Enhanced mRNA and pDNA delivery PEGylated solid lipid nanoparticles with an optimally balanced ionizable/cationic lipid content.

Non-viral gene delivery holds significant promise for the treatment of various diseases. Solid lipid nanoparticles (SLNs) are emerging as promising gene delivery vehicles due to their ease of manufacture and high stability. However, the development of efficient and safe SLNs remains a challenge.

Immune composition of the mononuclear cell fraction of human umbilical cord blood.

Despite its therapeutic potential and unique immunological properties, the immune composition of umbilical cord blood lacks consistent and comprehensive characterizations. Human umbilical cord blood (UCB) is often discarded after delivery and is difficult to obtain for research purposes. Furthermore, most research on UCB is focused on properties of CD34+ hematopoietic stem cells for transplantation.

Automated, aseptic sampling with small-volume capacity from microbioreactors for cell therapy process analysis.

Current workflows in autologous cell therapy manufacturing are reliant on manual processes that are difficult to scale out to meet patient demands. High throughput bioreactor systems that enable multiple cultures to occur in parallel can address this need, but require good bioprocess monitoring workflows to produce good quality cell therapy products. Commercial sampling systems have thus been developed for better feedback control and monitoring capabilities.

Functionally deficient UBOX5 variants and primary angle-closure glaucoma.

Primary angle-closure glaucoma is a major cause of irreversible blindness worldwide afflicting >20 million people. Through whole exome sequencing, we analysed the association between gene-based burden of rare, protein-altering genetic variants and disease risk in 4,667 affected individuals and 5,473 unaffected controls. We tested genes surpassing exome-wide significance (P < 2.

Optimizing T cell transduction: a novel transduction device for efficient and scalable gene delivery.

Background: Viral transduction is a critical step in the manufacturing of genetically modified T cells for immunotherapies, yet conventional transduction methods suffer from low to medium efficiency, high vector consumption, and limited scalability.

Methods: To address these challenges, we introduce the Transduction Boosting Device (TransB), an innovative, automated, and closed-system platform designed to enable efficient and scalable gene delivery and overcome the limitations of conventional transduction methods. TransB improves cell-virus interactions by facilitating proximity between target cells and viral vectors.

Functional expression and secretion of basic fibroblast growth factor in .

Introduction: Cultivated meat, produced by in vitro cell culture in bioreactors, offers a sustainable alternative to traditional meat sources. A significant challenge in its production is the high cost of mitogenic growth factors, which are essential supplements in serum-free media for cultivating meat cells. One strategy to reduce cost involves minimizing purification cost by using a food-grade host to secrete growth factors.

Maximising adherent cell production via customisable and dissolvable bio-polymer microcarriers.

Large-scale cellular production systems offer a significant and diverse benefit impacting the therapeutic (stem cell and vaccine production) and cellular agriculture (lab-grown meat) sectors. Producing desired cells at mass can improve production yield whilst reducing the environmental and ethical burden associated with industrialised agriculture and production of therapeutic goods. Many existing large-scale cultivation strategies of adherent cells leverage the use of microcarriers (MCs) within bioreactors.

Scalable, High-Density Expansion of Human Mesenchymal Stem Cells on Microcarriers Using the Bach Impeller in Stirred-Tank Reactors.

This paper describes the results of process developmental experiments to achieve higher cell densities in the manufacturing of hMSCs using the novel Bach impeller in a stirred-tank bioreactor. Engineering experiments have previously shown that the Bach impeller represents an efficient mixing device that suspends particles in fluids at low power inputs. To assess the impeller during biological experiments, the growth performance of Wharton Jelly (WJ)-hMSCs in a 1 L STR equipped with the Bach impeller was evaluated at a variety of culture conditions.

Functional lipid nanoparticles for safe delivery of macromolecular antibiotics to gram-negative bacteria.

Lipid nanoparticles (LNPs) hold great potential for delivery of macromolecular antimicrobials. Herein, we designed a series of anionic LNPs capable of delivering cationic polymyxin B (PMB) for effective and safe treatment of Gram-negative bacterial infection. The use of anionic lipid induced self-assembly of PMB, encapsulating cationic PMB molecules into LNPs via electrostatic interactions (PMB-LNPs).

