Pharmacokinetics and pharmacodynamics (PK/PD) were evaluated in vitro and in vivo. PK demonstrated that AGT mRNA and AGT protein maintained high expression levels for up to 48 hours. A single 2 mg/kg dose in AgxtQ84-/- rats achieved a 70% reduction in urinary oxalate. Toxicological assessment identified the highest nonserious toxic dose (HNSTD) as 2 mg/kg. These findings affirm the efficacy and safety of hAGT mRNA/LPP and support its clinical application in PH1 treatment.

Current Analytical Strategies for mRNA-Based Therapeutics

Recent advancements in mRNA technology, utilized in vaccines, immunotherapies, protein replacement therapies, and genome editing, have emerged as promising and increasingly viable treatments. The rapid, potent, and transient properties of mRNA-encoded proteins make them attractive tools for the effective treatment of a variety of conditions, ranging from infectious diseases to cancer and single-gene disorders. The capability for rapid and large-scale production of mRNA therapeutics fueled the global response to the COVID-19 pandemic. For effective clinical implementation, it is crucial to deeply characterize and control important mRNA attributes such as purity/integrity, identity, structural quality features, and functionality. This implies the use of powerful and advanced analytical techniques for quality control and characterization of mRNA. Improvements in analytical techniques such as electrophoresis, chromatography, mass spectrometry, sequencing, and functionality assessments have significantly enhanced the quality and detail of information available for product and process characterization, as well as for routine stability and release testing. Here, we review the latest advancements in analytical techniques for the characterization of mRNA-based therapeutics, typically employed by the biopharmaceutical industry for eventual market release.

Alkalinity exposure induced growth inhibition, intestinal histopathological changes, and down-regulated nutrient transporter expression in Nile Tilapia: The ameliorative role of dietary camel whey protein hydrolysates.

Alkalinity exposure induced growth inhibition, intestinal histopathological changes, and down-regulated nutrient transporter expression in Nile Tilapia: The ameliorative role of dietary camel whey protein hydrolysates.

Technological breakthroughs and advancements in the application of mRNA vaccines: a comprehensive exploration and future prospects.

mRNA vaccines utilize single-stranded linear DNA as a template for in vitro transcription. The mRNA is introduced into the cytoplasm via the corresponding delivery system to express the target protein, which then performs its relevant biological function. mRNA vaccines are beneficial in various fields, including cancer vaccines, infectious disease vaccines, protein replacement therapy, and treatment of rare diseases. They offer advantages such as a simple manufacturing process, a quick development cycle, and ease of industrialization. Additionally, mRNA vaccines afford flexibility in adjusting antigen designs and combining sequences of multiple variants, thereby addressing the issue of frequent mutations in pathogenic microorganisms. This paper aims to provide an extensive review of the global development and current research status of mRNA vaccines, with a focus on immunogenicity, classification, design, delivery vector development, stability, and biomedical application. Moreover, the study highlights current challenges and offers insights into future directions for development.

Normal embryo development needs MEIG1-mediated sperm formation.

Normal embryo development is a complex process that requires normal sperm to fertilize normal oocytes. Abnormal embryogenesis can be caused by either abnormal oocytes or abnormal sperm. However, the impact of sperm-associated factors is often underappreciated. Association between defects in sperm chromatin and poor embryo development has been consistently reported. In sperm cells, most histones are replaced by protamines to remodel sperm cell chromatin. However, the mechanism of nuclear protein replacement is largely unknown. Meiosis expressed gene 1 (MEIG1) plays a unique role in male fertility. The protein is recruited to the manchette at a late stage of spermatogenesis. The manchette is a unique structure only present in male germ cells, and one of the proposed functions is replacing histones with protamines. In this study, ICSI was conducted using sperm heads from the Meig1 KO mice. Significantly reduced fertilization was observed, and few embryos developed to blastocysts, which were associated with severe sperm DNA damage. Thus, we discovered an unexpected role for MEIG1 extending beyond spermatogenesis to include a role in embryogenesis, likely through remodeling sperm chromatin.

Glucocorticoid pre-administration improves LNP-mRNA mediated protein replacement and genome editing therapies.

