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 Alteration of Iris Color (Melanin Production) is Achieved via Tetracycline­Mediated Modification of OCA2 and HERC2 Expression in Human Iris Stromal Cells Abstract: The goal of this project is to alter the iris color as a cosmetic procedure by genetically controlling melanin production. HERC2 and OCA2 are the primary genes that contribute to eye color, specifically the varying alleles of their Single Nucleotide Polymorphisms associated with brown, hazel, green, or blue eye colors. HERC2 transcriptionally regulates OCA2 using a long range chromatin loop to activate or deactivate OCA2. OCA2 codes for the P protein which is the precursor to melanin; the degree and amount of melanin causes varying eye color. We will use an HSV­1 viral vector to replace these color determining alleles within the SNPs of both HERC2 and OCA2 in the uveal melanocytes of the eye. The vector will also contain a Tet­On system to promote and regulate the expression of these modified genes. The whole system will be activated by doxycycline, and when a color change is desired the system will be applied to the cells using eye drops. To change the melanin production in order to achieve a new iris color, the genes of the existing color will be repressed and replaced with the genes of the desired color. (Julia and Bailey) Background and Proof of Novelty: We will try to change eye color using gene therapy. The following process will be used on people mostly for aesthetic reasons. In our appearance­obsessed society where people are always searching for more permanent cosmetic changes, people may not want to the trouble of wearing colored contact lenses everyday, so they will opt for a more permanent solution by means of genetic therapy. For more practical reasons, some people may want to have darker eyes in order to be less sensitive to sunlight. (Tori) The sclera is a tough layer of cells that extends to completely cover the eye for protection. In the back of there is a layer of cells called the retina, which contains cones and rods which are photoreceptors which are involved in capturing light and converting it to neural impulses to transmit to the optic nerve. Behind the retina is also the choroid, a layer that nurtures and sustains the retina. The cornea is a layer of cells that covers the iris, pupil, and anterior chamber and is used for focusing light so that it goes through the pupil. The anterior chamber (not labeled on diagram) is located behind the cornea and before the iris and pupil, and it stores the aqueous humor (liquid found in the eye).​ ​The iris is the muscle behind that, and it is the colored portion of the eye. The pupil is a hole in the iris, in which light is able to enter. The lens is located behind the iris and pupil, and it helps the cornea by further focusing the light onto the retina. The vitreous gel is the portion of the eye that is composed of a jelly­like substance that is located between the lens and the back layers of the eye. The macula is the region of the retina that is directly behind the pupil. It produces vision that is very clear and allows people to see straight in front of them.The fovea is a pit in the center of the macula, and it produces the sharpest and most accurate vision. The optic nerve is at the back of the eye and it is a bundle of nerves that transmits neural information from the retina to the brain (Anonymous 2013). Our project will be focusing on targeting the iris of the cell because it is the region of the eye that contains the pigment that we are going to change. The iris is composed of two layers. One layer is called the iris pigment epithelium (IPE) and it is closer to the back of the eye. The other layer is called the iris stroma. There is melanin found in both layers of the iris. It is thought that the amount of melanin in the IPE is about the same in eyes of different colors, so we will be targeting the melanocytes (cells that produce melanin) in the stroma of the iris. In melanocytes, there are organelles called melanosomes which store melanin, and it is thought that the number and size of these melanosomes can cause differences in eye color, not the number and size of the melanocytes themselves (C 2007). (Samiha) Figure 1. Basic anatomy of the eye (Fox 2014) Melanin, a pigment produced by cells in the iris, controls eye color (as well as hair and skin color). Tyrosinase (TYR), Tyrosinase­related 1 (TYRP1), and Dopachrome Tautomerase (DCT) are the essential enzymes called melanosomes which produce pigment production in organelles. The two ­types of melanin associated with eye color are eumelanin (a dark black­brown) and pheomelanin (a red­brown). (researched and written by Tori and Julia) The outer­layer of the iris’s amount and ratio eumelinan and pheomelinan regulates eye color. Brown or darker eye colors in general contain a greater amount of melanin, specifically more eumelinan in relation to pheomelinan. Blue and green eyes, or lighter eyes in general, contain less melinan overall. Eyes of a color somewhere in between, such as hazel or bluish­brown, contain intermediate amounts of both. (Converse 2011) (researched and written by Tori and Julia) Numerous experiments and studies have been done to determine what exactly causes eye colors to differ. Eye color is not at all simple trait; there are many factors that contribute to it. Physically, the ratio of dark brown eumelanin to reddish pheomelanin (types of melanin) and the way in which they are distributed determines the color of the eye. Classifying eye colors into brown, blue, and green is an oversimplification as well. (McDonald 2011) In an experiment performed by Duffy et. al of Queensland Institute of Medical Research, all the OCA2 exons that encode P­protein were sequenced. The strongest associations between blue and non­blue eye colors were found for three SNPs, rs7495174 T, rs6497268 G, and rs11855019 T in the intron 1 haplotype block. (Duffy et. al. 2006, 241­252) Additionally, the OCA2 gene is located on chromosome 15 from the base pair 27,754,874 to 28,099,336. It is found on the long arm of this chromosome (15q). (Anonymous 2014a) (Tori) Figure 2. OCA2 controls the production of pigment. It is located on chromosome 15. (Anonymous 2014a) (Tori) Single nucleotide polymorphisms (SNPs) are considered to be the most common occurrence of genetic variation in humans, and they essentially are the differences in a single nucleotide. (Such as cytosine (C) with varying with nucleotide thymine (T) in a certain stretch of DNA in different people.) SNPs are normal for humans, and they generally occur approximately once in every 300 nucleotides. Therefore the human genome contains nearly 10 million SNPs. Such variations frequently occur in the DNA between genes. They sometimes pose as biological markers which can allow scientists to locate genes associated with diseases. SNPs are found within a gene or in a regulatory region close to a gene, affecting the gene’s function or expression. However the vast majority of SNPs do not affect health or development. such as toxins, and risk of developing particular diseases. (Anonymous 2014e) (Julia) In HERC2, rs12913832, located on intron 86, is arguably the most important SNP in the determination of eye color. As the genetics of eye color are extremely complicated, this SNP mainly controls whether a person has brown or non­brown eyes. AA and AG code for most likely brown eyes and GG codes for most likely non­brown eyes. (Yablonovitch et al. 2012) (Julia) Additionally, in a study performed by Sturm et. al. for the American Journal of Human Genetics, the experimenters found that rs12913832 in the HERC2 gene regulates the transcription of the OCA2 gene. Previously, researchers determined three SNPs in the OCA2 gene that they found to have a strong correlation that indicated the expression of brown eyes versus the expression of blue eyes. However, in this experiment, researchers also discovered that a single nucleotide difference in which there is a C allele in the HERC2 gene decreases the expression of the OCA2 gene, which they concluded lead to the expression of brown eyes over blue eyes (Sturm et. al. 2008). OCA2 seems to be responsible for the physical production of the P protein (which leads to the production of melanin), while the HERC2 gene seems to control the expression of OCA2. So, HERC2 is probably the main indicator of a person’s eye color. Therefore, a major part of our goal is to try to control the HERC2 gene, so that we can regulate the expression of OCA2 and thus regulate melanin production. (Samiha) HERC2 regulates OCA2 with a long range chromatin loop. So when HERC2 rs12913832 has the T­allele the transcription factors HLTF, LEF1, and MITF will bind to this region on the HERC2 enhance. The factors then create a long range chromatin loop with the OCA2 promoter. This ultimately amplifies the expression of OCA2 and leads to darker pigmentation in the eye. When HERC2 rs12913832 has the C allele these factors do not bind, no loop is established and the OCA2 expression is lessened; this is seen in lighter colored eyes (Visser 2012). (Bailey) Figure 