Various Viral Vectors Necessary for Gene Therapy Delivery Systems. Abstract

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1 Patibandla 1 Yamini Patibandla Dr. Rance LeFebvre COSMOS UC Davis Cluster 7 29 July 2013 Various Viral Vectors Necessary for Gene Therapy Delivery Systems Abstract A leading movement in today s times, gene therapy is a new strategy that has been created to manipulate our genes, the building blocks of heredity. In order to replace faulty genes with correct genes in the target cells, researchers use viruses as vectors to carry the correct DNA into our cells. This paper delineates four of the most common viral vectors that are studied, experimented on, and worked with- retroviruses, adenoviruses, adeno-associated viruses and herpes simplex viruses. In the mid-1970 s, the idea of gene surgery floated around in several scientific circles. The thought seemed fantastical, the idea of curing genetic disorders by playing around with the actual subunits that coded for our basic function of life. For many, this idea seemed so complex and far away, yet by 1990, this thought became a solid goal, as researchers jumped for their lab coats and raced to understand and utilize gene therapy to cure genetic disorders. These researchers decided to target genetic disorders that were caused by mutated genes that could be fixed with gene therapy. Gene therapy, as described by Johnny Huard, is a novel form of molecular medicine that can have a major impact on human health care in the future (Johnny Huard et al. 179). Gene therapy is a technique for correcting defective genes responsible for disease development ( Gene Therapy par.2). The most common way to correct these genes is to take out the abnormal, or faulty gene that is causing the disease and insert the correct gene into the genome. Initially, researchers tried to directly insert a gene in the form of naked DNA, but

2 Patibandla 2 this method proved to be unsuccessful as this method only works with certain tissues, requires large amounts of DNA, and only has a temporary effect. In order to replace these genes, researchers decided to use a vector, or a carrier molecule that would deliver the correct gene into the target cells. The most common vectors are viruses that [have] been genetically altered to carry normal human DNA ( Gene Therapy par.4). With centuries of time, viruses have evolved into complex infectious agents capable of entering human cells, delivering their genes into the nucleus, and reprogramming the cells to code their genes and make their proteins, essentially transforming our cells into virus-making factories. Researchers have genetically modified these viruses to make them harmless, [taking] advantage of [their encapsulating] capability and [manipulating] the virus genome to remove disease-causing genes and insert therapeutic genes ( Gene Therapy par.4). Retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses are the four most-studied viral vectors that are making strides in intensive gene therapy research for their unique molecular characteristics and their comprehensive roles in delivery systems. Retroviruses are a family of viruses that are defined by their ability to physically insert their genes into the host s genome. A well-known example of a retrovirus is the infamous Human Immunodeficiency Virus (HIV), which invades the CD4 cells in our immune systems and rewrites our DNA to produce more Human Immunodeficiency Viruses instead of defensive proteins that protect us from other infectious agents, eventually causing Acquired Immunodeficiency Syndrome (AIDS). Essentially all retroviruses operate in the same manner to invade our body cells - by integrating and rewriting the DNA in our body cells. The genetic material in retroviruses is in the form of RNA (ribonucleic acid) while the host s genetic material is in the form of DNA (deoxyribonucleic acid). In order to integrate its genetic material into