Glucocorticoid pre-administration improves LNP-mRNA mediated protein replacement and genome editing therapies.

Restoring hematopoietic stem and progenitor cell function in Fancc -/- mice by in situ delivery of RNA lipid nanoparticles.

Restoring hematopoietic stem and progenitor cell function in Fancc -/- mice by in situ delivery of RNA lipid nanoparticles.

Lung-targeted delivery of PTEN mRNA combined with anti-PD-1-mediated immunotherapy for In Situ lung cancer treatment.

Lung-targeted delivery of PTEN mRNA combined with anti-PD-1-mediated immunotherapy for In Situ lung cancer treatment.

Clinical development of therapeutic mRNA applications.

Clinical development of therapeutic mRNA applications.

High-density brush-shaped polymer lipids reduce anti-PEG antibody binding for repeated administration of mRNA therapeutics.

Messenger RNA lipid-nanoparticle-based therapies represent an emerging class of medicines for a variety of applications. However, anti-poly(ethylene glycol) (anti-PEG) antibodies generated by widely used PEGylated medicines and lipid nanoparticles hinder therapeutic efficacy upon repeated dosing. Here we report the chemical design, synthesis and optimization of high-density brush-shaped polymer lipids that reduce anti-PEG antibody binding to improve protein production consistency in repeated dosing. Brush-shaped polymer lipid parameters, including side chain length, degree of polymerization, anchor alkyl length and surface regimes on lipid nanoparticles modulate anti-PEG antibody binding affinity and control their blood circulation pharmacokinetics. Compared to widely used 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, lipid nanoparticles containing brush-shaped polymer lipids generate superior therapeutic outcomes in protein replacement therapy and genome editing models, reformulating structure-activity guidelines for the design of PEG lipid substitutes. Overall, these findings contribute to the general effort in the development of lipid nanoparticles with low immunogenicity to overcome current roadblocks to nucleic acid medicines.

The RNA Landscape of In Vivo-Assembled MS2 Virus-Like Particles as mRNA Carriers Reveals RNA Contamination from Host Viruses.

Virus-like particles (VLPs) are attractive systems for packaging and delivering therapeutic RNA molecules in vaccine development, protein replacement therapy, and gene editing. Different VLPs carrying target functional RNA have been biosynthesized and demonstrated for biomedical purposes. However, little attention has been paid to what other types of RNA, besides the target RNA, are encapsulated into VLPs, leading to a lack of knowledge of the landscape of RNA cargoes. In this work, we engineered the widely used MS2 VLPs to encapsulate a model cargo mRNA in yeast, with the packaging efficiency and specificity being quantitatively tuned by the copy number of packaging signals. Transcriptome sequencing of the RNA in the VLPs revealed RNA contamination from the hosts and host viruses. This study highlights the necessity of precise VLP and cargo design and a clear background of production hosts to ensure specificity and safety.

The effect of therapeutic plasma exchange on the inflammatory response in septic shock: a secondary analysis of the EXCHANGE-1 trial

