Protein Replacement Research Articles

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.

Endothelial and neuronal engagement by AAV-BR1 gene therapy alleviates neurological symptoms and lipid deposition in a mouse model of Niemann-Pick type C2

BackgroundPatients with the genetic disorder Niemann-Pick type C2 disease (NP-C2) suffer from lysosomal accumulation of cholesterol causing both systemic and severe neurological symptoms. In a murine NP-C2 model, otherwise successful intravenous Niemann-Pick C2 protein (NPC2) replacement therapy fails to alleviate progressive neurodegeneration as infused NPC2 cannot cross the blood–brain barrier (BBB). Genetic modification of brain endothelial cells (BECs) is thought to enable secretion of recombinant proteins thereby overcoming the restrictions of the BBB. We hypothesized that an adeno-associated virus (AAV-BR1) encoding the Npc2 gene could cure neurological symptoms in Npc2−/− mice through transduction of BECs, and possibly neurons via viral passage across the BBB.MethodsSix weeks old Npc2−/− mice were intravenously injected with the AAV-BR1-NPC2 vector. Composite phenotype scores and behavioral tests were assessed for the following 6 weeks and visually documented. Post-mortem analyses included gene expression analyses, verification of neurodegeneration in Purkinje cells, determination of NPC2 transduction in the CNS, assessment of gliosis, quantification of gangliosides, and co-detection of cholesterol with NPC2 in degenerating neurons.ResultsTreatment with the AAV-BR1-NPC2 vector improved motor functions, reduced neocortical inflammation, and preserved Purkinje cells in most of the mice, referred to as high responders. The vector exerted tropism for BECs and neurons resulting in a widespread NPC2 distribution in the brain with a concomitant reduction of cholesterol in adjacent neurons, presumably not transduced by the vector. Mass spectrometry imaging revealed distinct lipid alterations in the brains of Npc2−/− mice, with increased GM2 and GM3 ganglioside accumulation in the cerebellum and hippocampus. AAV-BR1-NPC2 treatment partially normalized these ganglioside distributions in high responders, including restoration of lipid profiles towards those of Npc2+/+ controls.ConclusionThe data suggests cross-correcting gene therapy to the brain via delivery of NPC2 from BECs and neurons.

Rational design and modular synthesis of biodegradable ionizable lipids via the Passerini reaction for mRNA delivery

The ionizable lipid component of lipid nanoparticle (LNP) formulations is essential for mRNA delivery by facilitating endosomal escape. Conventionally, these lipids are synthesized through complex, multistep chemical processes that are both time-consuming and require significant engineering. Furthermore, the development of new ionizable lipids is hindered by a limited understanding of the structure-activity relationships essential for effective mRNA delivery. In this work, we have developed a modular platform utilizing the Passerini reaction to rapidly generate large, chemically diverse libraries of biodegradable ionizable lipids. This high-throughput approach enables the systematic exploration of various lipid components-head groups, tails, and spacers-and their impacts on mRNA delivery efficiency. By investigating the hydrogen bonding potential between the lipid's head groups and the mRNA's ribose phosphate complex, we found that optimizing the methylene units between the lipid's head groups and linkages could enhance endosomal escape and, consequently, mRNA delivery efficiencies. Leveraging this insight, our platform has led to the identification of the biodegradable ionizable lipid A4B4-S3, which outperforms the current clinical benchmark, SM-102, in gene editing efficacy in mouse liver following systemic administration and demonstrates the promise for repeat-dose protein replacement treatments. This work not only offers a rapid, scalable method for ionizable lipid synthesis but also deepens our understanding of their structure-activity relationships, paving the way for more effective mRNA therapeutics.

Multiarm-Assisted Design of Dendron-like Degradable Ionizable Lipids Facilitates Systemic mRNA Delivery to the Spleen.

