Chad's Research

For some (Consider using people, more specific than some) suffering with a genetic disease, the only current treatment option is organ transplant followed by a life of immunosuppressant medication to prevent rejection of the transplanted organ. (Good opening sentence, but maybe a bit lengthy. Maybe put a period after transplant, and begin a new sentence like "These people receiving these transplants are plagued by a life of immunosuppressant...) Research scientists (Researchers) are currently exploring many options to treat genetic diseases at the source. One current treatment possibility is utilizing the viruses that have plagued mankind since the beginning of time to alter the incorrect genetic sequence in diseased individuals. This option is not only pleasing because of the ability to treat the individual without the use of a donor or immunosuppressant medications, but it also avoids conflicts such as embryonic stem cell use. Researchers have been able to isolate viruses, remove deadly genetic sequences, insert a useful treatment sequence and achieve positive results in animal models. (maybe a brief information on host vs. graft disease would be helpful to draw a reader in) I understand what you mean by this but you should try to explain a little bit more because in the first sentence is kind of confusing your introduction

Viral-mediated gene therapy utilizes the replication process of viruses in order to correct mutations to mutations in the human genome by inserting the corrected gene into the viral genome. In animal models, viral replication generally follows a five-step process: attachment, entry, synthesis, assembly, and release. In the attachment step, virion particles make contact with the cellular surface through random contact (virions are nonmotile.) The attachment of a virion particle with the surface of an individual cell is based upon the interaction between receptors on the surface of the cell and the particles composing the capsid or proteins on the surface of the virion.(Are there specific receptor-viral proteins for this interaction, or is it variable, just depending on the virus?) The next step of viral replication Entry differs depending upon the type of virus. For bacteriophages, inserting the viral genome into the host cell involves using the virion tail like a needle and then shooting the genome through the tail into the host cytoplasm. For animal viruses, there are three different methods of entry: direct penetration, membrane fusion and phagocytosis. Direct penetration inserts the genome in a similar manner to the bacteriophage, however, there is no tail for insertion. Membrane fusion utilizes a membrane envelope surrounding the virus to fuse with the membrane of the host, allowing the entire virion entry into the host cell. Phagocytosis also allows for (Consider deleting for) the entire virion to be incorporated into the host cell, but utilizes rearrangement of the host cytoskeleton to be brought into the cell instead of fusing with the host membrane 1.

Once the virion is inside the host cell, synthesis may begin to occur. Once again, different viruses have different ways of producing proteins needed for further steps of the replication process. For a dsDNA virus, the process of replication, transcription and translation of the DNA sequence is similar to that of the host genome. Replication of the ssDNA viral genome occurs in a slightly different manner. Once the ssDNA molecule has entered the cell, a complementary strand of DNA is synthesized and binds to the original ssDNA strand. Further synthesis is identical to the dsDNA model. Another category of viruses includes those that contain RNA as their genetic material as opposed to the DNA viruses. In an RNA virus, the genetic material is used as the mRNA strand to synthesize viral proteins, and to complete replication of the RNA strand. One type of RNA virus that does not follow this pattern is the retrovirus. Retroviruses complete their replication cycle by utilizing reverse transcriptase to convert the RNA genome into a DNA genome. This newly synthesized DNA genome is used as the pattern for future synthesis of viral proteins and replication back into the RNA genome 1.

Many researchers are interested in utilizing viral-mediated gene therapy for many genetic diseases. To develop an effective therapy , five main components need to be established: the gene, the vector, vector delivery, target tissue , and animal models 2. (a linked between these two sentences is needed) Current diseases being studied include dystrophin-deficient cardiomyopathy, prostate cancer, methylmalonic academia, brain tumors and cystic fibrosis among others.
Really feels like there needs to be a transition here just feels like it jump **
Muscular dystrophy is a genetic disease, which is currently only treatable through heart transplantation or symptom-relieving medication. As researchers analyze a genetic disease, a **one
main component for developing a vector is creationng of the DNA stretch that is to be inserted into the genome for genetic correction. In theory, one would expect that replacing the entire gene where a mutation occurs would clear all problems. As researchers analyzed muscular dystrophy, they have found that there are problems with using an entire gene for therapeutic reasons. One reason why an entire gene cannot generally be used is size constraints, often an entire gene is too large to fit into a viral vector. A second reason is that the entire gene cannot be used in many cases is due to the sequence of amino acids within the gene. In the dystophin (dystophin or dystrophin? i wasn't sure) gene, a region of the gene is a cleavage site for a viral protein 2.

