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Background and Significance

The ELP4 gene, found on chromosome 3 location 3q33 according to genome research, has been found to have a high correlation to children with Rolandic Epilepsy. It has been found that children with Rolandic epilepsy have a mutation of gene coding for the Elongator Protein Complex 4, which is involved in transcription and tRNA modification. It has been found that Elp4 is needed for histone acetyltransferase (HAT) activity which makes DNA more accessible for transcription. The lack of the Elp4/5/6 led to no HAT activity. The importance of HAT activity is the initiation of transcription as well as its assistance of RNA polymerase II in transcription elongation through chromatin and acetyl-CoA dependent pathways (Winkler 2002). The identification of ELP4 as the precipitating factor in the development of Rolandic epilepsy in children is a large step towards finding a cure for this disorder by genetic means. Although Rolandic epilepsy (RE), which has been observed as autosomal dominant with high penetrance (Bali 2007), develops around age 3 and disappears by age 12 there are serious problems that need to be addressed that occur while a child has RE. One of the major problems that can arise from RE is cognitive impairment. This occurs because of increased glucose uptake in cortical areas, most notably in the associative cortex (Strug 2009). The glucose disrupts the learning process and prevents the child form making the associations necessary to learn new things, which is how most human learning is achieved. By repairing the damaged gene that codes for the ELP4 protein the risk of cognitive issues and impairment would be greatly reduced. This topic is an important area of study because the youth that are affected by even benign Rolandic Epilepsy are at risk of developing cognitive dysfunction and at worst they may be left with significant impairment of their mental faculties, especially in the associative cortex, which is where learning takes place. This particular presentation affects about 15% of children with epilepsy and is more likely to affect boys (Holmes 2006). The proportion of those affected makes this treatment worth pursuing because of the risks that are involved with allowing a child to live with this condition even for a short while. Rolandic epilepsy, like other forms of epilepsy, has drug treatments through which a child's seizures can be controlled. While there are many treatments that have been found to reduce seizures in patients, mostly by blocking sodium and calcium channels. These drugs, however, also interfere with the endocrine, and on occasion, the immune system. In the endocrine system, it has been found that anti-epileptic drugs (AEDs) decrease secretion of many hormones, mostly the gonadal hormones. One AED, valproate, has been seen to increase insulin levels in the blood and raise body weight; while topiramate, another AED, has the opposite effect (Leskiewicz 2009). Most AEDs have been found to suppress the immune system as well. Valproate is an example of those that have been found to suppress activity, inhibit protein synthesis in lymphocytes, and cause changes or decrease plasma levels of some immunoglobulins (Basta-Kaim 2008). From these articles it can be seen that the use of AEDs, while decreasing the amount of seizures a person has, requires the patient to be on watch for malfunctions that may occur as a result, and possible cause permanent damage. An epilepsy treatment that is offered as an alternative to AEDs is surgery. A patient with epilepsy would have surgery if there condition has been found to be drug-resistant. Several different surgical techniques exist, and they are getting less invasive and most effective all the time. There has been a high success rate however they have been found to have palliative effects. Since the underlying reason for the epilepsy is not being solved, a patient can still have recurring seizures (Villanueva 2007). The Elongator Protein Complex (ELP) is what regulates the growth of cortical projection neurons. This means that it helps cortical neurons to exhibit dendrite branching and radial migration of neurons to form the close knit neural network of the cerebral cortex (Creepe et. al. 2009). If the ELP is not working properly or is not being expressed at the correct levels (too low) then the neurons in that region in particular would not be properly situated in relation to each other for proper brain activity. The expression of ELP and the fourth sub-unit (ELP4) in particular is the cause of Rolandic epilepsy and possibly other cognitive impairment later in life if the condition is severe enough or if it is not treated effectively. The use of adenovirus vectors, specifically AAV 2, can be used to introduce healthy ELP 4 genes into neurons. First the viral gene is altered to become mostly the healthy target gene; the 35 kb long viral genome can have 30 kb of it replaced by foreign DNA. Then the vector is injected into the brain, where its structure allows it to easily bind to neurons. It is then packaged in a vesicle and taken to the nucleus, where the viral DNA is released. This allows the cell to create proteins based on the healthy ELP 4 gene. This form of gene therapy is very successful due to its effective transduction into neuron cells (seen in animal testing). Furthermore, the vector generates little response from the organisms immune system, allowing it free passage into cells. The viral vector appears not to be pathogenic, and is able to stably bind to the host cells genome in a predictable manner. Therefore, the use of this virus in gene therapy is being tested on diseases such as cystic fibrosis, Parkinson’s, and Alzheimer's. So far the results have been promising. AAV has already shown good results in the gene therapy of Huntington’s disease. According to ScienceDaily, researchers at Rush University Medical Center, Chicago, and Ceregene Inc., San Diego, have successfully used gene therapy to preserve motor function and stop the anatomic, cellular changes that occur in the brains of mice with Huntington’s disease (Rush University Medical Center). The AAV modified to carry a gene for glial-derived neurotrophic factor, which would strengthen and protect the brain cells that the disease would kill. The AAV was injected into the brains of mice. The vector had been engineered to carry the gene to the specific areas of the brain where it was needed. The mice with the treatment were in considerably better condition than those that did not receive the gene therapy. Future work on this genetic modification would be to do it to young children that suffer from rolandic epilepsy. Chromosome 11 holds the ELP4 gene. This would be the desired next step for this research because it would work towards relieving the suffering of so many children. Research Plan Goal: Replace a mutated ELP4 gene in a rat with a normal functioning one that reduces seizures.

