Viral Vector Mediation for Gene Therapy: An Immunological Overview
Gene therapy is the introduction of new genetic material into a cell as a means to correct a known mutation that causes a disease. Viral vectors are viruses with new or modified genomes that remove the virus' pathogenicity. By replacing pieces of a viral genome with a known genetic sequence, harnessing the viral lifecycle allows the capability to carry the new viral sequence across the cell membrane and bring the modified genome in contact with a defective cellular genome. In order to be successful at transforming the defective genome, viral vectors must overcome physical barriers and immune responses. Viruses have evolved several methods to avoid immune system recognition, including breaking down major histocompatibility (MHC) molecules, interrupting cytokine signaling and avoiding antibody binding. Combining knowledge of immunological responses to viruses and viral evasion of the immune system has led to the creation of several viral vector models targeted to overcome the normal immune response. While human treatments have shown disappointing results such as leukemia or overactive immune response, animal models have shown promising results including almost entire correction of the mutated genome in affected cells. With the aid of more advanced models and a broader base of immunological knowledge, correction of genetic diseases currently untreatable may someday be achieved through the use of viral vectors. In time, viral vectors may be utilized as the main source of treatment for genetic diseases.
For 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. Research scientists are currently exploring many options to treat genetic diseases at the source through a method called gene therapy. Gene therapy is the introduction of new genetic material into cells of an individual in order to produce a therapeutic outcome in the diseased individual (Bunnell & Morgan, 1998). One current gene therapy treatment possibility is utilizing viruses to alter the incorrect genetic sequence in diseased individuals. This option is not only attractive because of the ability to treat the individual without the use of a donor or immunosuppressant medications, but it also avoids ethically controversial approaches such as embryonic stem cell use. Therapy involving embryonic stem cells involves the culturing and differentiation of stem cells from an embryo, as human stem cells are often difficult to culture in a lab setting (Choumerianou et al., 2008). Many of the ethical issues that arise from embryonic stem cell research and treatment settle around the argument of whether or not an embryo is truly a human, and whether using these embryos to treat a disease is disvaluing human life (Choumerianou et al., 2008). Through the analysis of current research in viral vector mediated gene therapy, it is possible to evaluate the practical and theoretical application of gene therapy through a viral vector as a treatment option for genetic diseases.
Researchers have been able to isolate viruses, remove pathogenic genetic sequences, insert a useful treatment sequence and achieve positive results in animal models. Viral-mediated gene therapy utilizes viral replication in order to correct mutations to genes in the human genome by inserting the corrected gene into the viral genome. In order to utilize the viral replication cycle to treat diseases, it is necessary to understand the viral replication cycle.
Viral replication generally follows a five-step process: attachment, entry, synthesis, assembly, and release. In the attachment step, virions make contact with the cellular surface through random contact. The attachment of a virion particle with the surface of an individual cell is based upon the interaction between cell surface receptors and the particles composing the capsid or proteins on the surface of the virion. Following cellular attachment, it is necessary for the genome of the virus to be moved inside the cell. Viruses utilize a wide variety of methods to move the genome across the cell membrane. Bacteriophages penetrate the cell membrane with a needle like tail and pass the genome across the membrane through this tail. Eukaryotic infecting viruses utilize other methods including direct penetration, phagocytosis or membrane fusion. 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 the entire virion to be incorporated into the host cell, but requires rearrangement of the host cytoskeleton to be brought into the cell instead of fusing with the host membrane (Bauman, 2004).
Once the virion is inside the host cell, viral protein synthesis may begin to occur. This process, called transduction is defined as the cellular uptake and expression of the viral genome product without replicating the viral genome (Kay, Glorioso, & Naldini, 2001). Once again, different viruses have different ways of producing proteins needed for further steps of the replication process. For a double stranded DNA (dsDNA) virus, the process of replication, transcription and translation of the DNA sequence is similar to that of the host genome. Replication of the single stranded DNA (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 a positive-sense ssRNA 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 (Bauman 2004).
Viral Evasion of the Immune System
Viruses are recognized by both the innate and acquired immune system as potentially harmful and foreign. While both acquired and innate immune responses do exist, viruses have evolved novel mechanism for evading these elaborate responses. If a virion enters a cell, it is capable of being digested by proteases, which allows fragments of the virus to be presented on the cellular surface through major histocompatability (MHC) type I molecules. Several viruses have shown capabilities of avoiding this response by producing proteins that either break down the MHC molecule or by producing proteins that compete for MHC peptide binding (Hewitt, 2003).
