Gut Microbiota Composition Assessment Via Targeted Radical Polymerization (TRAP)

Drug delivery platforms that target the deposition of therapeutics to sites of disease are needed to increase efficacy and decrease off-target effects of current interventions. To address these issues, we developed a probiotic-based strategy capable of directly secreting therapeutic proteins into the gut lumen. Our chosen chassis E. coli Nissle 1917 (EcN) is a human probiotic with a proven safety record in human subjects. To enable EcN to secrete heterologous proteins into the gut lumen, we engineered it to encode a type III secretion system modified to secrete proteins into its surroundings rather than into host cells. Next, we developed variants of this strain, referred to as T3EcN, that secrete functional nanobodies, variable domains of heavy chain only antibodies (aka VHH), isolated from immunized alpacas and identified to sequester and neutralize target proteins of interest. This was done by appending the N-termini of the nanobody with a type III secretion sequence. To enable T3EcN to stably colonize and constitutively secrete nanobodies within the mammalian gut, nanobody-coding genes were placed under the control of constitutive synthetic promoters and introduced onto a plasmid that can be maintained in the absence of antibiotic selection. Notably, we observe that nanobody-secreting T3EcN variants stably colonize and maintain secretory activity within the mouse gastrointestinal tract. Furthermore, using a bioluminescent reporter for T3SS activity, we have demonstrated that T3EcN’s T3SS is active in the colon and small intestine. Ongoing work in preclinical murine models is focused on establishing proof-of-concept for this novel targeted drug delivery platform.