Effects of Zinc Finger Domain Quantity in Zinc Finger Nucleases on Homologous Recombination Frequency

Kurt Whittemore
Custom designed zinc finger nucleases have been used to create double strand breaks in the genomes of several different organisms. When homologous DNA is introduced into a cell with a double strand break, the cell can use homologous recombination machinery to incorporate this DNA fragment into the genome at the site of the break. This process may be useful for gene therapy applications, allowing for the successful integration of exogenous DNA into a host genome. Although incorporation of foreign DNA into a host genome using zinc finger nucleases has been demonstrated, the success rate is low. In fact, zinc finger nucleases have generally shown a great amount of cytotoxicity. The number of DNA binding domains (“fingers”) present in zinc finger nucleases may affect the degree of cytotoxicity, and the frequency of successful homologous recombination could be measured against the number of domains present in a zinc finger nuclease.

Background and Significance Gene therapy is the technique of inserting foreign genes into the cells of an organism in order to correct a problem such as a hereditary disease caused by a nonfunctional gene. The field of gene therapy research is still in its infancy, and there are many details that need to be researched before the technique can be widely used. One of the details that must be better understood involves how to deliver the foreign DNA. Researchers have tried using viral vectors such as retroviruses (1), adenoviruses (2), and envelope protein pseudotyping of viral vectors (3). Resesearchers have also tried using non-viral vectors such as naked DNA (4), lipoplexes (4), polyplexes (4), and dendrimers (5) in order to introduce foreign DNA into an organism. Another detail that must be understood before gene therapy is widely used deals with how to maintain the genetic material as an episomal fragment (separate from any chromosome) or incorporate the genetic material into the host genome once the foreign gene(s) is in the cell. Once these details are well understood, gene therapy may be capable of curing many inherited diseases. The potential for gene therapy is great, as several successes have already demonstrated, but there have also been some major failures.

Many research groups have obtained a great deal of success with gene therapy. Researchers at the The University of Texas M. D. Anderson Cancer Center and the University of Texas Southwestern Medical Center were able to suppress human tumor growth in the lungs of mice with two tumor suppressor genes delivered with lipid based nanoparticles (6). Another gene therapy trial used an adenovirus to deliver a gene (AtohI) to stimulate hair growth in order to successfully restore the hearing of deaf mammals (7). One research group was able to treat sickle cell anemia in mice (8). Yet another research group was able to restore vision in RPE65-deficient dogs using gene therapy (9). These are just a few examples which demonstrate the huge potential of gene therapy to cure diseases and alleviate human suffering. Although there have been some successes achieved using gene therapy, there have also been many failures. Human gene therapy trials were tried in France to correct Severe Combined ImmunoDeficiency Disease (also known as “bubble boy syndrome”). This disease is caused by a defective IL2R-gamma gene, which is involved with the immune function of white blood cells. However, these human trials were later halted when one of the patients of the gene therapy developed leukemia (10). Since leukemia is a cancer usually attributed to the excessive proliferation of white blood cells, the researchers felt that the leukemia may have been caused by the gene therapy trials. Another tragic event occurred in 1999 when 18 year old Jesse Gelsinger died of multiple organ failures after four days of a gene therapy treatment with an adenovirus intended to treat a liver problem (11). These events were a major setback for gene therapy researchers, and gene therapy is still recovering from the stigma it acquired during these failed trials. There is no doubt that gene therapy has resulted in some tragic outcomes, but researchers are beginning to identify some of the problems with these previous gene therapy attempts. One of the major problems deals with the vector chosen to deliver genes to cells. The immune system often responds adversely to the vectors used in gene therapy research up to the current time. Once the vector problems are overcome, researchers must ensure that the genetic material inserted into a cell is properly