Another hurdle to in gene therapy is where to deliver the modified viruses in order to achieve optimal results. For some diseases, the answer it seems clear that the affected tissue is the only area that needs the corrective therapy. When considering dystrophin correctional therapy of the heart, the answer is not as simple. For some organs, such as the heart, associated tissues such as skeletal muscle and vascular smooth muscle have dystophin (dystrophin?)components that also play a role in normal heart function. Without treating these tissues in addition to treating the heart, it is possible that the treatment will not have the most positive results possible 2. This issue also gives rise to another question when treating specific tissues for a mutation. When treating a tissue, how much of that tissue needs to be corrected before a treatment can be considered successful or before a tissue can function properly?I dont think you should have questions in this paper

There are several options for viral mediated gene therapy in cancer cells. (wait, how did we get to cancer?) (I would like to see somewhere here that cancer is caused by genetic mutations) These options include correction, toxin release, recruitment of immune response, and designing a virus that recognizes and lyses preferentially (preferrentially lyses)tumor cells. Correction to the mutated sequence would eliminate the uncontrolled replication of these cells and return the cell to normal function. Releasing toxins would not only target the infected cell for death, but would also affect surrounding cells thereby eliminating larger tumors or cancerous cells with a minimal dose. Recruiting the immune system to the site of cancerous cells could be mediated through the production and cellular expression of immune response stimulating molecules. The last option of preferentially targeting and lysing tumor cells may seem to be the most advantageous. This method would give the possibility of eliminating tumor cells with the least damage to surrounding tissue, allowing for fully functional tissue during and immediately following treatment 3.

The adenoviral vector has been considered for treatment in cancer patients. A recent study has outlined the ability of utilizing tissue specific promoters when designing adenoviral vectors to lyse cancer cells. Adenoviral vectors naturally express the capability of lysing cells through a protein labeled the adenovirus death protein. By increasing the expression of this protein in this study, researchers were capable of effectively treating cancer through a viral vector specified for cancer within one tissue. Tissue selection was accomplished by removing the viral promoter, inserting the tissue specific promoter, and by deleting a gene that allowed viral replication in non-dividing cells. This added safety feature in mediator design ensures that non-rapidly dividing cells (such as neural cells) will not be damaged by this treatment option. Another safety precaution taken by this research team was to delete a gene that blocked targeting of the immune system. While this lowers the efficiency of the treatment, it also ensures that treatment is controlled to a specific area of the body, and that if random mutation occurs, which allows the viral vector to lyse other cell types, the body will be able to fight off the viral infection naturally4. sounds really interesting

Although most cancer cells can be targeted due to their constant multitude of s-phase dividing cells, prostate cancer is one type of cancer that only has about 5% of its cells dividing at one time. Therefore, targeting dividing cells would be a highly inefficient method of deleting these cancerous cells from the body. Viral mediated gene therapy could hold the answer for prostate cancer because it is not limited to targeting only dividing cells, but is capable of targeting non-dividing cells. The genome is generally also non-integrating, therefore, silenced insertion into the genome is not possible, and will not create another cancerous cell line in the prostate gland. Because this cancer is centralized in the prostate gland, a viral dose could be inserted directly into the gland, which has been shown to minimize immune response in animal models. 3.

Another option that has been considered for treatment of genetic diseases is the use of microRNA’s (miRNAs) to regulate the expression of proteins at the cellular level. A miRNA is a small non-coding segment of RNA that regulates RNA activity through RNA-mediated gene silencing. While the use of miRNAs in theory to silence the over-expression of specific genes in the cell seems reasonable, it is thought that one miRNA is capable of interacting with hundreds of mRNA strands. The inability to differentiate between the control of separate mRNA sequences within the cell currently makes the use of miRNAs unrealistic 5.How does this fit in with viral vectors

A class of viral vectors that has come into critical question for gene therapy is the retrovirus family. Designed retroviral vectors have the ability to remove themselves after the genetic material has been inserted into the genome, which avoids problems associated with other viral vectors. Some of these associated problems include recombination of the inserted sequence with helper viruses or associated viruses, which could lead to mutational insertion. One major downfall to utilizing a retroviral vector is how the vector inserts the gene of interest into the genome. Retroviruses by nature insert randomly into the genome, thereby increasing the likelihood of cancer formation after treatment6. (also, would reverse transcriptase be problematic?)

