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Precision Gene Editing
mpthompson writes "NewScientist.com is reporting that scientists at Sangamo Biosciences have developed a method of editing DNA mutations with unprecedented precision without weaving in potentially harmful foreign genetic material. Different combinations of amino acids are designed to latch on and cut the DNA at exactly the place where the mutated gene lies. This triggers the body's natural repair process which corrects the gene where the DNA was cut. The technique will be used to target diseases caused by single-gene mutations such as combined immune deficiency (X-SCID) - or bubble boy disease - and sickle cell anaemia."
TFA noted that the zinc fingers cue in on two sets of 6 base pairs to find the site that needs correction. Assuming randomness in the base-pair sequences, this 12 base-pair key will bind with approximately 1 out of every 16.8 million (actually 1 out of every 8.4 million due to complementarity of the base pairs). Given that the human genome has about 3.2 billion base pairs, this means that the modifier will match in 381 positions more or less.
Thus, this method will fix the error in one place and introduce an error in 380 other locations. The key needs more than 16 base pairs to be statistically assured of homing in on a unique mutation (depending on the statistics of DNA, it may need more or less).
Two wrongs don't make a right, but three lefts do.
I have not read the article, but repair processes can be "error prone". That is, the mechanisms cells use to repair DNA often involve high error rates.
/.ers may not appreciate is that typically, it is VERY, repeat VERY hard to get chemcial reaction specificity of anywhere close to 1e9 for reactions invovling DNA.
The human genome is 3e9 BP long (roughly..not counting indels, the unsequenced centromeres, etc etc)
So the chemical process of identifying the one single mutated basepair has to have a chemical specificity of >>1e9, because there are >>1e6 cells that are exsposed. That is, lets say you feed the reagent to a person. Millions of cells, each with 1e9 bp, are expsosed. Say the process has an error rate of 1e10 - many, many cells will have incorrect repairs done
This is just like error rates in, say, reading data from a harddrive: the larger the file, the lower the error rte has to be
What
I will rtfa,
In order to answer your question, i'm going to have to give a little background...
contrary to popular belief, 99.99% of the body's cells don't keep dividing. The somatic cells of the body are replenished by stem cells and progenitor cells which act as the main copy from which all the "backup" cells are made. These cells specialize into skin cells, blood cells, and possibly nerve cells. The only way to have a permanent effect with this treatment would be to fix the mutation in the stem cells/progenitor cells, so that future specialized cells will all have the fix incorporated.
To make this change heritable, you need to fix the mutation in the sperm or egg which is eventually used to create an embryo. Otherwise, the mutation will be passed on.
From what the article says, there's only an 18% transformation efficiency so of all of the cells treated (this would never be the entire human body, just the cells collected), only 18% will be fixed.
We are a long way off from doing the 100% effective gene therapy you see on Star Trek.
Highly efficient endogenous human gene correction using designed zinc-finger nucleases
FYODOR D. URNOV1, JEFFREY C. MILLER1, YA-LI LEE1, CHRISTIAN M. BEAUSEJOUR1, JEREMY M. ROCK1, SHELDON AUGUSTUS1, ANDREW C. JAMIESON1, MATTHEW H. PORTEUS2, PHILIP D. GREGORY1 & MICHAEL C. HOLMES1
1 Sangamo BioSciences, Inc. Pt. Richmond Tech Center 501, Canal Blvd, Suite A100 Richmond, California 94804, USA
2 Department of Pediatrics and Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas 75390, USA
Correspondence should be addressed to M.C.H. (mholmes@sangamo.com) or M.H.P. (matthew.porteus@UTSouthwestern.edu); requests for materials should be addressed to M.C.H.
Permanent modification of the human genome in vivo is impractical owing to the low frequency of homologous recombination in human cells, a fact that hampers biomedical research and progress towards safe and effective gene therapy. Here we report a general solution using two fundamental biological processes: DNA recognition by C2H2 zinc-finger proteins and homology-directed repair of DNA double-strand breaks. Zinc-finger proteins engineered to recognize a unique chromosomal site can be fused to a nuclease domain, and a double-strand break induced by the resulting zinc-finger nuclease can create specific sequence alterations by stimulating homologous recombination between the chromosome and an extrachromosomal DNA donor. We show that zinc-finger nucleases designed against an X-linked severe combined immune deficiency (SCID) mutation in the IL2Rbold italic gamma gene yielded more than 18% gene-modified human cells without selection. Remarkably, about 7% of the cells acquired the desired genetic modification on both X chromosomes, with cell genotype accurately reflected at the messenger RNA and protein levels. We observe comparably high frequencies in human T cells, raising the possibility of strategies based on zinc-finger nucleases for the treatment of disease.
Most human monogenic disorders remain difficult to treat because therapeutic transgenes do not undergo homologous recombination (HR) into the mutated locus1, 2, and gene addition by virus-driven random integration remains a challenge owing to transgene silencing, improper activity or misintegration3, 4. Furthermore, targeted alteration of DNA sequence in vivo--in principle, a powerful basic research technique for studying genome function--in mammals requires sophisticated targeting vectors and drug-based selection1, 2, which limits the use of this approach5-7.
The C2H2 zinc-finger, originally discovered in Xenopus8, is the most common DNA binding motif in all metazoa9. Each finger recognizes 3-4 base pairs of DNA via a single alpha-helix10, 11, and several fingers can be linked in tandem to recognize a broad spectrum of DNA sequences with high specificity12-14. Engineered zinc-finger protein (ZFP)-based DNA binding domains with novel specificities have been extensively applied in vivo to target various effector domains12, 15. Work from the Chandrasegaran laboratory has shown that a ZFP can be coupled to the nonspecific DNA cleavage domain of the Type IIS restriction enzyme, FokI, to produce a zinc-finger nuclease (ZFN)16, which then cuts the DNA sequence determined by the ZFP16, 17. An important specificity mechanism derives from the requirement that two ZFNs bind the same locus, in a precise orientation and spacing relative to each other, to create a double-strand break (DSB; Fig. 1a)17. One mechanism by which eukaryotic cells heal DSBs is homology-directed repair (Fig. 1b)18-20, which transfers information missing at the break from a homologous DNA molecule (Fig. 1b). Work from the Jasin laboratory21, followed by that of others22, 23, demonstrated that the endonuclease I-SceI can potentiate HR into loci previously engineered to contain its own recognition site, and the Carroll24, 25 and Baltimore26 laboratories have shown that a ZFN-invoked DSB increases the rate of HR in model systems.
Figure