cared for. There are currently four known possible outcomes for this genetic material. The DNA can be degraded by the cell, it can be maintained as an episomal fragment, it can be used as a template for the repair of a double strand break using homologous recombination, or it can integrate randomly into the genome (12). Gene therapy could be improved by researching techniques to address each one of these outcomes. In other words, if one could keep the DNA from being degraded, discourage the maintenance of an episomal fragment (if integration with the genome is the desired outcome), enhance/ aid homologous repair machinery, and inhibit random integration machinery, then gene therapy could potentially be much more effective. In this paper, the focus will be on how to aid homologous repair machinery by creating precise double strand breaks in the genome. An excellent tool for creating a precise double strand break is a zinc finger nuclease. Zinc finger nucleases are modified zinc finger proteins. Zinc finger proteins are a type of transcription factor commonly found in eukaryotes. The structure of a typical zinc finger protein is characterized by an alpha helix which interacts with 3 bp segments of DNA (13). A typical zinc finger protein can have multiple “finger” domains which each act with successive 3 bp segments independently of one another (14). Each finger is a fairly small globular domain which lacks a large stabilizing hydrophobic core. Instead, zinc fingers stabilize their small structure with positively charged zinc ions which interact with negatively charged histidine and cysteine residues (15). The modular nature of the zinc fingers allows them to be interchanged so that zinc fingers can bind a wide variety of unique DNA sequences. In fact, there are some natural occurring zinc fingers that contain over 60 zinc fingers, allowing them to recognize a unique 180 base pair sequence (15). In order to make a zinc finger nuclease from a zinc finger protein, a research group attached a restriction domain to a zinc finger protein. FokI is a restriction enzyme, which unlike many other restriction enzymes, contains a non-specefic restriction domain which is independent of the DNA sequence binding domain. Kim and Chandrasegaran created a new nuclease by designing a zinc finger protein with this non-specific restriction domain attached (16). In order to create a double strand break,

the restriction domain must form a dimer with another identical restriction domain (17). Therefore, in order to cut the genome in a specific place, two zinc finger nucleases must be designed which recognize DNA sequences near one another. There must be 4-6 base pairs of separation in order for these two different nucleases to interact with one another favorably (18). An interesting feature of these nucleases is that they can accomplish a double strand break without the requirement that they must bind to the same recognition sequence. Therefore, it is not necessary to cut the DNA at a palindrome sequence (19) even though this is often performed in practice. The feature that makes zinc fingers so versatile is their modularity. Each finger can bind to its DNA codon independently of the others, making it possible to design a zinc finger which can bind to virtually any sequence. There are currently some limitations on this versatility though. There are only known zinc fingers that bind to 49 of the 64 possible codons. All of the 5'-GNN-3', many ANN, many CNN, and some TNN can be bound by known zinc finger domains (20). In order for zinc fingers to be capable of binding to any sequence, it will be necessary to design or discover zinc fingers which bind to the other codons as well. Currently 6 zinc fingers can be linked together in order to bind an 18 bp sequence, not 160 zinc fingers as exist in some natural zinc finger proteins (21). Also, although zinc fingers are modular and customizable, zinc finger design has not yet reached the point at which a nonexpert could design one of these versatile transcription factors. The company Sangamo, which now has a monopoly on the market, has arisen in order to provide the service of designing complex zinc finger proteins (22). Research may progress more quickly in the future when zinc finger protein design is so well understood that a non-expert can create his/her own zinc finger proteins. Despite the current limitations of zinc finger protein technology, a wide variety of work has been accomplished with them. Zinc fingers can be fused to activation domains, repression domains, methylase domains, and cleavage domains (21). These different forms of zinc fingers make it possible to turn genes on and off at will, permanently inactivate certain genes, and cut at specific locations respectively. Cutting at specific locations with a zinc finger nuclease has a few different applications.

One group has used zinc finger nucleases to knock out genes in C. elegans (23). This process works by allowing the cell to repair the double strand break through the error prone non-homologous end joining method. However, nucleases can also be used to incorporate foreign DNA fragments. This is done by introducing the zinc finger proteins to some genomic DNA as well as a foreign DNA fragment flanked by regions of the sequence which are homologous to the genomic DNA at the site of the double strand break. After the double strand break occurs, the cell can use its homologous repair machinery to incorporate the foreign fragment into the genome. Erica A. Moehle et. al. has demonstrated that even fragments as large as 8 kb can be incorporated using this method (24). Although zinc finger nucleases have been used to incorporate foreign genetic material into a host genome, the homologous recombination frequency is still low and the cytotoxicity of zinc finger nucleases is still present. Some of the best reported homologous recombination frequencies reported are as high as 18 % (25). However, this percentage could be greatly improved. A common belief among researchers seems to be that the cytotoxicity of zinc finger proteins is due to the amount of specificity they have. If a zinc finger protein is not very specific, then there is a high probability that the nuclease could cut multiple locations in the genome, ultimately causing cell death. It may be possible to make zinc finger nucleases less cytotoxic by increasing their specificity through an increase in the number of fingers used. Proposed Experiments A series of experiments could be done to directly establish whether or not there is a link between zinc finger number and cytotoxicity. It is reasonable to think that increasing the number of fingers used would decrease cytotoxicity and increase homologous recombination frequency. The cytotoxicity would decrease because the zinc finger nuclease would be more likely to cut at the intended location and not anywhere else in the genome. Homologous recombination frequency would increase because there would be less of a chance for the exogenous DNA to incorporate at the incorrect location. However, until an actual experiment is performed, these assessments are just reasonable