3. Determination of eye color based on the regulation of OCA2 (Sturm et al 2009). Additionally, a haplotype, or group of genes, strongly associated with blue eye color is located on 3’ region of HERC2. A study has shown that rs1129038 and rs12913832 exactly associate with blue eyes. This suggests that this haplotype is a founder mutation, which interestingly may have originated in the North West part of the Black Sea region, and it was possibly naturally selected for its rarity and desirability. Independent genome wide association studies and a genomewide linkage study including close to 3000 people from the Netherlands were done, and it was found that the chromosome 15q13.1 region is the most important region associated with human’s iris color. SNPs in the HERC2 gene and, to a lesser extent, in the neighboring OCA2 gene were each independently involved with variation in iris color (Converse 2011).
HERC2 goes back and forth between the nucleus and cytoplasm and functions as an E3 ubiquitin ligase for the “ubiquitination and degradation of target proteins”. It works as an activator of other E3 ubiquitin ligases, and also adapts for the assembly of DNA damage response proteins. (Converse 2011) OCA2 and HERC2 work together to create the production of pigments. As previously stated, the SNP located in the HERC2 intron 86 is the most important factor in determining whether eyes will be brown or not brown, while a single halotype in the 3’ region of the HERC2 region that directly causes blue eyes. (Yablonovitch et al. 2012)
Specifically the OCA2 gene, located on the 15q11.2­15q12 chromosome, controls P­proteins and plays a major role in eye color. OCA2 associates with HERC2 to control pigment production. Often polymorphisms, (natural variations in genes, DNA sequences, or chromosomes), within the OCA2 gene account for the differences among human’s eye color. (Yablonovitch et al. 2012) The most specific and important function of OCA2 is its ability to code for the P­protein, which transports tyrosine, the precursor of melanin within melanocytes. It also plays a role in regulating the pH of melanosomes and the maturation of melanosomes. Additionally it is believed to control the post­translational tyrosinase process in melanin synthesis (Converse 2011). In California, a scientist named Gregg Homer is developing a way to turn brown eyes blue using lasers. The general idea of the procedure is that laser energy will be absorbed by the pigment, the pigment tissue will change, and changed tissue will be shed and not regenerated. This removes the pigment from the surface of the iris and turns brown eyes blue in two to three weeks. This procedure is still being researched and a release date has not been estimated. Critics have raised concern that this procedure may lead to pigmentary glaucoma, and the permanence and efficacy of the procedure remains unknown. Therefore, we have developed a different approach to this problem that may be favorable. Also, our procedure has a wider array of color change options, and it is inducible. (Homer 2012) (Tori) Scientists specializing in dermatology at the University of Cincinnati College of Medicine examined ocular pigment in comparison to cutaneous pigments, which are found in the skin. It was found that unlike skin, melanin is not produced in the iris as a result of UV exposure. In fact the alpha­melanocyte­stimulating hormone (alpha­MSH) that helps regulate the melanin production resulting from UV light does not significantly affect ocular melanocytes. The presence of tyrosine and other proteins would have indicated melanocyte proliferation from UV exposure. However, a Northern blot analysis proved that these proteins were not present. This study applies to our gene therapy as it determines what will not work for signaling an amplification in melanin in the eye when transforming light colored eyes to a darker one. Pigmentation in the eye is not regulated by a melanocyte activating hormone related to UV light (Li et al. 2006). Departments of Ophthalmology and Visual Sciences and Biostatistics at the University of Wisconsin Medical School investigated if the concentration of melanocytes varies between eye colors. There is not a significant difference in the number of melanocytes the concentration of melanocytes between eye colors. It was found that 65.9% of the iris stroma is composed of uveal melanocytes which works out to be that the average melanocyte number is 778±196 per 5­μm section of the stroma. (Wilkerson et al. 1996) Therefore, it seems that the amount of production of the melanocytes is what