Plasmid delivery

[Ema]

Hello :) 

If I have plasmid DNA that I want bacteria to pick up, how should I encapsulate it to make it attractive for them? At the same time, I want the capsule to be immuno-invisible to the human system.  

Do you know of any particular techniques? Possibly companies/researchers who focus on this? 

Bacteria exchange the plasmids between each other, but would they also pick them up from the environment, if the plasmids were, say, encapsulated in a micelle? 

Curious to hear your knowledge and thoughts. Thank you! 

[CharonY]

Bacteria generally transfer plamids via conjugation. Uptake of free DNA is generally only possible for relatively small fragments. Considering constraints in size and transport mechanisms, I do not think that bacteria can take up whole plasmids outside of in vitro settings (e.g. electrporation, chemical transformation etc.). 

What you are thinking about is probably more applicable to eukaryotic cells, which actually do phagozytosis. That being said, at least in theory you could probably encapsulated plasmids in a liposome and force a fusion with a bacterial cell (depending on the cell envelope structure of the bacterium in question). These are not active uptake mechanisms, but rather an attempt to increase perneability of small molecules, such as antibiotics. These methods have been discussed since at least the late 80s but I am not sure whether folks have tried e.g. with whole plasmids. It has seen a revival together with nanomaterials as a means for drug delivery. However, it does not seem to have seen much in progress toward clinical utility, and much of the data is in vitro.

[Ema]

Thank you so much for taking the time to answer so thoughtfully! This is really helpful to me. I wish you all the best. 

[Dagl1]

@CharonYIs it possible to, as OP is asking, provide a protein-encoding plasmid that is immuno-invisible? I feel like it shouldn't as that protein could always become an antigen for T cells to present right?

Edit: OP isn't specifically asking for a protein-encoding plasmid, but I could see how non-coding plasmids could be immuno-invisible, so the question is mostly interesting when talking about protein-encoding plasmids

 

Edited December 3, 2019 by Dagl1

[Ema]

@Dagl1 The protein on the plasmid should ideally only be induced by a promotor when needed, but until then it should preferably be immunoinvisible - while not producing the protein. I was hoping there could be an encapsulation made out of e.g. micelle membrane that would encapsulate it, be non-immunogenic itself and the plasmid would only be activated once fused with a cell that can produce it. The problem is, how to find such a capsule and make the cells take it up even from these 'plasmid vesicles'. 

Also, @CharonY, I only signed in yesterday and didn't know you could tag people in replies. So thank you both for sharing your ideas. 

[Dagl1]

Alright I see; so you want to transmit some DNA to bacteria present within the human body (and this DNA should not be detected as to not destroy it through immune defences I presume) which then should produce a protein inside those bacteria. As Charon said previously, researchers have used several small molecule / nano containers for drug delivery, you may be interested in reading further into nano-containers: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5636659/ . One other thing is to make sure that if your target for protein production are bacteria and not humans, you have to choose the right promoter etc. 

At university I once had to give a presentation about polymer nanocontainers, and while looking back at the presentation its pretty bad, but here are some references used:

These are mostly regarding drug delivery

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4322773/
https://www.ncbi.nlm.nih.gov/pubmed/20394391 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4626985/
https://www.ncbi.nlm.nih.gov/pubmed/19825408

For gene therapy:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449975/
https://www.sciencedirect.com/science/article/pii/B9780081005576000043
https://www.ncbi.nlm.nih.gov/pubmed/19488722

However, of course these methods are mainly for eukaryotic cells, I think exogenous RNA/DNA delivery into bacteria present within the body is something not researched very much.
 

[CharonY]

16 hours ago, Dagl1 said:

Edit: OP isn't specifically asking for a protein-encoding plasmid, but I could see how non-coding plasmids could be immuno-invisible, so the question is mostly interesting when talking about protein-encoding plasmids

 

I should add that DNA itself can be antigenic, too.

[Dagl1]

3 minutes ago, CharonY said:

I should add that DNA itself can be antigenic, too.

Yes but if encased and it doesn't circulate for too long, it shouldn't be too much of a problem, as it then (should) enter the bacteria right?

[CharonY]

On 12/4/2019 at 9:09 AM, Dagl1 said:

Yes but if encased and it doesn't circulate for too long, it shouldn't be too much of a problem, as it then (should) enter the bacteria right?

It is actually fairly complex. (Nano-)encapsulation has been under research for a very long time and the literature has expanded in several bursts. However, the  response of the immune system to these complexes is not as well researched.  I should also expand a bit and not limit the issue to antigenicity. Especially when it comes to nanomaterials we also have to look broader and include all cellular and humoral immune responses (i.e. immunogenicity). For example, the mentioned liposomes alone generally only elicit weak to no immunogenic responses. Yet certain formulations and depending on what they contain as load, various groups actually identified liposome-specific antibodies. Or take PEG, which is often used as a hydrophilic coating and to prevent immune recognition. However, in some studies it was found that repeated treatment with PEGylated products resulted in faster clearance from blood, suggesting eventual recognition.

The tricky bit is that to identify immunogenicty it is often not enough to just inject  and look for antibodies. Often many parameters have to be considered, such as the animal type, dosage, number of treatments, interval of treatment, the specific composition of carrier and cargo and so on.