One class of viruses that are of particular interest for gene therapy are "hit and stay" viruses (Tortorella, Gewurz, Furman, Schust, & Ploegh, 2000). This viral class infects a host cell and continues a prolonged infection within the same host. Unlike other viral classes, these viruses must avoid both innate and acquired immune response due to their constant, prolonged presence in the host (Tortorella et al., 2000). Protein products of these viruses have been known to limit molecule presentation by MHC molecules, interrupt cytokine signaling molecule production, and avoid antibody binding (Tortorella et al., 2000),(Hilleman, 2004).
One of the first innate immune responses a virus must evade is the attachment of interferons. A serotype of herpesvirus, a classic example of a hit and stay virus, is capable of producing a viral interferon regulating factor that is homologous to a cellular interferon regulating factor (Joo et al., 2007). By binding the cellular interferon-regulating factor, interferons cannot be activated and the cell cannot be marked for apoptosis (Joo et al., 2007). Zhu et al. showed that a product of the vaccinia virus, A46R, is capable of interfering with the Toll Like Receptor (TLR) pathway; this pathway has been shown to be critical for innate immune recognition of viruses such as the adenovirus(Zhu, Huang, & Yang, 2007). One ultimate result of blocking this pathway is stopping the production of interferons (Stack et al., 2005). This wide variety of immune evasion mechanisms could be utilized by researchers to produce a viral vector capable of avoiding natural immune responses in order to effectively transduce cells without allowing the immune system to lyse transduced cells.
Diseases Being Researched
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 (Duan, 2006).
As researchers analyze a genetic disease, a main component for developing a vector is creation 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 (Duan, 2006). One reason why an entire gene cannot generally be used is size constraints, as often an entire eukaryotic gene is too large to fit into a viral vector. A second reason the entire gene cannot be used in many cases is due to the sequence of amino acids within the gene. In the dystrophin gene, a region of the gene is a cleavage site for a viral protein (Duan, 2006).
A class of viral vectors that has come into question for gene therapy is the retroviruses. Designed retroviral vectors have the ability to remove themselves after the genetic material has been inserted into the genome (Russ, Friedel, Grez, & von Melchner, 1996), which avoids problems associated with other viral vectors. Some of the problems associated with other viral vectors include recombination of the inserted sequence with helper viruses or other viruses, which could lead to mutational insertion (Russ, Friedel, Grez, & von Melchner, 1996). One major downfall to utilizing a retroviral vector involves 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 treatment (Russ, Friedel, Grez, & von Melchner, 1996).
Another hurdle to gene therapy is where to deliver the modified viruses in order to achieve optimal results. For some diseases, the answer 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 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 optimal results (Duan, 2006). 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?
Cancer Treatment through Gene Therapy
While there is an abundant amount of research focused on mutating a viral vector in such a way that limits its replication capabilities in order to contain infection while only transducing those cells necessary for protein expression, some researchers are harnessing the life cycle of the virus to defeat a different type of genetic disease. Cancer is becoming a leading cause of death in the present world, and while many treatment options exist, many treatment options produce unwanted side effects.
There are several options for viral mediated gene therapy in cancer cells. These options include correction, toxin release, recruitment of immune response, and designing a virus that recognizes and preferentially lyses tumor cells (Lupold & Rodriguez, 2005). 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 (Lupold & Rodriguez, 2005).
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 (Doronin et al., 2001). By increasing the expression of this protein researchers are capable of effectively treating cancer through a viral vector specifically targeted 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 (Doronin et al., 2001). This novel 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 naturally (Doronin et al., 2001).
By modifying the adenovirus to specifically target cancer cells, researchers have developed a novel idea for cancer control. Once targeting is specified for cancer cells, researchers are capable of increasing expression of Adenovirus Death Protein and interferon alpha which both act to lyse the cell upon viral replication (Shashkova, Kuppuswamy, Wold, & Doronin, 2008). If this viral targeting can be limited specifically to cancer cells without damaging unmutated somatic cells, this treatment option could provide a welcomed alternative to current cancer treatment options.
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 (Collis, DeWeese, Jeggo, & Parker, 2005). Therefore, targeting dividing cells would be a highly inefficient method of deleting these cancerous cells from the body. Healthy tissues such as the intestinal epithelial, white blood cell stem cells of the bone marrow and hair follicles have an abundance of dividing cells, which are also targeted by conventional cancer treatments. 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 the possibility of mutation resulting in another cancerous cell line will be limited. Because this cancer is centralized in the prostate gland, a viral dose could be injected directly into the gland; this method has been shown to minimize immune response in animal models (Lupold & Rodriguez, 2005).