the genetics control, not the number of melanocytes. By decreasing the production levels the amount of melanin should eventually result in a noticeable change in eye color. We do not need to produce more melanocytes just regulate the melanin production in melanocytes. Research has been done to explore the effectiveness of a tetracycline regulated systems on the genes of eukaryotic cells. It is known that the system work on a wide range of species including mammals, making this applicable to controlling human genetics. The Tc system can be useful as it makes genes inducible, either on or off. However, the use of the mechanism must be exact as the effectiveness of the promoter depends on the location and correct delivery. Still, the Tet On/Off system is stable and reliable once it has been properly applied. Most commonly the virion protein 16 domain of the HSV­1 integrated with a human cytomegalovirus promoter. The system was tested at the Center for Molecular Biology of Heidelberg University using HeLa cells to control the expression of a luciferase gene. With the presence of Tc, luciferase was induced and expressed, and will not be produced when Tc is absent (Gossen 1992). We will be using a Tet­On System in our procedure to promote the expression of our modified genes. (Bailey) Several viral vectors have been used over the course of gene therapy research as vehicles by which new genes can be delivered to cells. Some viruses, such as oncoretrovirus and lentivirus, are favored due to their capacity to integrate their full genomes with the cell’s own genome. Others, such as AAV and adenovirus, are employed because they introduce genetic material as separate “episomes” that affect the cell’s gene expression differently. A fifth virus type, Herpes Simplex Virus, has drawn recent interest due to the quantity of unique beneficial characteristics it contributes to gene therapy (Thomas et. al. 2003). Herpes Simplex Virus (HSV­1) is a pathogen about which much has been discovered. It is an excellent viral vector for a number of reasons: it has a high genetic storage capacity and is very infectious. The facility with which it infects the nervous system has made it a promising tool in neurobiological gene therapy, but in this particular case this high rate of infection presents a specific difficulty (cf. Project Description). Two glycoproteins expressed by the virus, gB and gC, bind readily to proteoglycans on the cell surface and allow a third glycoprotein, gD, to initiate the virus’ passage into the cell. Viral reproduction is lytic in most cells, but it exhibits a latency when it infects neurons (Lim 2013). A number of HSV­1 modifications have been suggested in hopes of producing a variation of the virus that is most efficient as a viral vector. One method involves the inactivation of one of two genes expressed during the IE (immediate early) phase of viral infection, designated ICP4 and ICP27. Unfortunately, these viruses remain cytotoxic specifically because the expression of one of these genes is still sufficient to compromise the cell (Johnson et. al. 1992). Other methods targeting multiple genes have seen more success (Wu et. al. 1996). A more effective alternative treatment, however, has been attempted using HSV­1 with modifications to both these genes and a third gene called VP16, which helps induce the IE cascade (Preston et. al. 1997). (Jesse) Project Description: We chose to alter eye color in humans for aesthetic purposes. Certain SNPs have high associations with different eye colors. We plan on altering the already existing SNPs in the OCA2 and HERC2 genes in order to create the desired color. (Tori) The version of the HERC2 gene that a person carries is a strong indicator of whether a person will have blue eyes or brown eyes. However, there are other variations in the OCA2 gene which can also affect the expression of eye color. Therefore, we will be using SNPs to change nucleotides in both the HERC2 and the OCA2 genes, to better increase the chances of changing the patient’s eye color. We could also try to apply this project to change eye color in people with heterochromia, which is a condition in which a person has a different eye color in each eye. There are various causes of heterochromia, some environmental and some genetic, which can produce this condition (Kaneshiro et. al. 2013; Iko and Walter 2013). If it is a purely a genetic cause and the patient was born with heterochromia, we could simply give them an eye drop in the eye of which they wish to change color. (Samiha) In order to make make blue eyes, we