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Therapeutic use of zinc fingers in serious diseases


The use of zinc fingers in permanent gene therapy
Aaron Klug Scientist
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They took this SCID gene. Now the SCID gene it's known what it is. And it's a single monogenic disease but it occurs in different places and different patients. It isn't like sickle cell anaemia where there's... every single patient has the same mutation, in the very same place, but the mutations tend to be clustered, they tend to be clustered in one of the X arms which is 100 base pairs along but you can make zinc fingers for any particular target. As I said they are cheap to make. So in order to do research on it, there's a system of cells called K562 cells in which the SCID mutation has been transplanted into human kidney cells. So this is a model system but it's got the SCID mutation in. And this K562 cells are basically leukemic cells. If you introduce them into mice, you can put the human cells into mice and then mice get leukaemia. So this is the object of the experiments at Sangamo.

So the K562 cells were... Sangamo made... Michael Holmes made a zinc finger nuclease using four zinc fingers, two twos, remember I said things were made in twos? Four zinc fingers attached... to a catalytic domain to target... if this is the mutant site... four zinc fingers to bind to one side carrying the catalytic domain which then would be in position over the mutant, over the mutants of the desired site. Another group of four zinc fingers with another catalytic domain and the two catalytic domains interact, they make homodimers, they homodimerize. Each makes a single strand you cut them together you make a double stranded cut. So they made double stranded cuts and then they introduced an extra chromosomal DNA donor, a DNA donor which carried the right sequence and this was a piece of DNA which is several hundred bases long, with the correct sequence, put into a plasma which is a thousand bases. And they did this, of course this was all experimental medicine really. They did this with different amounts of donor DNA, extra chromosomal donor DNA rather than then donor... so the donor is now the piece of DNA rather than the chromosome because the sister chromosome also has, of course, the same, it's a homozygous disease, also has this... also has this defect. And the... and now so you have something which is a pretty unique target, you've got four zinc fingers, four zinc fingers, that's twelve base pairs on either side and they are a certain fixed distance apart. And the chances of that occurring anywhere else in the genome is negligible. One of the chaps there calculated it, it's ten to the minus 22, or something which means it's negligible. So they created this and they said... they did rough and ready experiments with different amounts of dosage, different amounts of nuclease and so on. And they found that as they improved the efficiency, they were getting anything from 15% to in the end 21% correction. So 21% of all the cells are corrected. In some cases, the 21% or the 20% is 16% on one chromosome and 4% on the other. So this is correcting, basically they can correct... you only have to correct one chromosome really, but that would be enough, so that showed. And they also did study to make sure there would be no disruptions anywhere else in the genome. The referees were pretty hard in all this, you had to demonstrate that.

Now, the other thing about this editing is that in the model experiments, which I talked about earlier, but done by Carroll and Porteus the corrections weren't stable gradually they, so to speak, died out and these things reasserted themselves. So by southern blotting, which looks at the genome sequence of the... they showed that after a month, Sangamo showed that these corrections in the K562 cells were stable. And after six months they're still stable so it looks as if they've made permanent modification of the mutations in these genes and it's now more than six months now but I think they would have to report if they've now reverted their lifetime. So this means they're in a position to apply to the FDA for, probably, for a licence to start clinical trials. And for clinical trials they'd have to use the precursor cells, the original cells from the patients. So what they've done in the meantime, although that hasn't been published, so they have reported it at meetings, they've taken what are called CD34 cells which are immunoglobulin cell line and these are primary cells, they're not artificial cells they're really immunoglobulin precursor cells and they've corrected those and they haven't published that because that would require a whole lot of extra labour. I don't know what the complications are... they haven't published that. So it seems that... and they're in touch with a man at the Children's Hospital, UCLA who has SCID patients and I think that... anyway we'd have to start handing it over to the medics, to the consultants, and the... one worry is... is an adverse immune reaction because this after all is, in some sense it's foreign, it's not very foreign because they're so many zinc fingers in the nucleus anyway and the paths of these zinc fingers are all exactly the same. Clearly the exact amino acid sequence isn't the same as any natural sequence because these genes aren't switched on by zinc fingers, so there'd have to be some kind of testing, maybe they'll be required to be done in animals, I don't know enough about it. But it seems to me that... there's a good chance... that... permanent gene therapy will be achieved in the not too distant future, and this has been done by zinc fingers so I find it very gratifying to see these things put to this use.

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, Michael Holmes

Duration: 7 minutes, 25 seconds

Date story recorded: July 2005

Date story went live: 24 January 2008