Biology subjects

Waymouth, R. M.

Publications and source records attributed to Waymouth, R. M..

5 recordsLinked to original sources

bioRxiv2025

In vivo mRNA delivery to the lung vascular endothelium by dicationic Charge-Altering Releasable Transporters

Endothelial cells (EC) comprise the pulmonary vascular bed and play a significant role in health and disease. Consequently, the EC niche represents an attractive therapeutic target for treating a wide range of pulmonary vascular diseases. We have identified a new class of dicationic Charge-Altering Releasable Transporters. These single-component transporters selectively deliver mRNA to the lung upon intravenous administration without the use of a targeting ligand. Significantly, the number and spatial array of cationic charges within the repeating units of the CART polymer are found to control both mRNA delivery efficacy and tissue tropism. High-resolution imaging revealed efficient mRNA delivery to endothelial cells in pulmonary arteries, veins and capillaries. The selective lung tropism of these new CARTs, coupled with the efficient and tunable synthesis of this new family of CART amphiphiles, represents an enabling platform for research and clinical applications.

bioengineering↗

bioRxiv2021

Biocatalytic Formation of Novel Polyesters with para-Hydroxyphenyl groups in the Backbone - Engineering Cupriavidus necator for production of high-performance materials from CO2 and electricity

Synthetic materials are integral components of consumable and durable goods and are indispensable in the modern world. Polyesters are the most versatile bulk- and specialty-polymers but their production is not sustainable and their fate at end-of-life is of great concern. Bioplastics, though highly regarded, often lag behind conventional plastics due to poor material properties and a competitive market. This has limited the success of sustainable replacements at scale. Enabling the production of bioplastics with superior properties from waste-derived feedstocks could change that. To this end, we created a synthetic entry into the metabolic pathway of bio-polyester synthesis of Cupriavidus necator H16 employing heterologous hydroxyacyl-CoA transferase and mutant PHA synthase. The resulting microbial cell factories enabled the co-polymerization of a range of aliphatic and aromatic hydroxy carboxylates, including a hydroxyphenylic and a hydroxyfuranoic acid, for the first time incorporating aromatic rings in the backbone of biological polyesters. The latter were structurally analogous to synthetic polyesters like PET and PEF, as well as PBST and PBAT. The obtained bio-polyesters underwent characterization for specific physicochemical properties, complemented by the prediction of uncharacterized physicochemical and mechanical properties. In a further advance, the transgenic strain was cultivated in a bio-electrochemical system under autotrophic conditions, enabling synthesis of aromatic bio-polyesters with in situ generated H2+O2, while assimilating CO2. Employing elementary flux-mode analysis, metabolic modeling confirmed the feasibility of producing an extended range of aliphatic, arylatic, and aromatic PHAs de novo from various feedstocks. This comprehensive study paves the way toward sustainable bio-production of advanced high-performance thermoplastics and thermosets. Significance StatementBiomaterials can help the chemical industry transition to a carbon-neutral and circular economy, reducing the accumulation of greenhouse gases and plastic waste by developing biological replacements for fossil carbon-based plastics and implementing end-of-life strategies. Accomplished via the genetic engineering of a microbe that can assimilate carbon dioxide, this work demonstrates the first biocatalytic polymerization and incorporation of aromatic building blocks into the backbone of polyhydroxyalkanoates (PHAs). Employing a bio-electrochemical system for cultivation of the microbes, oxyhydrogen is formed and consumed in situ, thus avoiding explosive gas mixtures. The obtained aromatic PHAs are structurally similar and functionally analogs to synthetic bulk- and high-performance polymers such as PET (polyethylene terephthalate) and PEF (polyethylene furanoate) as well as PBST (polybutylene succinate-co-terephthalate) and PBAT (polybutylene adipate-co-terephthalate). Graphical AbstractThe biosynthesized novel aromatic polyhydroxyalkanoates described in this study are structurally similar to incumbent synthetic polyesters such as poly(ethylene terephthalate) (PET, orange box) and poly(ethylene furan-dicarboxylate) (PEF, yellow box). While PET and PEF may be bio-based to varying degrees, they are always synthesized chemically. In contrast, not only the monomers of poly(hydroxybutyrate-co-phloretate) and poly(hydroxybutyrate-co-hydroxymethylfuranoate) (green box) are fully bio-derived, but also the entire polymers are synthesized biologically. This was demonstrated by employing the autotrophic bacterium Cupriavidus necator H16. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/472320v3_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@20714borg.highwire.dtl.DTLVardef@1ee1c06org.highwire.dtl.DTLVardef@171cee7org.highwire.dtl.DTLVardef@c6bc21_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFirst-ever biocatalytic formation of polyesters with aromatic rings in the backbone C_LIO_LIMicrobial production of aromatic PHAs in a bio-electrochemical system on CO2 and H2+O2 C_LIO_LIExpression-level of PHA synthase and molecular weight of polyesters are inversely correlated C_LIO_LIIn silico design and pathway analysis of bio-polyesters from low-cost carbon-feedstocks C_LIO_LIPrediction of material properties for novel aromatic co-polyesters and structural analogs C_LI