Viral mediated gene therapy has also been considered for preventative medicine. One current study showed that insertion of a heme oxygenase gene into a recombinant adeno-associated virus protected myocardial cells from long-term damage. This type of preventative measure could be taken to protect cells of those with who have the potential of developing coronary ischemic events. This new idea of preventative care could allow expression of genes to increase a normal response under stress by expressing multiple genes encoding for the same protein 7.

Foamy viruses are also being considered for preventative treatment of another kind. Although HIV is not a genetic disease, gene therapy may help in the prevention or decrease the severity and spread of the disease within the body. Inserting a combination of three anti-HIV transgenes into the foamy viral vector allowed a significant decrease in HIV infection by blocking HIV replication in macrophages. By limiting the replication capabilities of HIV in vivo, it is possible that this therapy may not only be capable of treating HIV, but preventing HIV transfer between partners. It is also important to note that inserting three anti-HIV genes into this viral vector limits HIV resistance to the viral vector due to the multiple mutations necessary to overcome the anti-HIV properties newly inserted into the human macrophages 8.

One of the greatest hurdles to overcome before viral mediated gene therapy can become a reality is overcoming the immune response to the viruses that deliver the genetic material to the cell. Because many viruses used as genetic therapy vectors are common and infectious to humans, completely eliminating the immune response to viruses would allow for ease of a viral infection to the human body reword. Also, many viral vectors are recognizable not only by the innate immune system, but also by the acquired immune system. For example, nearly every adult has antibodies present to the adenovirus, a virus commonly used as a viral vector in research 3. One option for overcoming the immune response to viruses would be to utilize miRNAs and silence transgene expression in the cells. Transgenes have been to known to allow specific immunity to viruses. 5

An investigation done on reducing the immune response to viral vectors centered on modification of the vector through the use of synthetic polymers. Many research facilities currently working on viral-mediated gene therapy utilize the adenovirus or a variation of this virus. As a highly successful gene delivery mediator, it is important to develop a mechanism to protect this virus from the immune system in order to effectively deliver a substantial amount of designed DNA into a large population of mutated dividing cells. Adenoviral vectors are often targeted by the innate immune system, which limits gene expression in models due to deleterious effects of the immune system as a direct response to the virus’ presence. By attaching a polymer (such as polyethylene glycol) to the capsid surface of the virus, the virus is capable of not only avoiding an innate immune response, but also avoiding anti-viral antibodies. Avoiding antibody recognition is a valuable asset to gene therapy because many viruses used as mediators are common viruses that the general public is exposed to on a regular basis 9.

One method for avoiding the innate immune response to viral vectors would be to eliminate or mutate capsid proteins that could be recognized by the immune system which are targeted for degradation. Mutating or eliminating these proteins does come with limitations. Viral vectors utilize receptors on the cell surface for forced re-arrangement of the cytoskeleton and phagocytosis to be brought into the targeted cell. Removal of the receptors, which could be recognized by the immune system, would be removal of the proteins that allow incorporation into the cell. Once the receptor was removed from the vector, a new receptor which can be recognized by the targeted cells but not the immune system must be added to the viral vector for functional therapeutic use 3.

A recent study may cast shadows upon the ability to remove antibody recognized capsid proteins and retain cellular receptors on the same capsid. One of the most widely used vectors for gene therapy is the adeno-associated virus because most of the genetic material of the virus can be removed and replaced with the designed sequence. A detailed look into the atomic structure of the adenovirus shows the possibility of overlap between receptor-binding sites and antibody recognition sites.10. Deletion of the antibody recognition site may lead to deletion of at least a part of the receptor-binding site, rendering the vector useless for future therapy. (Are there any methods to prevent the immune system from recognizing viral dna/rna?)