will need to perform multiple site­directed point mutations on the OCA2 gene. In the first intron of chromosome 15 there are three SNPs that favor blue eyes. The existing nucleotides on rs6497268 will have to be switched to AC, on rs11855019 to AG, and on rs7495174 to AG. These SNPs have high associations with the color blue, although the last has an association with blue/green which could introduce variability in the outcome, so people could decide to not switch that particular SNP in order to have more certainty that the final color would be blue. (Yablonovitch et al. 2012) These associations are for caucasians. In order to make brown eyes, we will perform a site­directed point mutation on rs1800401for CT. (Yablonovitch et al. 2012) This will then favor brown eyes. It is located in exon 7 at amino acid position 305 of the OCA2 gene. (Anonymous 2014d) For a green/hazel color, rs1800407 must have the nucleotides AG. It is located in exon 13 at amino acid 419 of the OCA2 gene. However, only about 4 percent of eye color variation is explained by this nucleotide position, so it would have to be in addition to another nucleotide mutation. (Anonymous 2014d) These associations are for caucasians. In intron 86 of the HERC2 gene, rs12913832 is an important SNP for eye color determination. If this SNP has AA or AG, then eyes will most likely be brown. If this SNP has GG, then eyes will most likely non­brown. (Yablonovitch et al. 2012) This gene is likely more significant in determining eye color than OCA2, so SNPs from both genes will need to be altered to ensure the correct color is produced. (Tori) Thus, we will utilize a site­directed point mutation to change the nucleotide sequence (a single base change), specifically we will use the oligonucleotide synthesis method. (Tori) Oligonucleotides, which are essentially short, single­stranded​ DNA​ molecules produced in a laboratory, can be created with any desired sequence; thus, they are vital for​ artificial gene synthesis​ and the regulation of advanced gene expression. The custom synthesis is useful because the created oligonucleotide will only bind to the region of DNA that is complementary to our necessary sequence, allowing amplification and or restriction of certain DNA segments. Additionally, the custom oligonucleotides are most often 15­20 bases long. (Holmberg 2003) (Julia) We will add nucleotides to the oligonucleotide chain in the required order to properly make the product. The new chain that we are making will be identical to the old one, except it will have changes in these particular alleles of the SNPs. The number of potential errors increases with longer oligonucleotide chains, so it is best to keep it shorter. The products can then be isolated by high­performance liquid chromatography in order to have high purity of our desired oligonucleotides. This will be ordered from a company, and we must desalt the heterogeneous mixture to obtain the desired oligonucleotide. (Anonymous 2014b) (Tori) The materials needed include: a Commerical Nucleic Acid Synthesizer, a solution of the four DNA phosphoramidite monomers, or bases, because each of the 5’­hydroxyl groups must be blocked with a DMT group for all four bases​, ​a cyanoethyl group to block phosphorus linkages​, blocking solutions​, ​reaction chamber and a type of solid support (we will use controlled pore glass) already prepared with the desired first base already attached, via an ester linkage at the 3’­hydroxyl end, dichloroacetic acid or trichloroacetic acid​, ​tetrazole​, ​acetic anhydride and N­methylimidazole, dilute iodine in a water/pyridine/tetrahydrofuran solution,​ ​concentrated ammonia hydroxide, and materials for one desalting method. (Holmberg 2003) (Julia) Figure 4. The phosphoramidite oligonucleotide synthesis cycle (Anonymous 2014b) (Tori) The method of choice is phosphoramidite oligo synthesis. It proceeds in the 3′­ to 5′­direction and one nucleotide is added per sequence. The 5’­DMT protecting group must be removed from the support­bound nucleoside through detrilytion. Next, activation and coupling occur in which the support­bound nucleoside reacts with phosphoramidite and is altered into a support­bound phosphite triester. Next is a capping step that works to prevent an accumulation of deletion mutations. Since the coupling cannot react 100%, there remains unreacted 5’­hydroxyl groups that are blocked in this step through acetylation (specifically, we will add acetic acid and N­methylimidazole that are dissolved in tetrahydrofuran and pyridine, and mix them on the DNA synthesizer). Then, we