Lipid nanoparticles (LNPs) have emerged as pivotal vehicles for messenger RNA (mRNA) delivery to hepatocytes upon systemic administration and to antigen-presenting cells following intramuscular injection. However, achieving systemic mRNA delivery to non-hepatocytes remains challenging without the incorporation of targeting ligands such as antibodies, peptides, or small molecules. Inspired by comb-like polymeric architecture, here we utilized a multiarm-assisted design to construct a library of 270 dendron-like degradable ionizable lipids by altering the structures of amine heads and multiarmed tails for optimal mRNA delivery. Following in vitro high-throughput screening, a series of top-dendron-like LNPs with high transfection efficacy were identified. These dendron-like ionizable lipids facilitated greater mRNA delivery to the spleen in vivo compared to ionizable lipid analogs lacking dendron-like structure. Proteomic analysis of corona-LNP pellets showed enhancement of key protein clusters, suggesting potential endogenous targeting to the spleen. A lead dendron-like LNP formulation, 18-2-9b2, was further used to encapsulate Cre mRNA and demonstrated excellent genome modification in splenic macrophages, outperforming a spleen-tropic MC3/18PA LNP in the Ai14 mice model. Moreover, 18-2-9b2 LNP encapsulating therapeutic BTB domain and CNC homologue 1 (BACH1) mRNA exhibited proficient BACH1 expression and subsequent Spic downregulation in splenic red pulp macrophages (RPM) in a Spic-GFP transgene model upon intravenous administration. These results underscore the potential of dendron-like LNPs to facilitate mRNA delivery to splenic macrophages, potentially opening avenues for a range of mRNA-LNP therapeutic applications, including regenerative medicine, protein replacement, and gene editing therapies.

Fishmeal intervention after short-term novel proteins stimulates compensatory growth and affects intestinal health in largemouth bass (Micropterus salmoides).

This research utilized a compensatory growth phenomenon aimed at reducing the use of fishmeal in aquatic animal feed. However, the compensatory growth triggered by fishmeal restriction with novel protein replacement has yet to be understood. Five isonitrogenous and isolipidic diets containing different proteins were manufactured, with fishmeal serving as the control and diets containing novel proteins, i.e., Chlorella (Chlorella vulgaris), cottonseed protein concentrate, Clostridium autoethanogenum, and yellow mealworm (Tenebrio molitor), serving as the experimental diets. Largemouth bass (Micropterus salmoides) with a starting body weight of 4.73 ± 0.04g were respectively fed these five diets for the first 29days (first stage), followed by a fishmeal diet for the remaining 29days (second stage). The diet of fishmeal, Chlorella vulgaris, cottonseed protein concentrate, Clostridium autoethanogenum, and Tenebrio molitor was referred to as FM, ChM, CSM, CAP, and TM, respectively. All novel protein groups showed a 60-73% higher weight gain compared to the fishmeal group during the second phase; however, they did not reach the weight level seen in the control group. In addition, the second stage of fishmeal intervention resulted in a reduction of approximately 35% in the area of positive Alcian blue (AB) staining of intestinal tissues in the CSM group, and about 40% in the CAP group, compared to the first stage. Furthermore, the area of intestinal apoptosis in TM was enlarged after fishmeal intervention while it was decreased in other experimental groups. At day 58, gene expression analysis of the CAP group fish at the end of the trial revealed increased levels of the anti-apoptotic gene bcl-2, as well as higher expression of intestinal inflammatory cytokines il-1β, tnf-α, nf-κb p65, and il-10, compared to the FM group. In terms of the biochemical indices, the levels of catalase (CAT) and glutathione peroxidase (GSH-Px) were reduced in CSM on day 58. An assessment of potential microbial function at end of trial demonstrated that CAP could reduce the lipid metabolism and amino acid metabolism, and increased carbohydrate metabolism pathways in the largemouth bass intestine. In conclusion, both CAP and ChM groups showed promise to reduce fishmeal with respect to intestinal health rather than growth. This study should be of value to practitioners wishing to use novel proteins to reduce fishmeal via the utilization of the compensatory growth phenomenon.

