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| ZFNs are also used to rewrite the sequence of an allele by invokeing the ] (HR) machinery to repair the DSB using the supplied DNA fragment as a template. The HR machinery searches for homology between the damaged chromosome and the extra-chromasomal fragment and copies the sequence of the fragment between the two broken ends of the chromsome, regardless of whether the fragment contains the original sequence. If the subject is homozygous for the target allele, the efficiency of the technique is reduced since the undamaged copy of the allele may be used as a template for repair instead of the supplied fragment. | ZFNs are also used to rewrite the sequence of an allele by invokeing the ] (HR) machinery to repair the DSB using the supplied DNA fragment as a template. The HR machinery searches for homology between the damaged chromosome and the extra-chromasomal fragment and copies the sequence of the fragment between the two broken ends of the chromsome, regardless of whether the fragment contains the original sequence. If the subject is homozygous for the target allele, the efficiency of the technique is reduced since the undamaged copy of the allele may be used as a template for repair instead of the supplied fragment. | ||
| ===Gene Therapy=== | |||
| The success of gene therapy depends on the efficient insertion of therapeutic ]s at the appropriate ] target sites within the human ], without causing cell injury, ] mutations or an ]. The construction of plasmid vectors is simple and straightforward. The construction of ] ]s is simple and straightforward. Custom-designed ZFNs that combine the non-specific cleavage domain (N) of ''Fok''I endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific DSB to the genome, and stimulate local homologous recombination by several orders of magnitude. This makes targeted gene correction or genome editing a viable option in human cells. Since ZFN-encoded plasmids could be used to transiently express ZFNs to target a DSB to a specific gene locus in human cells, they offer an excellent way for targeted delivery of the therapeutic genes to a pre-selected chromosomal site. The ZFN-encoded plasmid-based approach has the potential to circumvent all the problems associated with the viral delivery of therapeutic genes.<ref name= Kandavelou>{{cite book |chapterurl=http://www.horizonpress.com/pla|author= Kandavelou K; Chandrasegaran S|year=2008|chapter=Plasmids for Gene Therapy|title=Plasmids: Current Research and Future Trends|publisher=Caister Academic Press|id=}}</ref> | |||
| === Problems === | === Problems === | ||
Revision as of 11:00, 15 April 2008
Zinc finger nucleases (ZFNs) are protein chimera comprised of a zinc finger-based DNA-binding domain and a DNA-cleavage domain. They are able to introduce double-strand breaks (DSB; breaks at the same or very close points in both strands of a double-stranded DNA molecule) at specific locations within a DNA molecule which may subsequently be used to disable a specific allele or even rewrite the code it contains. They are undergoing development for use in gene therapy and research applications.
DNA-binding domain
The DNA-binding domain of a ZFN may be composed of two to six zinc fingers due to their supposed modularity (appositeness to be used interchangeably). Each zinc finger motif is typically considered to recognise and bind to a three-base pair sequence and as such, a protein including more zinc fingers targets a longer sequence and therefore has a greater specificity and affinity to the target site.
Depending upon the required specifications of the end-product, the included zinc fingers may be selected via a parallel, sequential or bipartite technique or through an in vitro cell-based technique.
(see: Zinc finger chimera for more info on zinc finger selection techniques)
DNA-cleavage domain
The non-specific nuclease domain of FokI is functionally independant of its natural DNA-binding domain and is therefore employed in the construction of ZFNs. Since the domain must dimerise to accomplish a double-strand break it is necessary that a nuclease is also bound to the opposite strand by virtue of another ZFN molecule bound to its target sequence as shown in the diagram. The two target sites need not be the same, so long as ZFNs targeting both sites are present.
Mode of action
In order to form a dimer, two ZFN molecules must meet with their respective recognition sites not less than 4-6 base pairs apart but also not so far apart that they may not dimerise. While one ZFN molecule binds its target sequence on one strand, another ZFN molecule binds its target sequence on the opposite strand, as shown in the diagram. The nuclease domains dimerise and each cleaves its own strand, producing a DSB.
Applications
Disabling an allele
ZFNs can be used to disable dominant mutations in heterozygous individuals by producing DSBs in the mutant allele which will, in the absence of a homologous template, be repaired by non-homologous end-joining (NHEJ). NHEJ repairs DSBs by joining the two ends together and usually produces no mutations, provided that the cut is clean and uncomplicated. In some instances however, the repair will be imperfect, resulting in deletion or insertion of base-pairs, producing frame-shift and preventing the production of the harmful protein.
Allele editing
ZFNs are also used to rewrite the sequence of an allele by invokeing the homologous recombination (HR) machinery to repair the DSB using the supplied DNA fragment as a template. The HR machinery searches for homology between the damaged chromosome and the extra-chromasomal fragment and copies the sequence of the fragment between the two broken ends of the chromsome, regardless of whether the fragment contains the original sequence. If the subject is homozygous for the target allele, the efficiency of the technique is reduced since the undamaged copy of the allele may be used as a template for repair instead of the supplied fragment.
