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Experiments with zinc fingers: 'The mouse ear model'


Zinc finger work with different viruses
Aaron Klug Scientist
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Vascular endothelia growth factor, and this is a growth factor which starts a cascade of gene expression to develop the vasculature in most of eukaryotes. It's the, like many of eukaryotic genes, it's spliced, there are six spliced isoforms of which three are pretty important. And so, one of the thing that has been done by many people, or tried by many people is to switch off VEGF in certain cancers because the tumours recruit blood vessels and they use VEGF, they also use other growth factors, FG, FBGF and so on, and so in the Sangamo lab they switched off the VEGF, in a number of cases, but I don't think it's reached therapeutic applications. The other thing we did here with Monika Papworth was to inhibit virus expression of herpes simplex virus, that was done here, using just six fingers. You needed six fingers to do it on rather than three fingers and we used our libraries. This is herpes simplex virus and we chose that deliberately because it's a very, highly infective. It carries with it its own VP16s, are activation they use in the virus particle and it carries six immediate early genes. And all of these, these genes interact with each other and there's a second set of genes which is the early genes, these are the immediate early genes, so as a kind of exercise we switched on, we decided to target a gene called 175K which is the most important of the immediate early genes. And using six fingers, using three fingers we were only able to reduce the infective titre by a factor of 20% but by six fingers we were able to reduce it by 90%, a ten-fold reduction, so that wouldn't be enough to... so if you wanted to use it as a therapeutic for virus you'd really have to target all the other immediate early genes. So you have to target at a very early stage because then by the time the virus starts replicating it's making more and more virus and you haven't got a hope. That's why we target the immediate early genes.

And we did the same, or rather I say we did, this time it was done by Yen Choo and Lindsey Reynolds at... Gendaq and they targeted HIV. And this was the, we made a lot of zinc finger constructs, partly they were made here, partly at Gendeq and you simply looked at the HIV promoter region and what you do, since we have now... zinc finger production is automated, high throughput automation, and they're cheap to make, you don't worry too much about looking for accessible regions in chromatin, which people did, what Sangamo used to do. It's like scatter bombing, you simply scatter enough zinc fingers around and you hope that some of them will be able to get through the chromatin structure. There was a lot of fuss made about determining chromatin structure. A man called Alan Wolffe, one of the leading people in the field who died unfortunately, advocated this, but it's not necessary. Because the promoters may be 300-bases long and you could usually find some way through. In fact, we found that you could switch off HIV expression in HeLa cells with the three-finger peptide. This surprised me no end until I discovered something about the life cycle of HIV. When HIV enters the cell, it picks up very cellular transcription factors including something called SP1, which is a three-zinc finger, a well-known three-zinc finger transcription factor, it uses the cellular factors. And by sheer chance, because we were simply doing, as I say, scatter targeting, this zinc finger we'd chosen bound the... obviously prevented the SP1 binding which meant that the other domain, the non-DNA binding domain of SP1 couldn't be put into play. So it stopped, actually, gave a pretty good result. And so we did things of this sort. So I think I decided that we could do more of these things, but we'd got enough proofs of principle and I think for virus diseases, anyway, you might use other targets, either vaccines or else small molecules and so on. So the... that's why I switched on to mitochondria.

Born in Lithuania, Aaron Klug (1926-2018) was a British chemist and biophysicist. He was awarded the Nobel Prize in Chemistry in 1982 for developments in electron microscopy and his work on complexes of nucleic acids and proteins. He studied crystallography at the University of Cape Town before moving to England, completing his doctorate in 1953 at Trinity College, Cambridge. In 1981, he was awarded the Louisa Gross Horwitz Prize from Columbia University. His long and influential career led to a knighthood in 1988. He was also elected President of the Royal Society, and served there from 1995-2000.

Listeners: John Finch Ken Holmes

John Finch is a retired member of staff of the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK. He began research as a PhD student of Rosalind Franklin's at Birkbeck College, London in 1955 studying the structure of small viruses by x-ray diffraction. He came to Cambridge as part of Aaron Klug's team in 1962 and has continued with the structural study of viruses and other nucleoproteins such as chromatin, using both x-rays and electron microscopy.

Kenneth Holmes was born in London in 1934 and attended schools in Chiswick. He obtained his BA at St Johns College, Cambridge. He obtained his PhD at Birkbeck College, London working on the structure of tobacco mosaic virus with Rosalind Franklin and Aaron Klug. After a post-doc at Childrens' Hospital, Boston, where he started to work on muscle structure, he joined to the newly opened Laboratory of Molecular Biology in Cambridge where he stayed for six years. He worked with Aaron Klug on virus structure and with Hugh Huxley on muscle. He then moved to Heidelberg to open the Department of Biophysics at the Max Planck Institute for Medical Research where he remained as director until his retirement. During this time he completed the structure of tobacco mosaic virus and solved the structures of a number of protein molecules including the structure of the muscle protein actin and the actin filament. Recently he has worked on the molecular mechanism of muscle contraction. He also initiated the use of synchrotron radiation as a source for X-ray diffraction and founded the EMBL outstation at DESY Hamburg. He was elected to the Royal Society in 1981 and is a member of a number of scientific academies.

Tags: Sangamo Therapeutics, Gendaq, Monika Papworth, Yen Choo, Alan Wolffe

Duration: 5 minutes, 15 seconds

Date story recorded: July 2005

Date story went live: 24 January 2008