Development and application of an uncapped mRNA platform

Background A novel uncapped mRNA platform was developed. Methods Five lipid nanoparticle (LNP)-encapsulated mRNA constructs were made to evaluate several aspects of our platform, including transfection efficiency and durability in vitro and in vivo and the activation of humoral and cellular immunity in several animal models. The constructs were eGFP-mRNA-LNP (for enhanced green fluorescence mRNA), Fluc-mRNA-LNP (for firefly luciferase mRNA), SδT-mRNA-LNP (for Delta strain SARS-CoV-2 spike protein trimer mRNA), gDED-mRNA-LNP (for truncated glycoprotein D mRNA coding ectodomain from herpes simplex virus type 2 (HSV2)) and gDFR-mRNA-LNP (for truncated HSV2 glycoprotein D mRNA coding amino acids 1–400). Results Quantifiable target protein expression was achieved in vitro and in vivo with eGFP- and Fluc-mRNA-LNP. SδT-mRNA-LNP, gDED-mRNA-LNP and gDFR-mRNA-LNP induced both humoral and cellular immune responses comparable to those obtained by previously reported capped mRNA-LNP constructs. Notably, SδT-mRNA-LNP elicited neutralizing antibodies in hamsters against the Omicron and Delta strains. Additionally, gDED-mRNA-LNP and gDFR-mRNA-LNP induced potent neutralizing antibodies in rabbits and mice. The mRNA constructs with uridine triphosphate (UTP) outperformed those with N1-methylpseudouridine triphosphate (N1mψTP) in the induction of antibodies via SδT-mRNA-LNP. Conclusions Our uncapped, process-simplified and economical mRNA platform may have broad utility in vaccines and protein replacement drugs. KEY MESSAGES The mRNA platform described in our paper uses internal ribosome entry site (IRES) (Rapid, Amplified, Capless and Economical, RACE; Register as BH-RACE platform) instead of caps and uridine triphosphate (UTP) instead of N1-methylpseudouridine triphosphate (N1mψTP) to synthesize mRNA. Through the self-developed packaging instrument and lipid nanoparticle (LNP) delivery system, mRNA can be expressed in cells more efficiently, quickly and economically. Particularly exciting is that potent neutralizing antibodies against Delta and Omicron real viruses were induced with the new coronavirus S protein mRNA vaccine from the BH-RACE platform.

Localized In Vivo Electro Gene Therapy (LiveGT)-Mediated Skeletal Muscle Protein Factory Reprogramming

Gene electrotransfer (GET) has gained significant momentum as a non-viral gene delivery method for various clinical applications, primarily in the cancer immunotherapy and vaccine development space. Preclinical studies have demonstrated exogenous gene delivery and expression in various tissues, including the liver, skin, cardiac muscle, and skeletal muscle. However, protein replacement applications of this technology have yet to be fully actuated. Plasmid DNA skeletal muscle delivery has been shown to maintain expression for up to 18 months. In the current study, we evaluated localized skeletal muscle delivery for protein replacement applications. We developed localized in vivo electro gene therapy (liveGT) protocols utilizing mono- and biphasic pulse sequences for localized pulse delivery directly to skeletal muscle with a custom monopolar platinum electrode. Plasmid DNA encoding human insulin and human glucokinase were chosen for this study to evaluate the liveGT platform for protein replacement potential. Initial in vitro GET was performed in mouse myoblasts to evaluate human insulin and glucokinase co-delivery. This was followed by liveGT-mediated reporter gene delivery in the skeletal muscle of Sprague–Dawley rats for pulse sequence selection. Protein replacement potential was evaluated in healthy (non-diabetic) rats with liveGT-mediated human insulin and glucokinase co-delivery to skeletal muscle. Human and rat insulin levels were measured via ELISA over the course of 3 months. Fed-state blood glucose measurements were monitored in correlation with serum human insulin levels. LiveGT-mediated skeletal muscle reprogramming successfully produced physiological levels of human insulin in serum over the course of 3 months. Hypo- and hyperglycemic events were not observed. Therefore, liveGT is a safe and viable platform for potential protein replacement therapies.

In vivo adenine base editing ameliorates Rho-associated autosomal dominant retinitis pigmentosa.

In vivo adenine base editing ameliorates Rho-associated autosomal dominant retinitis pigmentosa.

The Historical Evolution and Significance of Multiple Sequence Alignment in Molecular Structure and Function Prediction.

Multiple sequence alignment (MSA) has evolved into a fundamental tool in the biological sciences, playing a pivotal role in predicting molecular structures and functions. With broad applications in protein and nucleic acid modeling, MSAs continue to underpin advancements across a range of disciplines. MSAs are not only foundational for traditional sequence comparison techniques but also increasingly important in the context of artificial intelligence (AI)-driven advancements. Recent breakthroughs in AI, particularly in protein and nucleic acid structure prediction, rely heavily on the accuracy and efficiency of MSAs to enhance remote homology detection and guide spatial restraints. This review traces the historical evolution of MSA, highlighting its significance in molecular structure and function prediction. We cover the methodologies used for protein monomers, protein complexes, and RNA, while also exploring emerging AI-based alternatives, such as protein language models, as complementary or replacement approaches to traditional MSAs in application tasks. By discussing the strengths, limitations, and applications of these methods, this review aims to provide researchers with valuable insights into MSA's evolving role, equipping them to make informed decisions in structural prediction research.

