Science Interviews

Interview

Mon, 12th May 2014

Gene editing and repair kit

Jacob Geim Mikkelsen, Aarhus University, Denmark

Listen Now    Download as mp3 from the show eLife Episode 11: Pain, gene therapy, and regenerating worms

Aarhus University Denmark scientist Jacob Giehm Mikkelsen has found a way to use a modified form of HIV – the AIDS virus – to enter cells and quite literally edit mutations out of DNA, replacing them with the correct genetic message.  The way that this all-in-one repair kit works is that the team use the virus to bring into cells, proteins called nucleases which goes to the right place in the DNA and cut out the broken gene.  Then a strand of genetic material which is included inside the virus is pasted in to make good the repair.Retroviral gene therapy vector Chris Smith has been speaking to him...

Jacob -   We have been looking to find new ways to correct mutations within genes.  There are some conventional techniques on the market already and they are based on delivering genes that will encode proteins that will take care of the editing within cells.  So, what we’re trying to achieve here is a safer way by delivering the proteins themselves instead of delivering the gene for the protein.  So, what we have been doing is trying to develop viruses to deliver these foreign proteins into cells.

Chris -   Which virus are you trying to do that with?

Jacob -   In this case, we’re using HIV.  This is of course a sort of a controversial topic, but what we are trying to do is to deliver specific proteins by using these viruses that will take care of the correction process in the cells that are treated with the virus.

Chris - So, at the same time it’s delivering a genetic message in the conventional way of viruses and gene therapy vector, you're also able to exploit the fact that the virus does carry in some proteins as part of the virus and deliver the proteins at the same time.

Jacob -   Yes.  First off, what we tried to do was just to figure out whether it’s possible to deliver these proteins.  What we’re delivering here is proteins that will then lead to a specific cut in the genome of the cells.  But to be able to repair specific mutations inside a cell, people have seen in the past that it’s possible to do this by inducing a specific break in the DNA at some specific position that you have predetermined.  And then the cell will take care of the repair mechanisms.  So, if you, at the same time provide a small piece of DNA that had some homology to that particular region in the genome, then this small piece of DNA will be used as a template for the repair.

Chris -   This is rather like a puncture repair kit for a bicycle.  You’ve got a virus particle there which brings in all of the machinery to make good the damage.  It cuts open, rather like identifying the hole in the inner tube, and then it’s even got the sticky patch inside which is the piece of genetic material you want to paste in, in order to make good the abnormality.

Jacob -   Yeah, exactly.  You put it in a perfect way.  We have called it ‘scissors and glue’ or ‘the scissors and the patch’.  So that by providing these proteins that are able to make the cut in the DNA by providing those in the virus, we think we are providing the scissors for cutting in the DNA.  At the same time, you can then have these viruses to deliver this little piece of DNA and then this DNA will be able to act as a patch that will make the repair.

Chris -   Tell us the nuts and bolts of how this actually works though.  Where are the proteins that you're carrying in inside the virus particle and how do you then package up the piece of genetic material that you want to do the repair with?

Jacob -   Right.  So, that’s of course sort of the basics of this – how to get the protein into the virus particles.  What we’re doing in this case is to fuse this particular protein to the structural proteins of the virus.  So, the smart thing about the virus and how the virus is actually working in nature is that it’s packaging all its proteins into the virus as one big protein so to speak.  It’s what is called a polypeptide.  So, this polypeptide is packaged into the virus and after budding of the virus from the cell, this polypeptide will be cut into smaller pieces.  So, when it does that, this particular foreign protein is released.

Chris -   So, that will be floating around inside the virus particle and then when it mix, goes into the cell.  It will be released in the cell where it can do its job.

Jacob -   That’s true and when it gets into the cell, it is now ready to do its action.  So, that’s what we are seeing.  That we actually have quite low amounts of this protein in the cell, but it’s far enough to make the action.  The action in this case would be to find its position in the genome, make the cut in the genome, and induce this sort of repair.

Chris -   What is the evidence that this wonderful strategy does deliver?

Jacob -   Yes, so we’ve done several things.  One thing is first, to deliver the protein in the viruses.  Not going for repair.  Just to see whether this protein has actually able to cut inside the genome.  So, we can see what we call ‘gene disruption’ very efficiently after the delivery of this virus.  So, the next thing we did was to then include the patch for repair inside the virus particles so that we do what we call ‘all-in-one particles’ where we have both the scissors and the patch in the same virus particles.  For that, we developed a system where we could screen for specific repair mechanisms.  So, in this case, we used a reporter gene, the GFP gene that has a mutation.  We are trying to repair this particular mutation.  So, we can see when we deliver these virus particles both with the scissors and patch that we have quite significant repair of this particular GFP mutation.

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