A novel cancer treatment mechanism that has been recently developed involves modifying the natural acquired immune response to cancer cells to be more cytotoxic through delivery of an adenovirs upon contact. Cytotoxic T-lymphocytes specific for tumor-antigens have been shown to be able to cross tissues and attack malignant tumors. However, it has been shown that tumors have immune evasion systems that prevent recognition by the immune system. Modifying the cytotoxic T-lymphocytes genome by inserting two adenoviral genes into the lymphocyte genome resulted in lymphocytes capable of targeting lymphomas created by the Epstein-Barr virus. Upon recognition through the receptor, a viral product is released and tumor cell lyses occurs. This method increases cytotoxicity of cytotoxic T-lymphocytes without disabling function of their receptors and maintaining the ability to cross tissue types (Yotnda, Savoldo, Charlet-Berguerand, Rooney, & Brenner, 2004).
Preventative Treatment through Gene Therapy
Viral mediated gene therapy has also been considered for preventative medicine. Melo et al. 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 (Melo et al., 2002).
Foamy viruses, a family of retroviruses with two positive sense RNA strands, are 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 (Taylor et al., 2008).
Overcoming Immunological Hurdles
One of the first barriers to entry a virus must overcome is the physical barrier that separates the virus from the cells of interest. In cystic fibrosis patients, the airway epithelia that cause the symptoms of this debilitating disease lie in open contact with air and could easily come in contact with a viral vector. However, research has shown that because this epithelial layer does come into contact with so many microbes, the cell surface receptors for microbes lies on the basal side of these epithelial cells (Pickles et al., 1998). This adaptation decreases the uptake of the vector and hence decreased expression of the modified gene (Pickles, 2004). Disruption of the airway epithelia to expose the basolateral surface to the viral vector would only expose this surface to potentially dangerous pathogens already harbored in the lungs, which are in continual exposure to microbes in the air (Lee, Matthews, & Blair, 2005), (Zabner, Freimuth, Puga, Fabrega, & Welsh, 1997).
Evolution has also provided physical protection through mucous secretion by the ciliated cells to carry the pathogen away from the lungs, and also a slow rate of endocytosis in order to prevent possible infections (Pickles, 2004). While these physical barriers are helpful in protecting the body from dangerous pathogens, overcoming these obstacles has shown to be a difficult task in order to treat genetic mutations.
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 oppurtunistic viruses an open target. Oppurtunistic viruses could easily infect and overtake a host without an immune response to keep them in check. 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 (Lupold & Rodriguez, 2005), (Bangari & Mittal, 2006). One option for overcoming the immune response to viruses would be to utilize micro RNAs (miRNAs) and silence transgene expression in the cells. A miRNA is a small non-coding segment of RNA that regulates RNA activity through RNA-mediated gene silencing. Transgenes have been shown to allow specific immunity to viruses (Marquez & McCaffrey, 2007). Also, current treatments options require high multiplicities of infection in order to obtain optimal results, which only increases the inflammatory response to newly injected viral vectors resulting in cytotoxicity and failed gene expression (Bangari & Mittal, 2006),(Shayakhmetov, Papayannopoulou, Stamatoyannopoulos, & Lieber, 2000). Even if low multiplicities of infection could be obtained for some diseases in order to produce a corrected protein, some diseases such as cancer may always require a high multiplicity of infection in order to rapidly target cells to produce high levels of a specific protein in order to stop the spread of mutated cells(Kay et al., 2001).
Modifying Viruses for Vector Use
An investigation by Kreppel and Kochanek 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 (Kreppel & Kochanek, 2008).
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 used by the targeted cells to induce phagocytosis but which will not be recognized by the immune system must be added to the viral vector for functional therapeutic use (Lupold & Rodriguez, 2005).
A recent study by Xi et al. may cast doubt 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 (Xie et al., 2002). 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.
Although there is a high rate of mutation and wide variety of viruses that exist, there seem to be a limited number of viruses that can be used in viral mediated gene therapy. Not only does the lack of receptors play into the ability for a virus to efficiently transduce a cell, but also a current study shows that modification of a virus that has a receptor on the cellular surface also limits the efficiency of transduction. By converting a single-stranded genome into a double stranded intermediate, the efficiency of transduction decreased hindering the efficiency of use in gene therapy (Fisher et al., 1996). This data suggests that there is a very limited pool of viruses from which vectors could be made that would be useful in future research. Other research suggests that this pool may be expanded by utilizing related viruses that are specific to another mammal species. The canine adenoviral vector was capable of efficiently transducing murine airway epithelial even after exposure to human adenovirus (Keriel, Rene, Galer, Zabner, & Kremer, 2006). This data suggests that related viruses specific to a non-human species of mammal may be useful tools to avoid acquired immune responses to viral vectors.
Cross species vectors may prove to be useful for many aspects of gene therapy. One study done on bovine herpesvirus 4 showed that infection into the lateral ventricle of rat brains allowed for the transduction of migratory stream cells while not infecting neurons (Radaelli et al., 2008). Other studies have shown that differing serotypes of adenoviral vectors are capable of targeting specific tissues (Coura Rdos & Nardi, 2007). This cell type specificity of infection could be utilized to treat tissue-specific cancers or target specific tissues for gene therapy.
