The Development of an Oral Gene Delivery System using Bacterial Outer Membrane Vesicles

Madison Seefeld Author

04/02/2021 Added

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In this video I discuss my UCARE project where outer membrane vesicles were used as a nonviral vector for oral gene delivery.

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[00:00:01.080]My name is Madison Seefeld and I'm an undergraduate student in the biological
[00:00:04.770]systems engineering department. This past year,
[00:00:07.350]I worked on a project titled the development of an oral gene delivery system
[00:00:11.190]using bacterial outer membrane vesicles.
[00:00:14.280]Gene delivery is a delivery of external genetic material to cells to produce a
[00:00:18.510]therapeutic effect.
[00:00:20.220]Viral gene delivery is the most efficient method because viruses have evolved
[00:00:24.270]over billions of years to transport DNA into cells. However,
[00:00:28.650]viral gene delivery has several negative consequences,
[00:00:31.890]including limited gene size, unwanted immune responses
[00:00:35.460]and oncogenesis the development of tumors.
[00:00:38.910]While non-viral gene delivery is less efficient.
[00:00:41.340]It does not carry the side effects that viral gene delivery does,
[00:00:44.700]It can carry larger genes, several materials are available
[00:00:48.150]so it can be customizable for specific responses and it has lower
[00:00:52.860]immunogenicity.
[00:00:54.510]There are several ways to deliver non-viral systems
[00:00:57.036]but the most promising route
[00:00:58.530]is oral gene delivery.
[00:01:01.950]Oral gene delivery is advantageous as opposed to intravenous delivery
[00:01:06.150]because of high patient compliance, ease of administration and dosing,
[00:01:10.410]and the ability for local or systemic delivery.
[00:01:14.010]Many patients are already used to taking medications in their daily routine,
[00:01:17.610]so oral gene delivery would not be a new practice. However,
[00:01:22.230]there are many challenges to oral gene delivery,
[00:01:24.720]including the low pH of the GI tract,
[00:01:27.750]an abundance of degrading enzymes and the mucosal barrier and mucus
[00:01:32.190]turnover.
[00:01:33.600]Therefore the ideal oral gene delivery system will protect DNA through the harsh
[00:01:38.520]conditions of the GI tract, cross the mucosal barrier and have
[00:01:43.350]specific cellular targeting.
[00:01:47.760]The material that seems to check all three of these boxes is outer membrane vesicles
[00:01:52.800]OMVs are produced by blebbing from the outer membrane of bacteria. They can be
[00:01:57.060]considered bacterial sample packs because they contain most of the biological
[00:02:01.080]content of bacteria, just in non replicative form.
[00:02:04.410]They are used in cell to cell communication.
[00:02:06.870]They've been shown to cross the mucosal barrier.
[00:02:09.570]They retain biological function after administration suggesting they protect DNA
[00:02:14.370]throughout transit,
[00:02:15.810]and finally they are endocytosed by human epithelial cells.
[00:02:20.250]The OMVs used in this project. We're isolated from DH5-alpha E. Coli,
[00:02:24.480]They were isolated using the Vivaflow crossflow ultrafiltration
[00:02:29.340]concentrator following the provided protocol,
[00:02:32.310]The size of the OMVs were confirmed using a nanosight,
[00:02:35.400]which takes the OMV solution and runs it over a laser,
[00:02:38.490]which then produces a size distribution,
[00:02:40.590]which can be seen to the right. Then a BCA assay was used to determine the
[00:02:45.240]micrograms of OMV membrane protein per milliliter.
[00:02:48.930]This data was used to calculate the amount of OMV solution needed for
[00:02:52.620]each DNA to OMV ratio used in the project.
[00:02:55.880]Previously, my
[00:02:59.830]work focused on using electroporation to load OMVs with plasma DNA.
[00:03:04.540]Electroporation creates pores in artificial or cellular membranes due to an
[00:03:08.710]elevated transmembrane voltage, which can allow DNA to enter the OMV,
[00:03:13.060]creating a DNA OMV nanocarrier.
[00:03:16.840]While we were optimizing loading OMVs through electroporation,
[00:03:19.960]we discovered that not all of the loaded DNA was enclosed in the OMV.
[00:03:25.090]This let us try hypothesize that plasmid DNA associates with the surface of the
[00:03:30.690]OMV and could be used for oral gene delivery. First,
[00:03:35.130]we wanted to test if surface associated DNA is protected from DNase treatment,
[00:03:39.450]which we had been previously using to remove DNA
[00:03:41.940]not enclosed inside the DNA OMV nanocarrier. To test this,
[00:03:46.560]we ran a loading experiment comparing electroporated OMVs and non
[00:03:50.430]electroporated OMVs using Pico green, which can not cross the cell membrane
[00:03:55.290]so it will only recognize surface associated DNA. From the graph
[00:03:59.910]you can see that the data suggest that plasmid DNA associated to the surface of
[00:04:04.020]OMVs is protected from DNase treatment.
[00:04:08.220]In this project, various DNA to OMV ratios were used.
[00:04:12.300]These were determined by using the protein content from the BCA assay.
[00:04:16.980]These ratios were used to help determine the best formulation method.
[00:04:21.360]Once the DNA OMV mixtures were made,
[00:04:23.610]they were allowed to rest for one hour at various temperatures.
[00:04:27.480]After the rest period, the mixtures were treated with DNase to remove any plasmid
[00:04:32.130]DNA not attached to the OMVs. To quantify
[00:04:35.670]the DNA Hoechst dye was used,
[00:04:37.890]Hoechst is a fluorescent dye that binds to DNA.
[00:04:42.300]The first thing I wanted to test was how incubation temperature affects loading
[00:04:46.200]efficiency. From the graphs
[00:04:48.060]you can see that room temperature incubation leads to the greatest loading
[00:04:51.420]efficiency while 37 and four degrees Celsius, decrease loading efficiency.
[00:04:57.060]However, loading efficiency is only meaningful
[00:04:59.820]if it also translates to increased transfection efficiency. Transfection
[00:05:04.770]is the process of introducing genetic material to cells using non-viral methods.
[00:05:09.720]For this project we measured transfection by delivering a reporter plasmid that
[00:05:13.650]uses as luciferase transgene.
[00:05:16.740]Then a luminometer was used to measure the luminescence as a measure of
[00:05:21.030]transgene expression.
[00:05:22.650]We then normalized transgene expression to total protein content in the cells.
[00:05:29.190]To see if incubation temperatures lead to better transfection efficiency.
[00:05:33.210]I use the same loading parameters as previously described.
[00:05:36.840]As you can see from the graphs room
[00:05:38.640]temperature incubation does not increase transfection efficiency when compared
[00:05:43.350]to 37 and four degrees Celsius.
[00:05:48.840]In conclusion,
[00:05:49.680]surface associated DNA is protected from DNase treatment.
[00:05:54.090]Room temperature incubation leads to the greatest surface loading efficiency.
[00:05:58.020]However,
[00:05:58.880]incubation temperature does not significantly affect
[00:06:01.938]transfection efficiency.
[00:06:04.040]In the future.
[00:06:04.760]I plan to investigate new ways to increase services associated DNA loading by
[00:06:09.410]letting mixtures rest on a shaker plate and using calcium and using calcium ions
[00:06:13.790]to increase DNA binding to the surface of OMVs. Finally,
[00:06:18.123]I would like to thank the Pannier Lab and our funding sources for letting me be a
[00:06:21.830]part of this project.

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The Development of an Oral Gene Delivery System using Bacterial Outer Membrane Vesicles

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