Growth performance, rumen development, and sensorial meat quality of goat kids fed extruded and pelleted starters

The aim of this study was to determine the effects of supplementing extruded and pelleted starter feeds on growth performance, rumen development, carcass characteristics, and sensorial meat quality of goat kids fed an all-milk protein replacer. Forty-five new-born French Alpine kids (21 males, 4.93 ± 0.58 kg; and 24 females, 4.40 ± 0.60 kg) were randomly assigned to three treatment diets, milk replacer-only (RO), milk replacer supplemented with extruded starter feed (RE), or milk replacer supplemented with pelleted starter feed (RP) in a 3 x 2 factorial arrangement. After 35 days, the final weights, weight gain, body size measurements, organ weights, and hot and cold carcass weights were greater (p < 0.05) for goat kids in the RO group compared to those in the RE and RP groups. Milk replacer intake was reduced (p = 0.000) with starter supplementation, and intake of the extruded starter was higher (p = 0.001) than that of the pelleted starter. The rumen weight was greater (p = 0.019) in the RE group than in the RO group. Rumen pH was higher (p = 0.006) and acetic acid (p = 0.008) and total VFA (p = 0.012) concentrations were lower in the RO group than in the RE and RP groups. Mesenteric fat content was higher (p = 0.002) in the RO group than in the other groups. Meat odor of the RO group was preferred (p = 0.051) by the sensorial panel. Feeding an RO diet is beneficial for goat kids sold to restaurants. For replacement kids, an extruded starter feed may improve rumen development and facilitate adaptation to post-weaning dry feed consumption.

High plant protein diet impairs growth performance and intestinal integrity in greater amberjack (Seriola dumerili): Molecular and physiological insights

High plant protein diet impairs growth performance and intestinal integrity in greater amberjack (Seriola dumerili): Molecular and physiological insights

Effects of supplementation with freeze-dried Clostridium butyricum powder after replacement of fishmeal with cottonseed protein concentrate on growth performance, immune response, and intestinal microbiota of Litopenaeus vannamei

The present study was designed to investigate the effects of supplementation with freeze-dried Clostridium butyricum (CB) powder on the growth, immune function and intestinal health of Litopenaeus vannamei after replacing fishmeal in the diet with cottonseed protein concentrate (CPC). Six treatment groups were designed, namely the control group (CON, 25% fish meal) and five alternative groups (CPC replacing 40% fishmeal protein in the control group). Based on the alternative group, 0%, 0.065%, 0.26%, 1.04%, and 4.16% of freeze-dried CB bacterial powder (4.6 × 108 CFU/g) were added, recorded as CB 0, CB 0.065, CB 0.26, CB 1.04, and CB 4.16, respectively. Each treatment had 3 replicates of 40 shrimps (0.29 ± 0.01 g) each and breeding for 8 weeks. After the experiment, serum enzyme activities, muscle amino acids, and intestinal parameters (short-chain fatty acids, digestive enzymes, gene expression, and microbiota) were tested to explore the effects of freeze-dried CB powder in shrimp aquaculture. The results showed that the CB1.04 group had the highest final body weight, weight gain rate, and specific growth ratio (P > 0.05). Freeze-dried CB powder increased the activity of serum superoxide dismutase, glutathione peroxidase, complement 3, and complement 4. Muscle tyrosine, proline, and total essential amino acids were remarkably increased in the CB 1.04 group (P < 0.05). Propionic acid levels were elevated in the CB 1.04 and CB4.16 groups (P < 0.001). The relative expression of Dorsal, Relish, and Target of Rapamycin (TOR) genes was significantly increased in the CB 1.04 group (P < 0.01). Actinobacteria and Demequina abundance was significantly higher in the CB 1.04 group (P < 0.01). The results of the Vibrio parahaemolyticus challenge test showed the highest cumulative mortality rate (43.33%) in the CB0 group and the lowest cumulative mortality rate (20%) in the CB1.04 group. This study confirmed that freeze-dried CB powder alleviated the negative effects of CPC replacement of fish meal protein in Litopenaeus vannamei, and the optimum additive level was 2.11% (9.71 × 109 CFU/kg) as indicated by binary regression analysis of specific growth ratio.

Dietary fishmeal protein replacement with superworm (Zophobas morio) larvae meal: Effect on growth, feed utilization, survival, digestive enzyme activity, skin colour enhancement of juvenile goldfish, Carassius auratus

Dietary fishmeal protein replacement with superworm (Zophobas morio) larvae meal: Effect on growth, feed utilization, survival, digestive enzyme activity, skin colour enhancement of juvenile goldfish, Carassius auratus

Development of Plant-Based Burgers with Partial Replacement of Texturized Soy Protein by Agaricus bisporus: Effects on Physicochemical and Sensory Properties.