assumptions without any supporting evidence. An experiment used to test the correlation between zinc finger domain quantity and cytotoxicity could involve varying the number of zinc fingers and measuring the cytotoxicity. First, one would need the zinc finger nucleases for the experiment. Since zinc fingers are currently still fairly complicated to design, it would be best to order zinc finger nucleases from Sangamo. Three sets of zinc finger nucleases would be ordered. All of these zinc fingers would bind to exon 5 of the IL2R-gamma gene. This is a good region of the genome to target for several reasons. First, zinc fingers have already been designed for this region of the genome so the quality of the zinc fingers would be more ensured. Second, this region of the genome has successfully been targeted in HEK293 cells so the region of this genome must be accessible and not excessively bound to histones. Third, the IL2R-gamma gene is the gene which is defective in Severe Combined Immuno Deficiency Disease (SCID) so this work would have potential therapeutic applications. Each set of ordered zinc fingers would contain two zinc finger nucleases. One zinc finger would bind to the DNA upstream of the location of the cut. The second zinc finger would bind to the sequence downstream of the location of the intended cut so that there were six base pairs of separation between the two zinc finger proteins. The zinc finger nucleases in the first set would contain three fingers. The ZFNs in the second set would contain four fingers, the ZFNs in the third set would contain five zinc fingers, and the ZFNs in the fourth set would contain six zinc fingers. Once the zinc finger sets had been ordered and designed by Sangamo, they would be introduced into human cells (not a human patient). The best way to introduce these proteins would be to transfect HEK293 cells with the genes coding for the appropriate zinc finger nucleases. HEK293 cells would be good cells to use for this procedure because this particular cell line of human embryonic kidney cells is particularly easy to work with and has been well characterized. These same type of cells were used in the Erica A. Moehle et. al. experiment used to insert a gene at the same IL2R-gamma site (24). The cells would also be transfected by using an adenovirus to insert the gene for GFP flanked by stretches

of DNA which were homologous to the DNA at the site of the double stranded break at the IL2Rgamma site. After this process, there would be five groups of HEK293 cells. All groups would have the GFP with homologous sequences, but they would have different zinc finger nucleases. One would have 3 finger ZFNs, 4 finger ZFNs, 5 finger ZFNs, 6 finger ZFNs, and one group would be the control with no ZFNs at all. After the cells were allowed to live and grow for some time, the effects of the transfection would need to be determined. Two different features of the transfection could be checked. The cytotoxicity of each ZFN set could be checked using an MTT assay. The homologous recombination efficiency could be checked using two different complementary methods. One method would involve extracting the genomic DNA, isolating the region of interest with PCR, running the PCR product on a gel to check fragment length, and then sequencing. The other method would involve fluorescence activated cell sorting (FACS) to count the number of cells expressing GFP, which would indicate the number of cells that had undergone a successful homologous recombination. The cytotoxicity of each ZFN set would be checked using an MTT assay. An MTT assay measures cellular growth by exposing cells to yellow 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (26). This compound is reduced to purple formazan in the presence of mitochondrial reductase enzymes which are only active in a living cell. Therefore, by measuring the absorbance of a solution of cells with the compound at a certain wavelength of light, one can determine the viability of the cells. This would allow one to conclude the amount of cytotoxicity each ZFN set expressed. Homologous recombination would be measured using two different methods, the first of which would involve extracting the genomic DNA. Genomic DNA would be extracted from the cells using a kit. Then a PCR would be performed with primers to amplify the IL2R-gamma region. The product PCR DNA would then be run on a gel. The gel could be used to quickly determine the size of the DNA fragments between the two PCR primer recognition sequences. These fragments could then be