will perform iodine oxidation in the presence of water and pyridine to create a cyanoethyl group that will prevent undesirable reactions as the process moves forward. Next, the 5’­end of the resin­bound DNA chain will be removed by deprotection with trichloroacetic acid in dichloromethane. This must be done in order for the primary hydroxyl group to react with the next nucleotide phosphoramidite. This process is repeated until the desired oligonucleotide is produced (one base added per cycle). We can assess whether this process is working using commercially available DNA synthesizers that monitor the efficiency of synthesis in real time. When the primary hydroxyl group is cleaved off, an orange color is produced and its intensity can be used by these machines to determine efficiency. With a small number of bases(10­15), the coupling efficiency should be over 97%. (Anonymous 2014b) (Tori) The process of oligonucleotide synthesis will be repeated to make OPTC genes in order to ensure that they will be present to code for anti­opticin antibodies on the surface of the cell produced virions. The expression of the anti­opticin antibodies ensure that the virus will target only eye cells expressing opticin (the essential protein expressed in certain eye cells). (Julia and Jesse) We will therefore be introducing the changes before the introns are removed from the pre­mRNA, because the nucleotide in the rs12913832 of the HERC2 gene (which regulates transcription of OCA2) is in an intron (Sturm et. al. 2008). So, we need to insert the gene before the introns are spliced out before the translation of the gene. (Samiha) Table 1 below details the eye colors and their corresponding gene types, SNPs, and alleles. In the process of changing the eye colors, we will look to the existing color’s alleles (of the gene type’s SNP) and change each of them to the desired color’s alleles with the synthesized oligonucleotide through our viral vector (Anonymous 2008). (Julia) Color Gene Type SNP Allele Brown HERC2 Rs12913832 AA / AG Brown OCA2 Rs1800401 CT Brown OCA2 Rs1800401 CT Non­brown* HERC2 Rs12913832 GG Blue OCA2 Rs11855019 AG Blue OCA2 Rs6497268 AC Blue/green OCA2 Rs7495174 AG Green/hazel OCA2 Rs1800407 AG Table 1: Color variation depending on gene type, SNP, and specific allele *HERC2 is the dominant gene in eye color determination, so HERC2 must express “non­brown” in order for OCA2 to have any significant effect with its expression of colored eyes. (Yablonovitch et al. 2012) (research and made by Julia and Tori) The required genetic material will be transferred to the cells in question by way of modified Herpes Simplex Virus 1 (HSV­1), a common virus about which much is known, and which has the capacity to store approximately 150 kB of DNA in its genome (Burton et. al. 2002). Normal (non­modified) HSV­1 generally binds to cells by way of glycoproteins B and C (gB and gC, respectively), which in particular bind to proteoglycans on the cell membrane. Entry of the virus into the target cell is initiated as glycoprotein D binds to one of two proteins, known (among other names) as Herpesvirus Entry Mediator (HVEM) and nectin­1. Once the virus has passed through the cell membrane, its genes are added to the cell’s genome and expressed in three very specific “cascades.” The first genetic expression phase, called the IE (immediate) phase, is largely responsible for the infection and death of cells containing the HSV­1 genes. This is then followed by the E (early) and L (late) phases of gene expression, totaling somewhat greater than 80 expressed genes (Lim 2013). Multiple steps will be necessary in order to prepare the HSV­1 specimens for infection. An alternative method of viral preparation, described by Preston et. al., suggests that the VP16 gene (expressed in the IE phase), which is partially responsible for the destruction of infected cells during viral replication, can be altered such that the cells are not destroyed and virions can still be produced (Preston et. al. 1997). We recommend that, along with this mutation, the proteins expressed on the outer coat of the virus be altered. Our suggestion is that gB and gC be replaced by the opticin antibody, which will interact with the opticin protein expressed only by cells in the eye (Friedman et. al. 2002). Although some research initially suggested that opticin was also expressed in the optic nerve, this was later determined to be mistaken (Friedman et. al. 2002). The use of this antibody will ensure the specificity of HSV­1 by disabling its ability to bind globally to any cells expressing