This study aims to develop plant-based burgers with partial replacement of texturized soy protein (TSP) by Agaricus bisporus mushrooms at proportions of 5%, 10%, 15%, and 20%. The substitution was evaluated regarding its impact on the burgers' chemical composition, texture, color, cooking performance, and sensory properties. Chemical analyses showed a significant increase in moisture content starting from the 10% substitution level, contributing to improved juiciness. Protein content remained similar to the control until the 15% substitution level, while the fat content showed no significant variation among treatments. The texture profile indicated reduced hardness in burgers with mushroom enrichment, particularly at 5% and 10%, leading to a more tender product. Color analysis revealed a reduction in lightness (L*) and red intensity (a*) with increased mushroom levels. Sensory analysis showed that burgers with up to a 15% substitution level maintained consumer acceptance comparable to the control, with attributes such as "softness", "pleasant color", and "good appearance" positively correlated with consumer acceptance. The findings indicate that Agaricus bisporus mushrooms can be effectively used as a partial substitute for TSP in plant-based burgers, enhancing sensory properties without compromising quality. This substitution offers a promising approach to diversifying ingredients in plant-based products while maintaining desirable characteristics for consumers.

MRNA therapy effective treatment of rare hereditary diseases

mRNA therapy, or mRNA-based drugs that have emerged thanks to vaccines against SARS-CoV-2, have successfully passed preclinical tests and are currently at various stages of clinical trials in the treatment of many diseases, including rare metabolic disorders. In the case of rare genetic metabolic diseases, the concept of mRNA therapy can be considered as an alternative to protein replacement therapy, where exogenous mRNA leads to the production of a fully active protein instead of a non-functional one, and also delivers it to the desired cellular compartment, such as mitochondria or the cell membrane. Preclinical studies on animal models of some rare genetic diseases have fully confrmed the validity of this concept. In this mini-review, we examine and discuss the mentioned preclinical studies on efficacy and safety in several animal models. For all the diseases considered, mRNA therapy restored functional protein to therapeutically significant levels in target organs, led to stable and reproducible results after each dose of mRNA, and was well tolerated, as confirmed by functional liver tests evaluated in animal models, including non-human primates. These data convincingly confirm the potential of clinical development of mRNA therapy for the treatment of various rare metabolic disorders.

The Gut Microbiome Regulates FVIII Immune Responses

The Gut Microbiome Regulates FVIII Immune Responses

The complex pathophysiology and clinical management of epidermolysis bullosa: a comprehensive review

Epidermolysis bullosa (EB) represents a group of rare, genetically heterogeneous disorders characterized by the formation of blisters and erosions of the skin and mucous membranes in response to minor mechanical trauma. EB is classified into four major types-EB simplex, junctional EB, dystrophic EB, and Kindler syndrome, each associated with mutations in specific genes that encode structural proteins essential for skin integrity. The clinical spectrum of EB ranges from mild forms, presenting with localized skin involvement, to severe variants that lead to widespread blistering, mutilating scarring, and significant morbidity. The pathophysiology of EB is complex, involving disruptions in the adhesion between the dermis and epidermis, leading to compromised structural stability of the skin. Current therapeutic strategies focus on symptom management, including wound care, infection prevention, and pain control, as no definitive cure exists. Advances in gene therapy, stem cell therapy, and protein replacement therapy hold promise for future treatment paradigms. This review aims to elucidate the molecular underpinnings, clinical manifestations, and emerging therapeutic approaches for EB, providing a comprehensive overview for clinicians and researchers engaged in the management and study of this challenging condition.

Controlling Circular RNA Encapsulation within Extracellular Vesicles for Gene Editing and Protein Replacement.

Extracellular vesicles (EVs) are a population of vesicular bodies originating from cells, and EVs have been proven to have the potential to deliver different cargos, such as RNAs. However, conventional methods are not able to encapsulate long RNAs into EVs efficiently or may compromise the integrity of EVs. In this study, we have devised a strategy to encapsulate long circRNAs (>1000 nt) into EVs by harnessing the sorting mechanisms of cells. This strategy utilizes the inherent richness of circular RNAs in EVs and a genetic engineering method to increase the cytoplasmic concentration of target circRNAs, facilitating highly efficient RNA back-splicing to drive the circularization of RNAs. This allows target circRNAs to load into EVs with high efficiency. Furthermore, we demonstrate the practical applications of this strategy, showing that these circRNAs can be delivered by EVs to recipient cells for protein expression and to mice for gene editing.