Aim 1: Isolate and replicate the healthy ELP4 gene out of a rat. This step is crucial to our process. A healthy ELP4 gene is needed in order to create a vector that can replace a mutated ELP4 gene. After the gene is isolated it has to be replicated. Replication is needed to ensure that the mutated ELP4’s are replaced by healthy ones. Isolation will be done by first isolating the DNA. A lysozyme will be used to break down the cell wall, protease K will be used to digest the outer membranes. Phenol extractions get rid of all the extra things in the cell and the nucleic acids are precipitated in ethanol. With the pure DNA, PCR is performed. Two primers will bind the ends of the ELP4 gene ( 5’ atg gcg gcg gca, 3’ ctg atc ggt tag) and make sure it is the only thing copied. After many cycles, the ELP4 gene will be the only DNA present.

Aim 2: Create a vector that contains the ELP4 gene that is able to replace the mutated gene with a normal ELP4 gene. In order to achieve the goal of replacing the mutated ELP4 gene to reduce seizures a vector must be created. In order to create this vector a normal ELP4 gene must be isolated. The ELP4 gene is located in a rat genome is location at 1 through 270 on the long arm of chromosome 3 karotype 3q33. The promoter to express this gene is also isolated from the healthy rat genome, which is 4420 base pairs and is located between 91,359,266 to 91,364, 586. Once isolated the gene and promoter are implanted into the recombinant adeno-associated viral (rAAV) vector. This is accomplished by inserting the isolated ELP4 gene into the rAAV cassette. The gene was accompanied by the cytomegalovirus (CMV)-chicken β-actin recombinant promoter (CBA), the woodchuck hepatitis virus post-translational regulatory element and a bovine growth hormone poly(A) signal. The cassette was subcloned into the vector along with rAAV2 inverted terminal repeats and a rAAV1 capsid helper plasmid to produce enough of the vector.

Aim 3: Inject the rat with vector and change its genetic structure to reduce seizures. The final aim that must be achieved in order to successfully eliminate seizures in the rat is to introduce the created vector containing the genetic sequence into the rat, which will take the place of the mutation in the rat with Rolandic epilepsy’s genome. This will be done via injections that would directly expose the internal structure of the rat to the gene-containing vector. The new desired gene sequence would then be integrated into the rat’s genome, taking the place of the mutated sequence that is causing the seizures. It is expected that the genes will have undergone gene replacement, which will inactivate the seizures do to a now normal functioning ELP4 gene.