proteoglycans, and ensuring that it binds only to cells expressing opticin in their extracellular matrices. Viruses will initially be directly modified to express only anti­opticin, and will also be modified to contain genes for both eye color change and the expression of anti­opticin, simultaneously removing the genes responsible for the expression of gB and gC. This virus modification will then be grown in a HeLa cell line so that all viruses will express the appropriate proteins (Anonymous n.d.). Once the viruses are ready for treatment, they will be placed in a saline solution and dispensed into the patient’s eye as eyedrops. Care must be taken to ensure that the solution comes into contact only with the eye, and contact with the skin and hair especially should be avoided. (Jesse) We will be using a Tetracycline On System to promote expression of HERC2 for better control over the modified gene. The Tet­On system allows us to make the modified target gene (HERC2) dependent on being induced by a transcriptional activator. We will be using the reverse tetracycline­controlled transactivator (rtTA) which must be binded at tetracycline­responsive promoter element (TRE) in order for the target gene to be expressed (Anonymous 2014c). The TRE consists of the Tet operator (tetO) sequence combined with a minimal promoter sequence extracted from the human cytomegalovirus. The Tet­On system is ultimately controlled by the doxycycline (Dox) which is of the Tc class of antibiotics. Only if Dox is present can the rtTA recognize the tetO and bind, and then the target gene will be expressed. The Tet­On system will be incorporated into the Herpes Simplex virus vector we are using to modify HERC2. The mechanism described above will be on the VP16 domain, as it is a strong transactivating region of the viral vector (Baron et al. 2000). Dox is usually taken orally and is actually more effective as an inducer for the Tet­On System than using Tc. Dox will be used in our procedure as the concentration needed to activate the Tet­On 0.01–1 µg/ml is low and safer. Dox has a longer half life than Tc which makes it ideal for long term use, which therapy could potentially be (Bartlett et al. 1975). (Bailey) Figure 5. The Tet­On System activates expression of desired target gene in presence of Dox. There is no expression when Dox is absent. The Dox ( a Tc derivative) will be delivered using eye drops so that people can activate the changed eye color whenever they want to. However, when they do not apply the Dox, the eye color will revert back to its natural state, as the original genes with be expressed instead. We do not know the exact amount of time this change should take, but with the Tc regulated system it should more efficient and easier to control than if we did not utilize tetracycline. In summary, we will utilize tetracycline on the HERC2 gene to regulate the OCA2 gene. (Yablonovitch et al. 2012 researched by Samiha) OCA2 encodes for the P­protein which is involved in the transport of tyrosine which leads to melanin production. (Duffy et. al. 2006, 241­252) Thus, regulating HERC2 will therefore regulate melanin production and thus eye color. To test the efficacy of this gene therapy, we can determine whether the eyes have changed color from the original color (before altering any genes). (Tori) Ethical Concerns and Honest Reflection: This project idea does not directly improve lives or help people’s health, so although learning about the genetics of eye color is very interesting, it is not a particularly needed life­saving field. We also realize that there are already existing colored contacts that people can buy to temporarily alter the appearance of their eye color, so our project is only helpful for people who want to permanently change their eye color. (Julia and Samiha) Altering OCA2 is potentially dangerous because a procedure that does not function properly or as expected may cause genetic mutations. For example, oculocutaneous albinism could occur if there is a large deletion in the OCA2 gene, Angelman syndrome can develop if one copy of the OCA2 gene is missing in cells, and Prader­Willi syndrome can develop if the region of chromosome 15 that contains OCA2 is deleted. ​(Anonymous 2014a) The strong associations between variations in SNPs and differing eye colors are for predominantly caucasian populations. Therefore, the procedure may only be applicable to white populations. Also, due to the complexity of the genetics of eye color, it seems unlikely that we will be able to attain the optimal color gradient desired. (Tori) Lighter