BackgroundSepsis and septic shock, defined by a profound immune dysregulation, are among the leading causes of death in the intensive care unit (ICU). Despite advances in understanding the underlying pathophysiology, evidence for specific immunomodulatory treatment does not exist to date. Therapeutic plasma exchange (TPE) represents an adjunctive treatment approach to rebalance immune homeostasis. In the EXCHANGE-1 trial, we recently demonstrated a rapid hemodynamic improvement, possibly caused by the removal of harmful mediators and the replacement of protective plasma proteins. The aim of this secondary analysis is to further characterize the underlying immunomodulatory effects and to identify biomarkers that may predict treatment response.MethodsThis secondary analysis included patients in early septic shock (< 24 h duration) and a norepinephrine (NE) dose of ≥ 0.4 μg/kg/min. Patients were randomized 1:1 to receive standard of care (SOC) or SOC + one single TPE and plasma samples were collected before and after TPE. Within-group and between group effects of circulating levels of acute-phase proteins [CRP and Pentraxin3 (PTX3)], inflammatory mediators (IL-4, IL-6, IL-8, IL-10, TNF-α, IL-2Rα/CD25) and damage-associated molecular pattern (DAMP) [cell-free DNA (cfDNA)] were analyzed via paired t test or Wilcoxon signed-rank test and a mixed-effects model. Multivariate mixed‐effects modeling of NE and lactate reduction was performed to investigate if cfDNA could be associated with treatment response to TPE.ResultsTPE led to a significant reduction in circulating acute-phase protein levels (CRP p = 0.00976, PTX3 p = 0.0001). Pro-inflammatory cytokines, such as circulating TNF-α-, IL-6- und IL-8-levels, were significantly reduced in both groups with no significant difference between treatment groups except for IL-2Rα/CD25 (p ≤ 0.0001). In a multivariate mixed-effects model, rising cfDNA levels over the first 6 h indicated refractoriness to SOC treatment regarding NE (p = 0.004) and lactate (p = 0.001), whereas those receiving TPE demonstrated sustained reductions in both parameters.ConclusionsIn this secondary analysis of the EXCHANGE-1 trial adjunctive TPE is associated with the reduction of acute-phase proteins and IL-2Rα/CD25, however not with the reduction of pro-inflammatory cytokines. This phenomenon could contribute to the observed enhancement in hemodynamics among patients with septic shock. Furthermore, TPE may be particularly beneficial for patients with septic shock who exhibit rising levels of cfDNA.

IQ Survey Results on Current Industry Practices: Part 2-Quantitative Evaluations of Immunogenicity Assessment.

All biotherapeutics have the potential to induce an immunogenic response and generate anti-drug antibodies (ADAs), especially when administered as multiple doses over prolonged periods. However, a clinically meaningful effect of ADAs can be difficult to identify to communicate the impact of immunogenicity on drug exposure, safety and efficacy outcomes in product labels in a way that is useful for health care providers. The immunogenicity working Group, IQ Consortium (Clinical Pharmacology Leadership Group) has conducted a survey to understand the current practices in analyzing immunogenicity data generated during clinical development and its impact on pharmacokinetics, clinically relevant pharmacodynamic biomarkers, safety, and efficacy outcome measures. Information was collected for 93 drugs, spanning multiple drug classes and over the different phases of clinical development, including post-approval. The predominant drug classes reported included monoclonal antibodies or Fc-fusion proteins, endogenous protein replacement therapies, bispecific antibodies, and antibody-drug conjugates. The extent of quantitative evaluation varied and was influenced by several factors, including descriptive analyses, statistical approaches, and modeling. In addition to understanding current practices, this survey also highlights areas for future exploration in analyzing clinical relevance of ADAs which can facilitate the use for regulatory submissions and product labels.

Effect of dietary fishmeal protein replacement with decapsulated Artemia cysts meal on growth, survival, whole-body composition and pigmentation of Rosy barb, Puntius conchonius

Effect of dietary fishmeal protein replacement with decapsulated Artemia cysts meal on growth, survival, whole-body composition and pigmentation of Rosy barb, Puntius conchonius

Synthesis of hydrophobic-tagged 2′-deoxy-modified cap analogs and its effect on mRNA translation

Abstract Messenger RNA (mRNA) has been implemented as COVID-19 vaccines. Following their success, the application of mRNA in cancer vaccines and protein replacement therapies is highly anticipated. The translational efficiency of mRNA largely depends on the cap structure located at its 5′ end. In particular, the Cap-1 and Cap-2 structures are known to exhibit higher protein synthesis levels than the Cap-0 structure. Previously, we developed a method called the PureCap technique, which utilizes reversed-phase high-performance liquid chromatography (RP-HPLC) to purify highly capped mRNA with high precision, enabling accurate evaluation of translational activity. In this study, we synthesized cap analogs incorporating deoxyribose, and evaluated their mRNA synthesis efficiency and translational activity. Furthermore, by optimizing the transcription reaction conditions using the synthesized DNA-modified Cap series, we successfully improved the capping efficiency and mRNA yield. The deoxyadenosine-modified cap analog synthesized using the PureCap technique demonstrated 3.2-fold higher translational activity than the standard Cap-1 structure. Additionally, when using tetranucleotide cap analogs, consistent translational activity was observed, regardless of base sequence differences. This confirmed the general applicability of DNA-modified mRNA for translational activity.