Random insertion of a designed sequence of DNA into the host genome through a viral vector creates a large risk for viral mediated gene therapy. As previously discussed, random insertion has been shown to cause many diseases, and often leads to the formation of cancer. Correct placement of a designed DNA stretch into the genome to replace the mutated gene is critical for avoiding more mutations in the genome, and for production of proper proteins. This type of gene replacement can be accomplished through homologous recombination (HR). HR utilizes sequence specific binding of the designed DNA to insert into the mutated genome. The designed DNA stretch base pairs to the genome, and upon DNA replication, one daughter cell carries the corrected genome and one daughter cell retains the original mutated genome. This method has been shown to effectively reduce the rate of random integration into the genome, and thereby eliminating the side effects that come with random integration. 11

An alternative approach to viral mediated gene therapy is the use of plasmids to correct genetic mutations. Without the use of a vector to transport the genomic material across the plasma membrane, more work is required to bring the sequence into the cell. Electroporation is one method that uses electric shock to open pores in the plasma membrane and allows the sequence entry into the cell where it can bind to the mutated genome and repair the mutation. Because this method has a very low efficiency of transduction, scientists have developed further methods to bring sequences into the cell.

Another alternative approach to gene therapy utilizes a class of proteins, labeled cell-penetrating peptides, which are capable of transporting cargo across the cell membrane into the surrounding cells. Several of these proteins are known to exist, including the TAT protein of HIV, the antennapedia homeodomain protein can you explain what it is from Drosophila melanogaster, and the VP22 protein from herpes simplex virus. To test the ability of the VP22 protein to carry a corrective genetic sequence across cell membranes to correct a genetic mutation, researchers fused the VP22 protein with a corrective cDNA segment of the microdystrophin gene. Use of the VP22 protein showed an increase in expression of the protein and also in distribution of the protein, indicating that the fusion protein was capable of moving across cell membrane barriers and correcting surrounding tissue 12. Plasmid mediated gene therapy holds some advantages over viral mediated gene therapy due to a lack of any immune response. However, plasmid mediated gene therapy is still very experimental, and has yet to achieve the efficiency of viral mediated gene therapy.

Although the use of gene therapy is widely experimental and not well developed for clinical use, there have been several cases of gene therapy for corrective treatments in humans. While few treatments have obtained significant results, many treatments have resulted in either no response to treatment, disease development, or death. One common disease developed in response to gene therapy has been leukemia. Development of leukemia gives rise to many problems from a medical standpoint. Beyond tracking the development of normal function to tissues, it is necessary to track development of possible diseases over long periods of time after surgery. In one case, the leukemia development took two years, while other studies show that lymphoblastic leukemia can take longer than two years for symptoms to show13.
The immune response to the vector itself has been observed to cause damage to individuals. One death was caused solely from an immune response to the viral vectors. An option for overcoming this hurdle could be the use of immunosuppressant medications, which has a list of its own problems 13. (such as…)

Another recognized problem with the current gene therapy model is the effectiveness once the virus has been administered. In most diseases, only one type of tissue will show positive results towards correcting the disease once the mutation has been fixed. If the virus is injected into a mix of tissue, there is likelihood **you can use a probability or possibility" that the virus does not preferentially target one specific tissue type, and the viral correction is wasted on tissue that will not correct the phenotype seen in the disease. This problem could be resolved by utilizing tissue-specific promoters to drive the expression of the corrected gene 13. Random integration into any cell type also posses a problem that needs to be overcome. Mutations to already non-functioning cells could have major negative implications in the individual being treated. If stem cells are targeted for correctional therapy, this could raise larger issues than isolated tumors or cancer, but could lead to the dysfunction of an already non-properly functioning area of the body. Utilizing homologous recombination could eliminate the issue of random integration into the genome be base specific pairing. Gene expression could also be affected in non-mutated genes neighboring the inserted corrected gene. Researchers are currently looking at insulators to resolve this conflict. Insulators are small DNA segments that act as a barrier between the promoter action of the inserted gene and possible promoter and enhancer activity of neighboring genes 13.