eye colors such as blue, green, hazel eyes contain less melanin and are therefore more sensitive to the sun, much like lighter skin tones are. Our method of changing eye color through pigment production could theoretically be an adjustment for a well protected brown eyed person to become less protected if they become blue eyed. Because unlike skin, the eye will not produce more melanin as sun exposure increases. Therefore, an individual electing to lighten their eye color may need to take extra precautions in the sun, like wearing sunglasses. In the opposite situation, in which an individual wants more melanin production to create darker eye colors, there is a possibility for error. Uveal melanoma is a cancer of the eye in which uveal melanocytes proliferate unregulated and metasize. It leads to dark pigmented tumors in the eye that can metastasize, which kills about 50% of those with uveal melanoma. It is possible that our treatment, by increasing melanin will alter the melanocytes genetically in ways we cannot foresee. It could potentially, although not likely cause uveal melanoma. Decreasing the protective melanin in brown eyes could also put an individual at the same risk for developing melanoma as a naturally blue eyed person (Landreville et al. 2011). ( Bailey) We do not exactly know how long the whole process would take. For changing from blue eyes to brown eyes, this process often occurs naturally in children. Many children are born with blue eyes, but usually at about one year old, they begin to transition to brown eyes. This is because it takes a significant amount of time for the melanin to store up in brown eyes (Griffin 2013). So, theoretically this process could take 1­3 years if we just let it occur naturally. However, we believe that the Tet­ON system will speed up the process of changing eye color, so that we could start to see the desired results in a day or up to a few days. We know that we need to change a nucleotide on an intron to change the expression of our gene, but we are not very sure why we need to do this. Introns are spliced out of the mRNA during RNA processing, and so they should not contribute to the expression of the HERC2 gene. The exact pathway of how the introns affect HERC2 expression is still not very clear to us. (Samiha) Thomas et. al. refer to the immune response to viral vectors as the “achilles heel” of this gene therapy treatment option (Thomas et. al. 2003). With most viral vectors, the risk of immune response must be taken into account, and in the case of HSV­1 a particular difficulty will be the tendency of iris cells to initiate the inflammatory response when infected, a condition called ​iritis (Baldwin et. al. 2013). It is possible that further genetic alteration of the virus may possibly avoid this response by inactivating whatever initially sets off the response in the eye. (Jesse) Future Applications: An extension of this project could be to try to add a variety of eye colors that patients could choose from, rather than offering just natural eye colors. (Samiha) Also, these different eye colors could be manipulated to be triggered by the expression of other genes. For example, the eye colors could change based on a person’s mood (release of endorphins could trigger the expression of blue eyes). This extension is very complex, because constantly adding or taking away melanin in the stroma to change eye color may be harmful to the eye. (Tori) A similar process to ours could be used to trigger pigment changes in those with no production of melanin, such as a partial treatment for albinism. Oculocutaneous albinism type 2 is caused by the OCA2 gene or the P gene albinism, produce not enough P­protein for tyrosine or low activity of melanocytes because of OCA2 gene, so our therapy could potentially assist in treating some forms of albinism. There is a plethora of vision problems associated with the lack of pigment in the eye cause by this condition. One of which the therapy could treat in photophobia (sensitivity to light). This can cause functional problems in the eye as it attempts to see despite the fact the light is uncomfortable. Our treatment could potentially supplement current surgeries and devices that are being used to aid individuals with oculocutaneous albinism type 2 (Anonymous 2002). (Bailey) This gene therapy could possibly be applied to animals such as dogs or cats. The genes that control their eye color may be different than in humans, but the process of altering these genes once found would be similar. 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