Summary Statement Although the data and research acquired for this modification is mostly spurious, one may approach errors in both the cloning of the gene and in transfusion of this viral vector into a human subject. The ELP4 gene is one of the six protein subunits associated with the elongator protein complex which acts as subunit of the RNA polymerase II elongator complex, which is a histone acetyltransferase component of the RNA polymerase II (Pol II) holoenzyme and is involved in transcriptional elongation. The ELP4 subunit first may not be taken up or incorporated into the vector of interest. In this case, there are several vectors which can be replaced to replicate this experiment. Among these are: E. coli bacteria, yeast (primarily those of S. cerevisiae), stem cells, or even mammalian vectors. However, if the gene is successfully incorporated into the vector of interest but does not yield expression, or is unsuccessful in replacing the mutant strain of ELP4, one may seek for alternative genes through several ELP4 orthologs of various species. The Pan troglodytes (chimpanzee), with a human gene similarity of 99.29% is a prospective source, with several other orthologs following: (decreasing in percentage of human similarity) Canis familiaris (dog: 89.2%), Bos taurus (cow: 89.05%), and even that of the Mus musculus (mouse: 83.41%). With all of these, it is important to include the addition of Saccharomyces cerevisiae (baker’s yeast) subunit, which is required for the modification of wobble nucleosides in tRNA; which is thus required for Elongator structural integrity. The laboratory will be well marked as one which contains biohazard. In our experiment, we will use viral vectors (rAAV vector). The use of viral vectors has been suggested as a factor in the death of a US gene therapy patient and also in cases when leukemia-like symptoms were present (Postnote ‘05). All lab workers will be trained to know how to work with viral vectors safely. The laboratory follows university and OSHA biosafety guidelines scrupulously. Mice will be used in this experiment to test the vector containing EPL4 gene. Animal care will be supervised by an animal technician and we will consult with a veterinarian in the event of illness or injury. We will submit our protocol to the Internal Animal Care and Use Committee (IACUC) for review of the procedures. Future studies that would stem from our research proposal would include implementing the gene that codes for ELP4 into more advanced species such as primates and other highly developed mammals. The long term goal is to be able to genetically modify chromosome 11 of humans. Chromosome 11 is the location for ELP4 protein elongation in humans. For all of the children who suffer cognitive dysfunction and associative complex impairments due to Rolandic epilepsy, we would like to find a source of hope that could rid them of such detriments. Even for children who are not affected as dramatically, there still remains the risk of low self esteem from such a disorder. Children do not have the mental awareness to understand why their classmate shakes uncontrollably during various activities. This creates a difficult environment for the children with Rolandic epilepsy to make friends and feel good about themselves. These children are in an extremely delicate stage in life that will have a great impact on their personality and behaviors later on. If we can solve this problem with modification through gene therapy of the ELP4 gene, then we will be sure to make a difference in the lives of many children. This study would further open the window of genetic modification and the achievements that it has made. Success in this research would only encourage the study of other forms of epilepsy and even other diseases. Curing diseases by genetic modification heavily reduces tedious medication intake, substantially improves emotional health, and saves the valuable time of health professionals by reducing annual appointments for such illnesses.

Literature Cited (1) Benign Rolandic Epilepsy epilepsy.com/epilepsy/epilepsy_benignrolandic Topic Editor: Gregory L. Holmes, M.D. Last Reviewed: 10/21/06 (2)Strug, L. J., et al. "Centrotemporal Sharp Wave EEG Trait in Rolandic Epilepsy Maps to Elongator Protein Complex 4 (ELP4)." European journal of human genetics : EJHG (2009). (3)Bali, B., Kull, L. L., Strug, L. J., Clarke, T., Murphy, P. L., Akman, C. I., et al. (2007). Autosomal dominant inheritance of centrotemporal sharp waves in rolandic epilepsy families. Epilepsia, 48(12), 2266-2272. (4)Winkler, G. S., Kristjuhan, A., Erdjument-Bromage, H., Tempst, P., & Svejstrup, J. Q. (2002). Elongator is a histone H3 and H4 acetyltransferase important for normal histone acetylation levels in vivo. Proceedings of the National Academy of Sciences of the United States of America, 99(6), 3517-3522. (5)Villanueva, V., Carreno, M., Fernandez, J. L. H., & Gil-Nagel, A. (2007). Surgery and electrical stimulation in epilepsy - selection of candidates and results. Neurologist, 13(6), S29-S37. (6)Basta-Kaim, A, Budziszewska, B, & Lason, W (2008). Effects of antiepileptic drugs on immune system. Przegl Lek. 65, 799-802. (7)Leskiewicz, M, Budziszewska, B, & Lason, W (2008). Endocrine effects of antiepileptic drugs. Przegl Lek. 65, 795-780.

(8) Parliamentary Offic of Science and Technology(POST). PostNote : Gene Therapy June 2005