A Targeted Integration-Based CHO Cell Platform for Simultaneous Antibody Display and Secretion.

Objective: We developed a targeted integration-based CHO cell platform for simultaneous antibody display and secretion, enabling a streamlined transition from antibody library screening to production without requiring the re-cloning of antibody genes.

Methods: The platform consists of a CHO master cell line with a single-copy landing pad, a helper vector expressing FLPe recombinase, and bi-functional targeting vectors. Recombinase-mediated cassette exchange was utilized to integrate targeting vectors into the landing pad.

Streamlined purification of recombinant Alpha-1 antitrypsin from CHO cell culture using integrated chromatography within a single unit operation.

Recombinant Alpha-1 Antitrypsin (A1AT) produced from CHO cell culture is a promising alternative to the commercially available drug, which is derived from the limited supply of blood plasma. By developing and optimizing the purification strategy through the combination of appropriate chromatography mechanisms and buffer conditions, we successfully achieved end-to-end purification of A1AT from cell culture supernatant to the final drug substance leveraging integrated chromatography within a single unit operation, with quality comparable or even superior to commercial drugs. With comparable processing time and buffer consumption, the proposed approach-integrating 2 anion exchange chromatography (AEX) and 1 hydrophobic charge induction chromatography (HCIC) -achieved 99.

Replacing PEG-Lipid with Amphiphilic Polycarbonates in mRNA-Loaded Lipid Nanoparticles: Impact of Polycarbonate Structure on Physicochemical and Transfection Properties.

Since the remarkable breakthrough of COVID-19 mRNA vaccines, lipid nanoparticles (LNPs) have gained substantial attention as the most cutting-edge clinical formulations for mRNA delivery. PEGylated lipid (PEG-lipid) has been regarded as an essential constituent of LNPs that helps to prolong their systemic circulation by preventing particle aggregation in the blood and sequestration by the mononuclear phagocyte system. Herein, we synthesized a series of mRNA-loaded nanoparticles by replacing ALC-0159 (a PEG-lipid used in the Comirnaty formulation) with amphiphilic PEG-polycarbonate diblock copolymers (PC-HNPs).

Transition from manual to automated processes for autologous T cell therapy manufacturing using bioreactor with expandable culture area.

Transition from the manual processes that are performed during the initial research and development (R&D) stage to automated processes for later and commercial stage cell therapy manufacturing can be challenging. It often requires significant effort, time, and costs - which hinders the therapy's access to the clinic. To ease this transition, we have developed a novel and flexible manufacturing platform, Bioreactor with Expandable Culture Area (BECA), that aims to support both R&D and manufacturing to accelerate cell therapies from bench to bedside.

Optimizing viral transduction in immune cell therapy manufacturing: key process design considerations.

Immune cell therapies have revolutionized the treatment of cancers, autoimmune disorders, and infectious diseases. A critical step in their manufacturing is viral transduction, which enables the delivery of therapeutic genes into immune cells. However, the complexity of this process presents significant challenges for optimization and scalability.

Rewiring of SINE-MIR enhancer topology and Esrrb modulation in expanded and naive pluripotency.

Background: The interplay between 3D genomic structure and transposable elements (TE) in regulating cell state-specific gene expression program is largely unknown. Here, we explore the utilization of TE-derived enhancers in naïve and expanded pluripotent states by integrative analysis of genome-wide Hi-C-defined enhancer interactions, H3K27ac HiChIP profiling and CRISPR-guided TE proteomics landscape.

Results: We find that short interspersed nuclear elements (SINEs) are the more involved TEs in the active chromatin and 3D genome architecture.

Differential polyamine metabolism in CHO cell lines: Insights into cell growth and antibody quality.

Chinese hamster ovary (CHO) cell lines are widely utilized host cell lines in cell culture bioprocessing. Although they originated from a common ancestor, accumulated genetic mutations have led to significant heterogeneity in their behavior under specific conditions. This study investigates the cell line-specific impact of polyamine (PUT; putrescine) withdrawal on the growth, metabolism, and antibody production among three CHO clones derived from different parental cell lines: CHO-K1, CHO-S, and CHO-DG44.