extracted from the gel, and sequenced to further validate that the GFP gene was inserted without mutations. Since sequencing is a more costly procedure than some of the other methods, this would probably just be done on one or two fragments. The second homologous recombination check would involve the FACS method. The FACS method would take advantage of the fact that cells which had undergone successful homologous recombination would express green fluorescent protein and glow. Fluorescence-activated cell sorting is a special type of flow cytometry in which cells are sorted into different containers one at a time based on their fluorescent properties (27). The basic process works by running the solution of cells along with a stream of liquid which separates the cells so that the distance between them is much greater than their diameter. Then the liquid passes by a fluorescence detector which then imparts a charge on that region of the liquid based on the fluorescence. The stream of liquid is then broken into droplets which are deflected into different containers depending on their charge. As the cells pass by the detector in this experiment, the number of fluorescent cells would be recorded, which would indicate the number of cells that had undergone successful homologous recombination. Possible Results and Interpretations The preceding experiments could provide a great deal of information about the role the number of zinc fingers in a ZFN plays. If more zinc fingers lead to decreased cytotoxicity as predicted, then it is likely that zinc fingers are cytotoxic as a result of cutting at more than one place on the genome. The results of the MTT assay would reveal such information. If there is a very strong purple color, then the cells are alive and the ZFNs in that cell would not be very cytotoxic. Quantitative values for each group of cells would be determined using Beer's Law. Mathematical analysis would reveal if any of the differences in absorbance between groups of cells with different numbers of zinc fingers was statistically significant. The control group would act as a baseline, indicating how many cells dye naturally without any zinc finger proteins present. A great deal of data could be obtained from extracting the genomic DNA, amplifying the region

of interest with PCR, and running a gel. The gel would reveal how large the DNA fragment between the two primer locations around the IL2R-gamma gene became. If the GFP gene underwent homologous recombination with the IL2R-gamma gene, then the region of interest would be larger than normal. Therefore, when the DNA fragments were run out on a gel, the fragments from the cells with ZFNs should travel less far from the well than the fragments from the control cells without ZFNs. However, it is likely that the fragments from the HEK293 cells with ZFNs will have two bands on the gel. One band for cells that underwent homologous recombination, and one band for those that didn't. The relative intensity of these bands could be used to determine an approximate proportion of cells that underwent homologous recombination to those that did not. Sequencing a few of the bands would reveal how error prone the homologous recombination process was. Ideally there would be no errors. The FACS method should reveal similar information to the PCR method. Both methods would measure the amount of homologous recombination that occurred. The FACS method would reveal this information by counting how many cells in each group fluoresce, indicating how many underwent homologous recombination. The data will not be perfect for various reasons. One of the major reasons the data will not be totally accurate deals with the fact that cells can undergo random integration a small percentage of the time even without a double strand break (28). Therefore, even the control would likely have a few fluorescent cells. Some of the control cells may also maintain the GFP gene as an episomal fragment, which could also result in some fluorescence. Mathematical analysis of the data for the number of fluorescing cells in each group would reveal if any of the differences were statistically significant. If the groups with the highest number of zinc fingers had the greatest number of fluorescent cells, then the results would indicate that more zinc fingers lead to decreased cytotoxicity and/or more successful homologous recombination. One of the most important pieces of data that these experiments could produce is not just whether zinc finger number affects cytotoxicity and homologous recombination frequency. This data might also set the standard for how many zinc fingers are used in a zinc finger nuclease in future

experiments by other research groups. For example, if it turns out that four finger ZFNs lead to much lower cytotoxicity than three finger ZFNs but have no significant difference from five finger ZFNs, then the new standard might become four finger ZFNs. This number of ZFNs would provide the required specificity without requiring extra effort to design ZFNs with “extra” fingers. Once the optimal number of zinc fingers is found, any further improvements in homologous recombination frequency would need to come from understanding other mechanisms. For example, one might need to inhibit proteins that degrade foreign DNA, or increase the expression of the proteins responsible for homologous recombination. Deducing the optimal number of zinc fingers to use in a zinc finger nuclease would be very useful information for future researchers to have. Conclusion The potential therapeutic benefits of gene therapy are vast. However, there are still many details that must be understood before gene therapy can be widely used to cure human diseases. Some of these details involve the mechanisms required to transport genetic material into the desired cells. Other details deal with what happens to foreign DNA once it is inside the cell. Foreign DNA can incorporate into a host genome if there is a double strand break present. Zinc finger nucleases give researchers the tools needed to induce DSBs at specific sites in the genome, but are also cytotoxic. A series of experiments involving an MTT assay, a PCR experiment, and a FACS technique could reveal if there is a direct correlation between the number of zinc fingers and their cytotoxicity. Even more importantly, these experiments could reveal the optimum number of zinc fingers to use in ZFNs in future human gene addition experiments. References 1) Nienhuis AW. (2005) Lentiviral vectors for the treatment of Wiskott-Aldrich syndrome. Gene Ther. (7):555-6. 2) Shiba H, Misawa T, Iida T, Okamoto T, Futagawa Y, Sakurai M, Ohashi T, Eto Y, Yanaga K. (2008) Adenovirus vector-mediated gene therapy using iodized oil esters for hepatocellular carcinoma in rats. Anticancer Res. 28(1A):51-3. 3) Sinn PL, Burnight ER, Hickey MA, Blissard GW, McCray PB Jr. (2005) Persistent gene

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