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

Madison Seefeld Author

07/28/2020 Added

48 Plays

Description

This presentation discusses research done on developing a method for loading Outer Membrane vesicles isolated from an e. coli strain with DNA to create an effective oral gene delivery system.

Searchable Transcript

Search:

[00:00:00.000]My name is Madison Seefeld
[00:00:02.477]and I am an undergraduate student
[00:00:04.009]in the biological systems engineering department.
[00:00:06.404]This summer I worked on a project titled
[00:00:08.567]The Development of an Oral Gene
[00:00:10.164]Delivery System using Bacterial
[00:00:12.015]Outer Membrane Vesicles.
[00:00:14.201]Gene delivery is the delivery of external
[00:00:17.071]genetic material to cells to produce
[00:00:19.118]a therapeutic effect.
[00:00:20.595]Viral gene deliver is the most efficient
[00:00:22.901]method because viruses have evolved
[00:00:24.827]over billions of years to transport DNA
[00:00:26.902]into cells. However viral gene delivery
[00:00:29.599]has several negative consequences including
[00:00:32.499]limited gene size, unwanted immune responses
[00:00:35.508]and oncogenesis, the development of tumors.
[00:00:38.308]While nonviral gene delivery is less
[00:00:40.520]efficient it does not carry the side
[00:00:42.310]that viral gene delivery does.
[00:00:44.173]It can carry larger genes,
[00:00:45.919]several materials are availible
[00:00:48.157]so it can be customizable for specific
[00:00:49.927]responses and it has lower immunogenicity.
[00:00:52.986]There are several ways to deliver
[00:00:54.696]non-viral systems, but the most
[00:00:56.326]promising route is oral gene delivery.
[00:00:58.405]Oral gene delivery is advantageous as
[00:01:01.253]opposed to intravenous delivery because
[00:01:03.433]of high patient compliance, ease of
[00:01:05.333]administration and dosing and the ability
[00:01:07.753]for local or systemic delivery.
[00:01:09.861]Many patients are already used to taking
[00:01:12.071]medications in their daily routine so oral
[00:01:14.506]gene delivery would not be a new practice.
[00:01:17.084]However, there are many challenges
[00:01:19.624]to oral gene delivery including
[00:01:21.759]the low pH of the GI tract,
[00:01:23.673]an abundance of degrading enzymes
[00:01:26.343]and the mucus barrier and mucus turnover.
[00:01:29.340]Therefore the ideal oral gene delivery
[00:01:32.500]system will protect the DNA through
[00:01:34.304]the harsh conditions of the GI tract,
[00:01:36.282]cross the mucosal barrier
[00:01:38.369]and have specific cellular targeting.
[00:01:40.946]The material that seems to check all
[00:01:42.916]three of these boxes is
[00:01:44.325]outer membrane vesicles.
[00:01:46.002]OMVs are produced by blebbing
[00:01:47.913]from the outer membrane of bacteria.
[00:01:50.096]They can be considered bacterial sample
[00:01:52.360]packs because they contain most of the
[00:01:54.294]biological content of bacteria
[00:01:56.276]just in non-replicative form.
[00:01:58.059]They are used in cell to cell communication.
[00:02:00.661]They have been shown to
[00:02:01.961]cross the mucosal barrier.
[00:02:03.520]They retain biological function after
[00:02:05.738]administration, suggesting they
[00:02:07.517]protect DNA throughout transit
[00:02:09.534]and finally, they are endocystosed
[00:02:11.544]by human epithelial cells.
[00:02:13.463]The OMVs used in this project
[00:02:15.861]were isolated from DH5 alpha E. coli.
[00:02:18.708]They were isolated using the ExoBacteria
[00:02:21.238]OMV isolation kit following the provided
[00:02:23.631]protocol. The size of the OMVs
[00:02:26.500]were confirmed using a nanosight
[00:02:28.262]which takes the OMV solution and
[00:02:30.045]runs it over a laser, which then
[00:02:31.915]produces a size distribution seen to the right.
[00:02:34.919]Then a BCA assay was used to
[00:02:36.996]determine the micrograms of OMV
[00:02:38.915]membrane protein per milliliter.
[00:02:40.727]This data was used to calculate
[00:02:42.589]the amount of OMV solution needed
[00:02:44.384]for each DNA to OMV ratio
[00:02:46.294]used in this project.
[00:02:47.587]Electroporation is the means by which
[00:02:50.847]we loaded OMVs with DNA.
[00:02:52.921]Electroporation describes the appearance
[00:02:55.225]of pores in artificial or cellular membranes
[00:02:57.544]due to an elevated trans-membrane
[00:02:59.257]voltage. Since the membrane of the OMV
[00:03:02.707]and the DNA are negatively charged
[00:03:04.627]the DNA cannot pass through the
[00:03:06.227]membrane naturally.
[00:03:07.734]When the membrane of the OMVs
[00:03:09.498]is exposed to an external electrical field
[00:03:11.838]it causes the molecular structure