Cholesterol-Derived Mannosylated Polypeptide-Formed Lipid Nanoparticles for Efficient in Vivo mRNA Delivery.

Effective delivery of lipid nanoparticles (LNPs)-based mRNA vaccines to antigen presenting cells (APCs) is important for eliciting potent immune responses. However, most LNPs-based mRNA vaccines usually suffer from insufficient accumulation in APCs due to the lack of active targeting capability, leading to the limited efficacy and the need for high injection dose. In this study, a series of cholesterol-derived mannopolypeptide (CPSM) and cholesterol-conjugated mannose (CM) derivatives is synthesized to prepare mannosylated LNPs for efficient mRNA delivery.

Nucleofection-based screening of chimeric antigen receptor candidates in human natural killer cells.

Chimeric antigen receptor (CAR)-modified cell therapy products approved for clinical treatment of hematological malignancies have hitherto been based on T cells. NK cells represent a promising immune cell type that can be considered for CAR engineering due to their potential to be generated as off-the-shelf allogeneic cellular therapy. Viral transduction of NK cells with CARs has been fraught with challenges of long process time and poor CAR transduction efficiency.

Characterization of group I introns in generating circular RNAs as vaccines.

Circular RNAs are an increasingly important class of RNA molecules that can be engineered as RNA vaccines and therapeutics. Here, we screened eight different group I introns for their ability to circularize and delineated different features that are important for their function. First, we identified the Scytalidium dimidiatum group I intron as causing minimal innate immune activation inside cells, underscoring its potential to serve as an effective RNA vaccine without triggering unwanted reactogenicity.

A platform for Bioengineering Tissue Membranes from cell spheroids.

Cell spheroids are essential building blocks for engineering tissues like cartilage, bone, liver, cardiac, pancreatic, and neural tissues, but controlling their fusion and organisation is challenging. Spheroids tend to fuse into a larger mass, impeding nutrient and waste diffusion. To overcome this, we developed a method to assemble spheroids into a thin layer by using two mesh scaffolds to spread them evenly, and a solid frame with grid to secure the assembly.

Photopolymer Resins from Sulfenyl Chloride Commodity Chemicals for Plastic Optics, Photopatterning and 3D-Printing.

The development of a low-cost photopolymer resin to fabricate optical glass of high refractive index for plastic optics is reported. This new free radically polymerizable photopolymer resin, termed, disulfide methacrylate resin (DSMR) is synthesized by the direct addition of allyl methacrylate to a commodity sulfur petrochemical, sulfur monochloride (SCl). The rapid rates of free radical photopolymerization confer significant advantages in preparing high-quality, bulk optical glass.

Multilevel─Intact, Subunits, and Peptides─Characterization of Antibody-Based Therapeutics by a Single-Column LC-MS Setup.

A comprehensive characterization of biotherapeutics, mandated by regulatory authorities, requires analyses of a protein drug at multiple structure levels. Such multilevel characterization can be performed by mass spectrometry (MS), with established conventional MS-based assays of product quality attributes (PQAs) comprising intact protein and subunit middle-up MS with analytes resolved on a C4 column, and bottom-up peptide mapping with analytes resolved on a C18 column. Recent advances in MS have facilitated the increasing use of middle-down analysis, expanding the qualitative analytical capability of MS for protein characterization.

Hyper-porous encapsulation of microbes for whole cell biocatalysis and biomanufacturing.

Biocatalysis using whole cell biotransformation presents an alternative approach to producing complex molecules when compared to traditional synthetic chemical processes. This method offers several advantages, including scalability, self-contained co-factor recycling systems, the use of cost-effective raw materials, and reduced purification costs. Notably, biotransformation using microbial consortia provides benefits over monocultures by enhancing biosynthesis efficiency and productivity through division of labor and a reduction in metabolic burden.

Enhancing recombinant growth factor and serum protein production for cultivated meat manufacturing.

The commercial growth factors (GFs) and serum proteins (SPs) contribute to the high cost associated with the serum-free media for cultivated meat production. Producing recombinant GFs and SPs in scale from microbial cell factories can reduce the cost of culture media. Escherichia coli is a frequently employed host in the expression recombinant GFs and SPs.