Gene Therapy
The success of gene therapy depends on the efficient insertion of therapeutic genes at the appropriate chromosomal target sites within the human genome, without causing cell injury, oncogenic mutations or an immune response. The construction of plasmid vectors is simple and straightforward. The construction of plasmid vectors is simple and straightforward. Custom-designed ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific DSB to the genome, and stimulate local homologous recombination by several orders of magnitude. This makes targeted gene correction or genome editing a viable option in human cells. Since ZFN-encoded plasmids could be used to transiently express ZFNs to target a DSB to a specific gene locus in human cells, they offer an excellent way for targeted delivery of the therapeutic genes to a pre-selected chromosomal site. The ZFN-encoded plasmid-based approach has the potential to circumvent all the problems associated with the viral delivery of therapeutic genes.
Problems
Cytotoxicity
A lack of specificity of the zinc finger domains used in construction of the ZFNs leads to recognition of secondary degenerate sites that they were not intended to target. Recognition and cleavage of secondary sites may lead to the production of many DSBs which may overwhelm the reparation machinery. Gross mutations then result through the joining of broken ends of different parts of the same chromosome (leading to massive deletions) or even joining ends belonging to different chromosomes.
Increasing the specificity of the zinc finger motifs used is expected to reduce the production of unintentional DSBs and reduce toxicity. Since each zinc finger typically recognises three consecutive bases, increasing the number of zinc fingers present in the protein increases the specificity of the protein to a particular DNA locus.
Random integration
One possible result of a DSB accompanied by an extra-chromasomal fragment is that the DNA fragment is incorporated into the chromsome through two instances of NHEJ (an inherently mutagenic event). Thus preventing NHEJ as well as promoting the use of the extra-chromsomal fragment as an HR template are both issues that must be considered in order to rewrite an allele using this method.
Immunogenicity
Further information: Adaptive immune responseAs with many foreign proteins inserted into the human body, there is a risk of an immunological response against the therapeutic agent and the cells in which it is active. Since the protein will only need to be expressed transiently however, the time over which a response may develop is short.
Prospects
The current methods of zinc finger selection are currently prohibitively expensive and demanding in expertise, precluding many laboratories from generating their own complete zinc finger proteins. To these laboratories, the only option is simple assembly by parallel selection, producing zinc finger proteins in which the constituent zinc fingers were not selected within the context of each other. This tends to produce a ZFP of lower affinity and specificity since non-compatible zinc fingers may interfere with each other's DNA interactions. ZFNs produced through these methods may be suitable for research purposes, but therapeutic applications will be likely to require ZFPs of the quality produced using more expensive selection proceedures.
The first therapeutic applications of ZFNs are likely to involve ex vivo therapy using a patients own stem cells. After editing the stem cell genome, the cells could be expanded in culture and reinserted into the patient to produce differentiated cells with corrected functions. The initial targets will likely include the causes of monogenic diseases such as the IL2Rγ gene and the b-globin gene for gene correction and CCR5 gene for mutagenesis and disablement.
See also
References
- ^ Durai S, Mani M, Kandavelou K, Wu J, Porteus MH, Chandrasegaran S (2005). "Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells". Nucleic Acids Res. 33 (18): 5978–90. doi:10.1093/nar/gki912. PMID 16251401.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - Gommans WM, Haisma HJ, Rots MG (2005). "Engineering zinc finger protein transcription factors: the therapeutic relevance of switching endogenous gene expression on or off at command" (PDF). J. Mol. Biol. 354 (3): 507–19. doi:10.1016/j.jmb.2005.06.082. PMID 16253273.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - Bitinaite, J. (1998). "FokI dimerization is required for DNA cleavage". Proc Natl Acad Sci U S A. 95 (18): 10570–5.
{{cite journal}}: Unknown parameter|coauthors=ignored (|author=suggested) (help) - Mani M, Smith J, Kandavelou K, Berg JM, Chandrasegaran S (2005). "Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage". Biochem. Biophys. Res. Commun. 334 (4): 1191–7. doi:10.1016/j.bbrc.2005.07.021. PMID 16043120.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - Kandavelou K; Chandrasegaran S (2008). "Plasmids for Gene Therapy". Plasmids: Current Research and Future Trends. Caister Academic Press. ISBN 978-1-904455-35-6.
{{cite book}}: External link in|chapterurl=|chapterurl=ignored (|chapter-url=suggested) (help)CS1 maint: multiple names: authors list (link)
Further reading
- Mandell, J. G. and C. F. Barbas, 3rd (2006). "Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases." Nucleic Acids Res 34 5978-5990.
External links
Zinc finger selector Zinc Finger Consortium website
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