Base-modified factor VIII mRNA delivery with galactosylated lipid nanoparticles as a protein replacement therapy for haemophilia A.

The bleeding disorder hemophilia A (HemA) requires systemic functional factor VIII protein infusions on prophylactic schedules. Recently, chemically modified mRNAs have emerged as promising protein replacement therapies to reduce repeated infusions and improve safety profiles. However, the influence of base modifications on mRNA translation kinetics to specific cell types remains unexplored. In this study, towards developing mRNA therapeutics for haemophilia A, we synthesized chemically modified mRNAs with commercially available base modifications of adenine, guanine, uridine, and cytidine, and evaluated in vitro transcription yield and translation kinetics in hepatic cell lines using reporter eGFP mRNA. Our findings demonstrated that mRNA with N1-methyl pseudouridine (m1Ψ) showed a 5-12-fold increase in translation efficiency in both hepatic and endothelial cell lines. As a proof of concept for developing mRNA therapy for HemA, where FVIII is deficient, we developed a m1Ψ modified functional FVIII mRNA with our liver-targeting lipid nanoparticle (Gal-LNP) system. We evaluated its delivery efficiencies in both hepatic cell lines and the HemA mouse model. The m1Ψ-FVIII mRNA showed high therapeutic efficacy up to 15 days in vivo in the HemA mouse model. Gal-LNPs were found to be safe for systemic administration. Our study reveals that incorporating m1Ψ base modifications on mRNAs could improve therapeutic efficacy in liver- and endothelial-based therapeutics. Optimized mRNA synthesis for superior expression kinetics in hepatic cells and its delivery with liver-targeted nanoparticles may emerge as protein replacement therapies for monogenic liver disorders.

Determination of dose-response calibration curves for gamma radiation using gamma-H2AX immunofluorescence based biodosimetry.

Background: Gamma-H2AX immunofluorescence assay has gained popularity as a DNA double strand break marker. In this work, we have investigated the potential use of gamma H2AX immunofluorescence assay as a biological dosimeter for estimation of dose in our institution. Materials and methods: Seven healthy individuals were selected for the study and the blood samples collected from the first five individuals were irradiated to low doses (0-10 cGy) and high doses (50-500 cGy) in a telecobalt unit. All the samples were processed for gamma-H2AX immunofluorescence assay and the dose-response calibration curves for low and high doses were determined. In order to validate the determined dose-response calibration curves, the blood samples obtained from the sixth and seventh subjects were delivered a test dose of 7.5 cGy and 250 cGy. In addition, time and cost required to complete the assay were also reported. Results: The goodness of fit (R2) values was found to be 0.9829 and 0.9766 for low and high dose-response calibration curves. The time required to perform the gamma-H2AX immunofluorescence assay was found to be 7 hours and 30 minutes and the estimated cost per sample was 5000 rupees (~ 60 USD). Conclusion: Based on this study we conclude that the individual dose-response calibration curves determined with gamma-H2AX immunofluorescence assay for both low and high dose ranges of gamma radiation can be used for biological dosimetry. Further, the gamma-H2AX immunofluorescence assay can be used as a rapid cost-effective biodosimetric tool for institutions with an existing confocal microscope facility.

An ultrastructural report of human articular cartilage resident cells in correlation with their phenotypic characteristics.

The recent discovery of progenitors based on their differential fibronectin-adhesion (FAA-CPs) and migratory-based (MCPs) assay has evoked interest due to their superiority in terms of their efficient chondrogenesis and reduced hypertrophic propensity. This study aims to isolate and enrich three articular cartilage subsets, chondrocytes, FAA-CPs, and MCPs, and compare their undifferentiated and chondrogenic differentiated status, using in-vitro phenotypical characterization in correlation with ultrastructural analysis using Transmission Electron Microscopy (TEM). Following informed consent, cartilage shavings were procured from a non-diseased human ankle joint and cultured to obtain the three subsets. Chondrocytes exhibited higher CD106 and lower CD49b and CD146 levels. Following chondrogenic differentiation, corroborative results were seen, with the MCP group showing the highest GAG/DNA ratio levels and uptake of extracellular matrix stain as compared to the FAA-CP group. TEM analysis of the chondrocytes revealed the presence of more autolytic cells with disintegrated cytoplasm and plasma membrane. The differentiated FAA-CPs and MCPs displayed higher collagen and rough endoplasmic reticulum. The results presented in this study provide novel information on the ultrastructural characteristics of cartilage resident cells, with the chondrocyte group displaying features of terminal differentiation. Both progenitor subtypes showed superiority in varied contexts, with greater collagen fibrils and greater GAG content in MCPs. The display of preferential and differentiation traits sheds insight on the necessity to enrich progenitors and coculturing them with the general pool of constituent cells to combine their advantages and reduce their drawbacks to achieve a regenerative tissue displaying genuine hyaline-like repair while limiting their terminal differentiation.

