Light-regulated release of recombinant Nerve Growth Factor from Bacterial Hydrogels
Final Report Abstract
Nerve growth factor (NGF) is a highly versatile protein that promotes the growth and survival of several types of nerve cells apart from regulating various processes in epithelial, immune and stem cells. Approaches for therapeutic application of NGF have been explored for decades with greatest success achieved in treating topical eye and skin diseases. However, clinical translation of NGF-based therapies is limited by three major obstacles: (i) High cost - as a large and complex protein, cost of production, purification, storage and delivery is very high and impractical for chronic diseases, where prolonged availability is required, (ii) Low stability - NGF is degraded by proteases in the body due to which large doses and repetitive administration is often required, leading to side-effects, (iii) Inefficient delivery - The loading capacity of NGF in drug-release materials and the control over release is very poor. The project aimed to address these issues by developing bacterial hydrogels as living therapeutic materials that can produce and deliver NGF in a controlled manner. Since such a technology is of interest to be applied in various organs that can suffer neurological disorders like the skin and eyes, it was decided to use probiotic bacteria compatible with several body sites. A promising probiotic lactobacillus strain, L. plantarum WCFS1, was accordingly identified. As lactobacilli suffer from poorly developed genetic toolboxes, the project initially dedicated efforts to expanding the toolbox for the selected strain. Consequently, novel genetic parts were discovered, including the strongest promoters reported to date, strong repressors, toxin-antitoxin-based plasmid retention systems, and signal peptides (SPs) for protein secretion. Using these genetic parts, the production and secretion of bioactive NGF was achieved. Furthermore, the recombinant lactobacilli were encapsulated in core-shell alginate beads and long-term protein secretion from these constructs has been demonstrated. Currently, the final demonstration of active NGF release from bacterial hydrogels is being validated.
Publications
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Expanding the genetic programmability of Lactiplantibacillus plantarum. Microbial Biotechnology, 17(1).
Blanch‐Asensio, Marc; Dey, Sourik; Tadimarri, Varun Sai & Sankaran, Shrikrishnan
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In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1. PLOS ONE, 18(2), e0281625.
Blanch-Asensio, Marc; Dey, Sourik & Sankaran, Shrikrishnan
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Novel genetic modules encoding high‐level antibiotic‐free protein expression in probiotic lactobacilli. Microbial Biotechnology, 16(6), 1264-1276.
Dey, Sourik; Blanch‐Asensio, Marc; Balaji, Kuttae Sanjana & Sankaran, Shrikrishnan
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Discovery of a high-performance phage-derived promoter/repressor system for probiotic lactobacillus engineering. Microbial Cell Factories, 23(1).
Blanch-Asensio, Marc; Tadimarri, Varun Sai; Wilk, Alina & Sankaran, Shrikrishnan
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PEARL: Protein Eluting Alginate with Recombinant Lactobacilli. openRxiv.
Tadimarri, Varun Sai; Blanch-Asensio, Marc; Deshpande, Ketaki; Baumann, Jonas; Baumann, Carole; Müller, Rolf; Trujillo, Sara & Sankaran, Shrikrishnan
