Novel Lung Targeting Cell Penetrating Peptides as Vectors for Delivery of Therapeutics

Rationale: Cell-penetrating peptides are able to cross membranes and deliver cargoes in a functional form. Our prior work identified a 12-amino acid, cardiac targeting peptide (APWHLSSQYSRT). Studies into its mechanism of transduction led to the identification of two lung targeting peptides (LTPs), S7A and R11A. Here we report on a) the comparative lung uptake of S7A versus R11A, b) complete biodistribution of R11A, c) show that cyclic versions are -100-fold more efficient than linear counterparts, d) uptake is via a non-endocytic pathway, and e) cyclic R11A's (cR11A) ability to deliver siRNA targeting structural proteins of SARS-CoV-2 and act as an anti-viral. Methods: Linear LTPs were synthesized with N-terminal labeled with Cyanine 5.5 (Cy5.5). Cyclic versions were synthesized with lysine added to the N-terminus, cyclized through a peptide bond, with a side NH-group labeled with Cy5.5. cR11A was conjugated to siRNA duplexes via a DTME linker. Wild-type, CD1 mice, were injected with S7A or R11A at 10, 5, and 1mg/Kg, peptides allowed to circulate for 15mins, mice euthanized, lung along with multiple other organs dissected and imaged using In Vivo Imaging Systems (IVIS, Perkin-Elmer) followed by confocal microscopy. CD1 mice were injected with R11A, 5mg/Kg, and euthanized at different time intervals for biodistribution studies. Endocytosis studies were done using serum-starved human bronchial epithelial cells (HBEC) incubated with fluorescently labeled transferrin and LTP-S7A or LTP- R11A. Lastly, anti-viral activity was tested in HBECs pre-treated with cR11A-siRNA followed by viral infection. Results: Mice injected with LTP-S7A or LTP-R11A showed robust uptake of the peptides by lung tissue, with R11A showing an increasingly favorable lung:liver ratio with decreasing dose. Lung uptake of R11A peaked at 120mins with complete dissipation of fluorescence by 24 hours. In Vitro studies in HBECs showed no co-localization of transferrin with LTPs, ruling out endocytosis as a mechanism of uptake. Comparison of linear versus cyclic peptides using FACS showed cyclic peptides had -100-fold increased transduction efficiency over their linear counterparts. cR11A conjugated to ant-spike, and anti-envelop proteins showed an anti-viral effect with EC90 of 0.6uM and 1.0μM, respectively. Conclusions: We have identified two novel lung-targeting peptides capable of acting as delivery vectors. Peak uptake of R11A occurred at 120mins. Furthermore, this uptake was not via endocytosis, and cyclic versions were -100-fold more efficiently taken up. Lastly, as proof of concept, we show cR11A acts as a vector and delivers siRNA to HBECs in a functional form, and act as anti-virals.

Sara Alert: An enduring national resource to support public health monitoring &disease containment

Background Public health agencies had to respond swiftly to the novel coronavirus that emerged in 2019 (COVID-19) to try to contain the virus, which requires early identification of new cases. Monitoring exposed individuals is labor intensive and available tools are often limited. MITRE, a company that operates federally funded research and development centers for the U.S. government, rapidly developed a configurable monitoring tool that allows public health authorities to monitor potentially exposed individuals in their jurisdictions. Methods A team, including public health leaders, field epidemiologists, software engineers, and health communication specialists, was quickly assembled to design and develop an open source, disease-independent monitoring tool called Sara Alert. Outreach to key public health stakeholders, including partner organizations and local and state health departments, was conducted early for requirements gathering and to validate assumptions. Public health law experts were consulted regarding data privacy and security. Results By four weeks, a minimally viable monitoring tool was available for testing by public health partners. Exposed individuals can be enrolled and reminded daily to enter a temperature and any symptoms by web or mobile interface, SMS messaging or phone. Public health officials monitor and can quickly take action if symptoms consistent with COVID-19 are reported of if there is failure to report within a configurable time frame. Dashboards provide insight into aggregated data appropriate to level of view. Conclusions Sara Alert serves as a force multiplier that supports disease containment and allows resources to be directed where they are most needed. Successful development was possible because key stakeholders across public health practice were consulted early. Sara Alert is available, free, to state and local public health departments and serves as an enduring resource easily configured for the next public health emergency. Key messages Sara Alert serves as a force multiplier that supports disease containment and allows resources to be directed where they are most needed. Sara Alert serves as an enduring resource easily configured for the next public health emergency.