Poster W19 - Supramolecular Peptide Gel for Delivery of Locally Sustained Enzyme Therapeutics

Introduction: Functional biomaterials fabricated from supramolecular self-assembly are of interest for many biomedical applications. However, to be useful for applications such as catalysis, whole protein must be installed to provide the biomaterial with this functionality. This can be challenging because covalently grafting proteins onto post-assembled biomaterials can alter protein bioactivity and is often non-specific and irreproducible. Instead, the protein can be installed via recombinant covalent fusion to an assembly peptide in the pre-assembled state. The CATCH (Co-Assembly Tags based on CHarge complementarity) biomaterial system consists of a pair of oppositely charged variants of the synthetic beta-sheet fibrillizing peptide, Q11. Separately, the CATCH(4K+/6E-) peptides do not aggregate due to electrostatic repulsion, but once combined, they co-assemble into nanofiber-based hydrogels. CATCH peptides recombinantly fused to folded proteins can be incorporated into the nanofibers during assembly, endowing the hydrogels with functionality. Immobilizing therapeutic enzymes to CATCH nanofiber-based hydrogels can locally prolong availability and functional efficiency of enzyme therapeutics. Localized delivery can decrease the rate of renal clearance and improve therapeutic outcomes, whilst also reducing off-target effects and administered dose. The overall goal of this work is to show the versatility of this biomaterial system to be utilized in localized and sustained delivery of enzyme-based therapeutics. Specifically, we show the functional incorporation of NanoLuciferase for reporter activity, uricase for the treatment of gout, and indoleamine 2,3-dioxygenase (IDO) for immunomodulation.

Materials and

Methods: Protein Expression/Purification: Three fusion proteins, CATCH-NanoLuciferase, CATCH-Uricase, and CATCH-IDO, were expressed in an E. coli host system and purified via nickel affinity chromatography.
CATCH Hydrogel Fabrication: CATCH peptides were dissolved in aqueous buffer and combined in equimolar ratios, with the addition of fusion protein, to final gel volumes of 10-40uL and peptide concentration of 12mM.
CATCH-NanoLuc Retention/Activity in vivo: 50nM CATCH-NL was injected into the subcutaneous space in the top of the hind paw in female C57BL/6J mice, either in a soluble formulation or in a hydrogel. Furimazine was administered shortly after and IVIS imaging was conducted to measure bioluminescence. Furimazine injections and IVIS measurements were repeated over a 22-day time period.
CATCH-Uricase Hydrogel: CATCH-uricase hydrogels, or blank hydrogels, were incubated with uric acid crystals and imaged on the microscope for crystal depletion. Female C57BL/6 mice were treated with either 20uL of PBS, wild type uricase, or hydrogels with or without uricase injected subcutaneously into the top of the hind paw. Two days later, 0.5mg MSU was subcutaneously injected into the same paw. Paw inflammation was subsequently assessed with digital calipers.
CATCH-IDOHydrogel: Female C57BL/6 mice were treated with 20uL hydrogels with or without IDO injected subcutaneously into the hock. 24 hours later LPS was locally injected. 2 hours later tissue was harvested for H&E histology and inflammatory gene expression gene expression.

Results, Conclusions, and Discussions: The overall goal of this work is to show the versatility of this biomaterial system to be utilized in localized and sustained delivery of enzyme-based therapeutics. We show the functional incorporation of three different CATCH-fusion proteins. Using NanoLuc as a reporter enzyme, we observed that the CATCH vehicle can prolong retention of active enzyme in vivo for 20 days. Uricase, a clinically used therapeutic enzyme, reduces inflammation in a mouse model of gout when delivered locally in the CATCH vehicle while locally delivered soluble uricase has no therapeutic effect. Finally, IDO provides localized immunomodulation in response to LPS-induced inflammation when delivered in the CATCH vehicle.

Through this work we have shown stable functional integration of three very different proteins into the CATCH hydrogel platform. This biomaterial system provides a modular, tunable, and reproducible method for the localized and sustained delivery of enzyme-based therapeutics.

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