synthetic biology↗

bioRxiv2021

An mRNA SARS-CoV-2 vaccine employing a novel delivery vehicle with a TLR-9 agonist induces neutralizing antibodies and T cell memory

The SARS-CoV-2 pandemic has necessitated the rapid development of prophylactic vaccines. Two mRNA vaccines have been approved for emergency use by the FDA and have demonstrated extraordinary effectiveness. The success of these mRNA vaccines establishes the speed of development and therapeutic potential of mRNA. These authorized vaccines encode full-length versions of the SARS-CoV-2 spike protein. They are formulated with Lipid Nanoparticle (LNP) delivery vehicles that have inherent immunostimulatory properties. Different vaccination strategies and alternative mRNA delivery vehicles would be desirable to ensure flexibility of future generations of SARS-CoV-2 vaccines and the development of mRNA vaccines in general. Here, we report on the development of an alternative mRNA vaccine approach using a delivery vehicle called Charge-Altering Releasable Transporters (CARTs). Using these inherently nonimmunogenic vehicles we can tailor the vaccine immunogenicity by inclusion of co-formulated adjuvants such as oligodeoxynucleotides with CpG motifs (CpG-ODN). Mice vaccinated with the mRNA-CART vaccine developed therapeutically relevant levels of RBD-specific neutralizing antibodies in both the circulation and in the lung bronchial fluids. In addition, vaccination elicited strong and long lasting RBD-specific TH1 T cell responses including CD4+ and CD8+ T cell memory.

immunology↗

bioRxiv2020

Charge-Altering Releasable Transporters enable specific phenotypic manipulation of resting primary natural killer cells

Natural killer (NK) cells are capable of rapid and robust cytotoxicity, making them excellent tools for immunotherapy. However, their recalcitrance to standard transfection techniques has limited both mechanistic studies and clinical applications. Current approaches for NK cell manipulation rely on viral transduction or methods requiring NK cell activation, which can alter NK cell function. Here, we report that non-viral Charge-Altering Releasable Transporters (CARTs) efficiently transfect primary human NK cells with mRNA without relying on NK cell activation. Compared to electroporation, CARTs transfect NK cells two orders of magnitude more efficiently, better preserve cell viability, and cause minimal reconfiguration of NK cell phenotype and function. Finally, we use CARTs to generate highly cytotoxic primary human chimeric antigen receptor NK cells, indicating potential therapeutic utility of this technique. To our knowledge, CARTs represent the first efficacious transfection technique for resting primary NK cells that preserves NK cell phenotype, and can drive new biological discoveries and clinical applications of this understudied lymphocyte subset.

immunology↗

bioRxiv2018

Ethane-dependent synthesis of polyhydroxyalkanoates by the obligate methanotroph Methylocystis parvus OBBP

Under conditions of nutrient-limited growth, Type II obligate methanotrophs oxidize C1 compounds, such as methane or methanol and accumulate intracellular granules of poly(3-hydroxybutyrate) (P3HB). Here, we report that, under same nutrient-limited conditions, the Type II obligate methanotroph Methylocystis parvus OBBP can use ethane as its sole carbon and energy source for synthesis P3HB granules, accumulating up to 35 {+/-} 4 wt% P3HB. 13C-NMR spectra of the P3HB confirmed incorporation of 13C from [13C2]ethane. Moreover, when valerate was added as a co-substrate with ethane, oxidation of the ethane supported synthesis of the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).\n\nIMPORTANCEThe presence of ethane in natural gas is often considered undesirable for methanotroph-based biotechnology due to the C1 specialization of obligate methanotrophs and concerns about inhibitory byproducts arising from methane monooxygenase-mediated cometabolism of ethane. This work establishes that co-oxidation of ethane and further metabolism in the absence of methane can support synthesis of the valuable polyhydroxyalkanoate bioplastics P3HB and PHBV.

microbiology↗