Even with a limited pool of viruses for gene therapy, scientists have found that certain viruses can avoid cellular and humoral immune responses to viral presence. When comparing the immune response to adeno-associated virus to the adenovirus, researchers found that transduction of dendritic cells was necessary for an immune response as opposed to the adenovirus which elicited an immune response from T-cells which ultimately resulted in the loss of transduced cells (Jooss, Yang, Fisher, & Wilson, 1998). While this study was limited to muscle fibers, it could have larger implications for a wider variety of diseases across the body.
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 of DNA has been shown to cause many diseases, and can lead to the development 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 (Ohbayashi et al., 2005).
Alternative Gene Therapy Methods
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, other methods are 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 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 (Xiong et al., 2007). 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. Also, delivery into a patient may be a difficult process. In vivo delivery generally involves electroporation for cellular uptake of the plasmid, conditions which cannot be replicated in a human patient.
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. While the use of miRNAs to silence the over-expression of specific genes in the cell seems reasonable in theory, it is thought that one miRNA is capable of interacting with hundreds of mRNA strands (Stark, Brennecke, Bushati, Russell, & Cohen, 2005). The inability to differentiate between the control of separate mRNA sequences within the cell currently makes the use of miRNAs unrealistic (Marquez & McCaffrey, 2007).
While alternative gene therapy methods may seem to hold some advantages over utilizing a viral vector to deliver a corrected genetic sequence, it is important to note how significant or insignificant these differences are compared to the advantages of viral vector mediated gene therapy. The viral life cycle is thoroughly studied and understood; therefore modifying this vector already holds an advantage over relatively unstudied vectors. Because viruses are pathogenic, the methods for controlling and removing the vector are also well studied. The immune response to viral vectors is very apparent, while the immune response to plasmids or vector proteins is relatively theoretical at best. Viral vectors, at least in the current setting, seem to hold many advantages as gene therapy vectors.
Recognized Problems with Current Gene Therapy Methods
Although the use of gene therapy is 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 (Porteus, Connelly, & Pruett, 2006). 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 appear (Porteus, Connelly, & Pruett, 2006).
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, but this has a list of its own problems including frequent bacterial infections from normal flora bacteria (Porteus et al., 2006).
Another recognized problem with the current gene therapy model is its 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, it is likely that the virus will 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 (Porteus et al., 2006).
As previously discussed, random insertion into any cell type also posses a problem that needs to be overcome. Adding additional mutations to already dysfunctional cells could have negative implications in the individual being treated. If stem cells are targeted for correctional therapy, further loss-of-function in an already damaged area of the body could occur. Any functioning role that these mutated cells may have been serving in the body may be lost, which could lead to even larger health problems for the patient. Utilizing HR could eliminate the issue of random integration into the genome by 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 (Porteus et al., 2006).
It is possible that gene therapy will never be able to replace the option of organ transplant for some diseases. A current study by Bemelmans et al. on rescuing cone function in Leber Congenital Amaurosis, a genetic mutation resulting in vision loss or blindness, 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 patients faced with this type of genetic disorder, organ transplant may be the only option to restore fully functioning tissue (Bemelmans et al., 2006).
Positive outcomes are becoming more prevalent in animal model studies. A recent study by Chandler et al. 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 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 effects associated with the delivery of the corrected gene into the host genome (Chandler et al., 2007).
More promising 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 acid sphingomyelinase activity in the body, which further leads to the build up of undegraded lipids in the central nervous system 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 (Passini et al., 2007).
Tumor and cancer research is becoming a prevalent focus of studies on gene therapy. A recent study by Chen et al. on brain tumors inserted into mice has shown a promising outlook for tumor treatment. The interaction between gancyclovir (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 (Chen, Shine, Goodman, Grossman, & Woo, 1994). While neural tissue in theory will not be harmed because they are comprised mostly of non-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.
In a world filled with genetic diseases and a rising rate of cancer, a need for a simple and effective treatment option has arisen. Current treatment options do not hold the answers for all diseases, and none target the root of the problem, but only alleviate the symptoms. By harnessing the life cycle of a disease-causing pathogen, it is possible that one day genetic mutations that are currently untreatable will be able to be corrected by viral vector mediated gene therapy. This treatment option directly targets the problem, a mutation in the genome. Once the mutation has been corrected, the body is capable of alleviating the symptoms of the disease without the need for further treatment.
Immunological barriers are the primary roadblocks to gene therapies utilizing viral vectors. Understanding the immune reactions to viruses and how viruses evade these responses can lead to a more effective vector. Similarly, a greater understanding on how viral genomes are incorporated into their hosts' genomes and evade host recognition will produce a more efficient treatment option.
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 promising 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.
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