[00:03:13.458]of the membrane to rearrange
[00:03:14.956]leading to the formation of pores
[00:03:17.206]increasing the permeability of the membrane
[00:03:19.208]allowing the DNA to enter.
[00:03:20.742]Once the electrical field is removed
[00:03:23.095]the membrane reforms and the DNA
[00:03:25.314]is enclosed inside the OMV
[00:03:27.124]creating a DNA-OMV nanocarrier.
[00:03:30.038]In this project various DNA to OMV
[00:03:33.599]ratios were used, these were determined
[00:03:35.898]using the protein content
[00:03:37.174]from the BCA assay.
[00:03:38.947]Various electroporation voltages were also
[00:03:41.859]tested, these were determined from
[00:03:43.774]literature on OMV loading studies.
[00:03:45.670]Using different ratios and voltages
[00:03:48.758]helped find the optimal method for
[00:03:50.346]formulation to achieve maximal DNA loading.
[00:03:52.926]Following electroporation, the DNA-OMV
[00:03:55.979]nanocarriers were treated with DNase
[00:03:58.005]to remove any DNA not enclosed by the OMV.
[00:04:01.039]To quantify the DNA hoechst dye was used.
[00:04:04.054]Hoechst is a fluorescent dye
[00:04:06.133]that binds to DNA.
[00:04:07.598]The first thing we wanted to look at
[00:04:10.107]was how voltages impact DNA loading.
[00:04:12.447]So we tested one microgram of DNA
[00:04:14.551]to one microgram of OMVs at
[00:04:17.032]at various voltages. From the graph
[00:04:19.018]you can see that 700 volts had the
[00:04:21.044]highest loading efficiency at 18%.
[00:04:23.469]This loading is comparable to values found
[00:04:25.946]in literature from other loading studies.
[00:04:27.741]Next we wanted to see how the different
[00:04:30.076]ratios affect loading efficiency.
[00:04:31.912]We predicted the one to one ratio
[00:04:34.166]would be a limiting factor
[00:04:35.399]because of the excess DNA present.
[00:04:37.432]From the graph you can see that the
[00:04:39.127]one to two DNA to OMV ratio had the
[00:04:41.412]highest loading efficiency at 32%.
[00:04:44.061]Finally, we wanted to look at how
[00:04:46.870]multiple pulses effected loading efficiency.
[00:04:49.450]We tested the different ratios at 700
[00:04:51.836]volts using two electroporation pulses,
[00:04:54.135]this means that pores will be
[00:04:55.630]formed twice instead of just once.
[00:04:57.792]From the graph you can see loading
[00:04:59.389]efficiency greatly increased for every
[00:05:01.603]ratio with one to four being
[00:05:03.242]the highest at 63%.
[00:05:05.329]However, loading efficiency is only
[00:05:07.846]meaningful if it also translates
[00:05:09.826]to better transfection rates.
[00:05:11.518]Transfection is the process of introducing
[00:05:14.830]genetic material to cells using
[00:05:16.985]nonviral methods.
[00:05:18.464]For this project we measured transfection
[00:05:21.364]by delivering a reporter plasmid
[00:05:23.044]that uses a luciferase transgene.
[00:05:25.382]Then a luminometer was used to measure
[00:05:28.214]luminescence as a measure
[00:05:29.585]of transgene expression.
[00:05:31.114]We then normalized transgene
[00:05:32.793]expression to total protein content
[00:05:34.571]in the cell.
[00:05:35.391]To see if different voltages have an
[00:05:37.579]effect on transfection rates we tested
[00:05:39.778]the one to one DNA OMV ratio
[00:05:41.857]at various voltages.
[00:05:43.318]From the graph you can see that
[00:05:44.880]varying voltages does not affect
[00:05:46.681]transfection of HEK 293T cells.
[00:05:49.263]In conclusion, electroporation voltage,
[00:05:52.255]DNA to OMV ratio and number of
[00:05:55.116]pulses impact loading efficiency
[00:05:56.996]into DNA-OMV Nanocarriers, DNA-OMV
[00:06:01.020]Nanocarriers are capable of mediating
[00:06:03.350]transfection in HEK 293T cells.
[00:06:05.714]Electroporation voltage does not
[00:06:07.789]significantly impact DNA-OMV
[00:06:10.334]nanocarrier mediated transfection in
[00:06:12.765]HEK 293T cells.
[00:06:14.382]In the future we would like to
[00:06:17.040]investigate transfection with other
[00:06:18.770]formulation parameters, loading efficiency
[00:06:21.113]with more pulses and how OMVS
[00:06:23.455]isolated from different commensal E. coli
[00:06:25.748]strains impact loading efficiency
[00:06:27.452]and transfection of DNA-OMV nanocarriers.
[00:06:30.774]Finally, I would like to thank the Pannier
[00:06:33.558]Lab and our funding sources for letting
[00:06:35.613]me be apart of this project!

The screen size you are trying to search captions on is too small!

You can always jump over to MediaHub and check it out there.

Comments

0 Comments

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

Description

Searchable Transcript

Comments icon comment

Related Channels

Comments