Human fetal cartilage-derived chondrocytes and chondroprogenitors display a greater commitment to chondrogenesis than adult cartilage resident cells.

Obtaining regeneration-competent cells and generating high-quality neocartilage are still challenges in articular cartilage tissue engineering. Although chondroprogenitor cells are a resident subpopulation of native cartilage and possess a high capacity for proliferation and cartilage formation, their potential for regenerative medicine has not been adequately explored. Fetal cartilage, another potential source with greater cellularity and a higher cell-matrix ratio than adult tissue, has been evaluated for sourcing cells to treat articular disorders. This study aimed to compare cartilage resident cells, namely chondrocytes, fibronectin adhesion assay-derived chondroprogenitors (FAA-CPCs) and migratory chondroprogenitors (MCPs) isolated from fetal and adult cartilage, to evaluate differences in their biological properties and their potential for cartilage repair. Following informed consent, three human fetal and three adult osteoarthritic knee joints were used to harvest the cartilage samples, from which the three cell types a) chondrocytes, b) FAA-CPCs, and MCPs were isolated. Assessment parameters consisted of flow cytometry analysis for percentage expression of cell surface markers, population doubling time and cell cycle analyses, qRT-PCR for markers of chondrogenesis and hypertrophy, trilineage differentiation potential and biochemical analysis of differentiated chondrogenic pellets for total GAG/DNA content. Compared to their adult counterparts, fetal cartilage-derived cells displayed significantly lower CD106 and higher levels of CD146 expression, indicative of their superior chondrogenic capacity. Moreover, all fetal groups demonstrated significantly higher levels of GAG/DNA ratio with enhanced uptake of collagen type 2 and GAG stains on histology. It was also noted that fetal FAA CPCs had a greater proliferative ability with significantly higher levels of the primary transcription factor SOX-9. Fetal chondrocytes and chondroprogenitors displayed a superior propensity for chondrogenesis when compared to their adult counterparts. To understand their therapeutic potential and provide an important solution to long-standing challenges in cartilage tissue engineering, focused research into its regenerative properties using in-vivo models is warranted.

Human milk oligosaccharides, milk microbiome and infant gut microbiome modulate neonatal rotavirus infection.

Neonatal rotavirus infections are predominantly asymptomatic. While an association with gastrointestinal symptoms has been described in some settings, factors influencing differences in clinical presentation are not well understood. Using multidisciplinary approaches, we show that a complex interplay between human milk oligosaccharides (HMOs), milk microbiome, and infant gut microbiome impacts neonatal rotavirus infections. Validating in vitro studies where HMOs are not decoy receptors for neonatal strain G10P[11], population studies show significantly higher levels of Lacto-N-tetraose (LNT), 2'-fucosyllactose (2'FL), and 6'-siallylactose (6'SL) in milk from mothers of rotavirus-positive neonates with gastrointestinal symptoms. Further, these HMOs correlate with abundance of Enterobacter/Klebsiella in maternal milk and infant stool. Specific HMOs also improve the infectivity of a neonatal strain-derived rotavirus vaccine. This study provides molecular and translational insight into host factors influencing neonatal rotavirus infections and identifies maternal components that could promote the performance of live, attenuated rotavirus vaccines.

Molecular analysis of the human faecal archaea in a southern Indian population.

Archaea are an important constituent of the human gut microbiota, but there is no information on human gut archaea in an Indian population. In this study, faecal samples were obtained from different age groups (neonatal babies, preschool children, school-going children, adolescents, adults and elderly) of a southern Indian population, and from a tribal population also resident in southern India). 16S rRNA gene sequences specific to Archaea were amplified from pooled faecal DNA in each group, sequenced, and aligned against the NCBI database. Of the 806 adequate sequences in the study, most aligned with 22 known sequences. There were 9 novel sequences in the present study. All sequences were deposited in the GenBank nucleotide sequence database with the following accession numbers: KF607113 - KF607918. Methanobrevibacter was the most prevalent genus among all the age groups accounting for 98% in neonates, 96% in post-weaning, and 100% each in preschool, school and adult population. In the elderly, Methanobrevibacter accounted for 96% and in tribal adults, 99% of the clones belonged to Methanobrevibacter genus. Other genera detected included Caldisphaera, Halobaculum, Methanosphaeraand Thermogymnomonas. Methanobrevibacter smithii predominated in all age groups, accounting for 749 (92.9%) of the 806 sequences. Archaea can be found in the faeces of southern Indian residents immediately after birth. Methanobrevibacter smithii was the dominant faecal archeon in all age groups, with other genera being found at the extremes of age.