It is possible that gene therapy will never be able to replace the option of organ transplant for some diseases. A current study on rescuing cone function in Leber Congenital Amaurosis mice showed that through both the use of an adeno-associated viral vector and the use of a Lentiviral vector failed to rescue cone function when injected post-birth. The model did show, however, that both the use of the adeno-associated viral vector and the Lentiviral vector were capable of rescuing the cone function in mice when injected in utero. Diseases where there is a small window of opportunity for preventative treatment may have very restricted use of gene therapy as a treatment option due to the restricted time frame that this therapy seems practical. For these individuals, organ transplant may be the only option to restore fully functioning tissue 14.

Positive outcomes are becoming more prevalent in animal model studies. A recent study on correction of methylmalonyl-CoA mutase in mouse models has shown that adenoviral correction of the mutated gene was possible. Linking the corrected gene to (a) GFP (?) to show positive transcription and translation in the cell resulted in the cells positively treated fluorescing green. Results were achieved through a small dose of concentrated virus, and results were replicated in human cell cultures. Another positive result of this experiment was the lack of side affects associated with the delivery of the corrected gene into the host genome 15.

More positive research has come in the form of combination therapy. The Niemann-Pick disease mouse model has shown promising results for treatment options for this specific disease. Niemann-Pick disease is characterized by the lack of ASM activity in the body, which further leads to the build up of undegraded lipids in the CNS and viscera. The combination treatment included injections of a viral vector to correct the mutation not only to the brain, but also through a systemic injection. Models treated with the combination therapy exhibited almost entire correction to the mutated protein compared to mice that were only injected at one site or systemically. This correction resulted in normal weight gain and normal motor performance. In addition to the positive results obtained toward the corrective therapy, there was no antibody production directed toward the vector, suggesting future treatments could be done with the same viral vector if deemed necessary 16.

Tumor and cancer research is becoming a prevalent focus of studies on gene therapy. A recent study on brain tumors inserted into mice has shown a promising outlook for tumor treatment. The interaction between ganciclovir (GSV) and herpes simplex virus thymidine kinase (HSV-tk) is known to be toxic at the cellular level. Insertion of the HSV-tk gene into an adenoviral vector allows specific targeting of dividing cells. Once inserted into the cell, the combination of HSV-tk and GSV acts as a chain terminator of DNA synthesis and can effectively eliminate dividing cells. This method effectively reduced tumor size compared to control groups, while showing no adverse side affects on the surrounding neural tissue 17. While neural tissue in theory will not be harmed due to the lack of dividing cells, more work will need to be done on the effects upon the dividing cells the cranial cavity before this treatment option can be fully pursued.

By harnessing the life cycle of a disease causing pathogen, it is possible that scientists may one day be capable of correcting genetic mutations that are currently untreatable. While viral mediated gene therapy is a relatively new medical development, preliminary models show promising results in the treatment of many genetic diseases such as muscular dystrophy. Viral mediated gene therapy may also be the cure to one of mankind’s most devastating diseases, cancer. However,as promising as the models may be, it is important to remember that the risks associated with viral mediated gene therapy are still very high, and more studies must be done before this type of therapy can be used regularly in modern medicine.

Chad- A wonderfully written paper. I thought at the beginning you were focusing your paper on host vs. graft disease, but then you discussed some diseases in more detail in relationship to the therapies. Maybe it would be good to mension that in your introduction somehow. If I was looked on a journal database for something about gene therapies and cancer I do not think I would pick up your paper just by reading the induction (but i think you paper would be something I would "want" to pick up). Paper flows expect for the sudden talk on cancer that continued for several paragraphs. You have an awesome start!!

Chad- I agree with the previous comments. Very interesting paper with excellent amount of supporting details and examples. A few tweaks to the beginning of your paper and it will be very solid. I liked your organization and your transitions.

Chad-This is a great paper and topic. The only thing i was a little confused about was how the whole host vs graft fit into gene therapy. Also I would suggest to go into a little more detail about the immune responses to specific viruses. I love how this topic fits in well with cancer treatment and it reads real well. great job

**Chad- Good Job. I personally think is a very good paper… I like your topic and the way you connect it we different viruses that concern the people today day… I really didnt see to many grammatical errors, the only part that i was kind of confuse was at the beginning… **

** I really like this topic it is very interesting and def enjoyed reading it… i think it is a very well written paper**

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