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Womens Health

Women's Health - Poster Session C

Poster L24 - Genetically Modified Extracellular Vesicles

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Women’s health conditions face a severe shortage of therapeutic options. Although recent advancements in nanocarrier technologies have significantly impacted the treatment of cancer, immune disorders, cardiovascular diseases, and infectious diseases, there has been minimal translational innovation in gynecologic and obstetric indications. This lack of progress contributes to ongoing disparities in women’s health. Drug delivery directly through the vagina is advantageous as it avoids the challenges that come with drugs delivered orally or systemically, as well as many side effects due to the ability to directly target the reproductive tract and deliver drugs. In the vaginal environment, the role of microorganisms to target diseases is a growing area of study. Recent work reveals the potential of bacterial extracellular vesicles (bEVs) as a promising approach to target diseases, and a better alternative to synthetic nanoparticle formulations which have proven to be low-throughput, time-consuming, and cost prohibitive for clinical translation. Here, we describe the development of synthetic biology tools for the genetic modification of bacterial strains to produce functionalized bEVs.

Materials and

Methods:  We are investigating the use of bEVs from the non pathogenic Escherichia coli ClearColi® BL21(DE3) to deliver drugs in the vaginal environment. As an example of genetic modification for loading proteins inside bEVs, we genetically modified ClearColi® BL21(DE3) to display the fluorescent protein moxNeonGreen on the inner membrane. To do so we fused moxNeonGreen to the C-terminus of OmpA, an abundant outer membrane porin often used as surface anchor for membrane integration. We modulated the amount of protein displayed by placing the fusion protein under the control of the inducible rhamnose (rha)-regulated t7 promoter and adding variable amounts of inducer in the culture media. The design of each plasmid was performed using the online software Benchling. DNA fragments were assembled through the Golden Gate Assembly method, using the Type IIS restriction enzyme BsaI. ClearColi® BL21(DE3) were transformed through electroporation and plated on corresponding antibiotic plates to confirm DNA uptake. Lastly, plasmids were miniprepped and sequenced to confirm accuracy of the plasmids and cells were observed with confocal microscopy. bEVs were isolated via ultracentrifugation and characterized via nanoparticle tracking analysis by our collaborators – the Zierden Lab at UMD.

Results, Conclusions, and Discussions: By tailoring plasmids whose expression levels could be controlled through varying levels of induction, different concentrations of fluorescent bEVs were produced (Fig. 1). By utilizing nanoparticle tracking analysis (NTA), bEV size and concentration of the total population were determined. The Zierden Lab houses a Particle Metrix NTA microscope with fluorescent filter capabilities, allowing for the determination of bEV sub-populations containing fluorescent markers. Using these measurements, loading efficiency of these fluorescent markers can be determined. bEVs loaded with fluorescent proteins were isolated from cultures of low, medium, and high induction levels of ClearColi® BL21(DE3). They produced a concentration of 1.93 x 108 ± 3.8 x 108 particles/mL, 5.81 x 108 ± 3.8 x 108 particles/mL, and 9.67 x 108 ± 3.8 x 108 particles/mL respectively. However, OD600, a marker of bacterial growth, shows differences in viability. OD600 values of cells were greater when induction levels were lower. This demonstrates that the level of protein display needs to be finely tuned with promoter strength as too much protein production can be toxic to the cell, resulting in greater bEV production. A greater understanding of bEVs and the use of genetic engineering will allow for the creation of novel nanomedicines and therapies.

Acknowledgements (Optional):  National Institutes of Health NIGMS MARC T34GM149472 to HK