siRNA: in vivo delivery - (Mar/27/2007 )

I work with control of viral infections by RNAi strategy. Currently I my searching ways to deliver siRNA sequences in vivo (infected animals). Does somebody has some experience about this? about some transfection agent?
I have read some interesting article about exosomes and owing to the capacity of these vesicles to deliver proteins to cells of the immune system (as lymphocytes) I thought on the possibility to use them to deliver siRNA sequences in virus-infected lymphocytes (the target of my virus). I would like to know some opinion about the possibility of to load siRNA (or plasmidial DNA) sequences in exosomes vesicles to be used as delivery way. Could somebody give me a opinion about this idea?

Sure, if you can get enough of it. Can you?

As genehunter pointer out, you will need a lots of it.

do you have any other options?

I have no idea but I know that there is a method to do it. From cell culture of monocytes enriched by interleukines you can collect, purify and concentrate the exosomes secreted into the supernatant. The problem is that I have to penetrate the lymphocytes. Do you have another sugestion?

I am not working on targeted delivery of macromolecules to lymphocytes. I think the idea is theoritically possible, but may be complicated due to several reasons. 1) The production and yield is a practical issue that should not be underestimated. 2) Also, how to entrap/absorb siRNA to these particles is another hurdle. 3) Assuming you can solve all these problems, how to trigger endosome release of the content will be the next question. You have thought about all these aspects.

I believe with the undersatnding of how exosomes interact with lymphocytes, one can come up with a design that can make artificial nanoparticles that can target these cells yet do not have production and potential antigenicity issues associated with exosomes. It is also much easier to formulate siRNA within these synthetic nanoparticles. Endosome release functionality (lipid and polymer-based) is also known.

In short, you maybe able to come up a nice paper with exosome as a siRNA carrier, how practical this approach is is a subject of discussion.

Thank for your contibution!
Yes, I'm sure that the production and purification processes of these vesicles are not easy, but to design artificial particle containing proteins to address them toward a specific cell group neither is evident (do you have experience with that?). My interest in this subject appeared after knowing about the clinical trials of anticancer therapies currently in progress using exosomes . I will try to contact some people that are working with exosomes in practice and take further information.

Using exosomes as antigen presentation and as drug carrier are quite different applications. Our immune system is known to amplify a signal many, many times, therefore not much protein antigen is needed. Rather a formulation that is capable of presenting antigen or stimulating immune cell is more critical.

As of exosomes as drug carriers, I dont know what type of drugs that you have in mind, but most likely, repeated dosings are needed. These are protein-containing vesicles and themselve are antigenic, which means, even if the injected vesicles go to the right cell type, DCs, macrophages, and possibly lymphocytes for your first injection, what about your next dosing?

You can do a search on cell-specific targeting. Quite a few works have been done in this direction.

A colleague tried to get cells from the bone marrow and transfect them with EGFP and later inject the same into the mouse. He got a decent number of the immune cells, but I donot know the exact percentages. he sorted them with FACS and then injected only the positive ones.

There is hope, I guess.

good luck !!!

Do you know if we can cross-link siRNA to peptides or proteins without affecting their activity?

siRNA conjugated to cholesterol or membrane transducing domain peptide have been done. You may need to incorporate a reducing bond to facilitate the release.