3 Patibandla 3 human DNA, retroviruses use the enzymes, reverse transcriptase and integrase, for reverse transcription ( Gene Therapy Vectors par.5). Instead of conventionally transcribing a messenger RNA molecule from the original DNA genome, retroviruses will use the enzyme reverse transcriptase to transcribe a DNA molecule from their RNA genome. Then integrase will take this DNA molecule and integrate it into the host cell s chromosomes. Once the genetic material of the retroviruses has been integrated into the chromosomes, the host cell will pass down the new genes to its daughter cells when it replicates. Retroviruses infect only dividing cells, so they cannot infect cells that have stopped dividing like neurons ( Vector Toolbox par.3). Additionally, retroviruses can be genetically engineered to recognize special proteins on surface of the target cells, which lends to more specificity. Unfortunately, the enzyme integrase adds the virus s RNA/DNA into the host cell s chromosomes in an arbitrary manner. Therefore, there is a chance that it will integrate into a place where it disrupts another gene, causing insertional mutagenesis ( Gene Therapy Vectors par.7). If the disrupted gene, for example, regulates cell division, the cell will undergo uncontrolled cell division, or cancer. Efforts are being made to curtail this malignant side effect, with the addition of zinc finger nucleases...[and] beta-globin locus control region to direct the site of integration to specific chromosomal sites ( Gene Therapy Vectors par.8). Another potent side effect of using retroviruses as viral vectors in gene therapy is the consequent immune response. Researchers have addressed this problem by removing the specific antigens located on the surface of the virus that trigger the immune response ( Vector Toolbox par.5). Unique for their reverse transcriptional ability, retroviruses stand out as one of the more promising ways to combat genetic disorders with gene therapy.

4 Patibandla 4 Another family of viruses that can be manipulated to serve as viral vectors for gene therapy is adenoviruses. Adenoviruses are responsible for the respiratory, intestinal, and eye infections in humans-especially the common cold ( Gene Therapy Vectors par.9). The genetic material of adenoviruses is in the form of double stranded DNA, compared to retroviruses that have RNA ( Vector Toolbox par.2). Adenoviruses, therefore, do not have to undergo reverse transcription to integrate their DNA into the nucleus of the host cell. Adenoviruses can be propagated relatively easily to produce high-titer stock and will express their genes in cells that are not actively growing and dividing (Thomas Shenk 162) as well as dividing cells. Like retroviruses, adenoviruses can be altered to recognize proteins on the target cell s surface with the use of genetic engineering. Adenoviruses bind to cells in two steps. First, the virus adsorbs to the host cell, and enters the cell through receptor-mediated endocytosis, and escapes into the cytosol (Shenk ). Then the virus breaks down into virions and the viral DNA reaches the nucleus. Unlike retroviruses, adenoviruses do not integrate their DNA into the host cell DNA. Instead, their DNA remains in the cell apart from the chromosomes. During transcription, the viral DNA will be transcribed along with the host cell DNA. However, when the host cell replicates, it will not replicate the viral DNA because it hasn t been integrated into the host cell DNA. Therefore, to strengthen treatment, researchers will have to transform the dividing target cells constantly, every 1 to 2 weeks, to maintain the correct gene s presence in the host cell ( Vector Toolbox par.3). Like the retrovirus, the adenovirus is susceptive to a strong immune response, but can stay hidden by removing the specific antigens that trigger the response ( Vector Toolbox par.7). Adenoviruses have proven to be very durable for transformation and gene delivery systems, but still pose a problem because their DNA doesn t integrate and

5 Patibandla 5 maintain a heavy presence in dividing cells: a problem that researchers are willing to look into and combat. Another family of viruses that would prove useful for gene therapy are adeno-associated viruses, also known as, AAV. Adeno-associated viruses, unlike retroviruses and adenoviruses, do not cause direct illness in their human host and consist of single-stranded DNA, compared to the RNA of retroviruses and double-stranded DNA of adenoviruses ( Vector Toolbox par.2). AAV also distinguishes itself from the other two types of the viruses by the way it infects the host cell. AAV can only infect the host cell with the assistance of a helper virus to replicate its DNA within the host cell. AAV, like retroviruses and adenoviruses, are capable of selecting specific cell types once they have been genetically engineered to recognize specific cell surface proteins of the target cells. Additionally, AAV can infect dividing and nondividing cells with equal efficiency ( Vector Toolbox par.2). Unlike adenoviruses, the original AAV will integrate its DNA, after [converting] to a duplex form (Terence R. Flotte et al. 1), with the host cell s DNA at a specific site on chromosome 19, so that a cryptic, latent infection ensues (Flotte et al. 1). However, the recombinant AAV virus with the selected gene will not integrate its gene into the host cell s chromosome 19 with 100% accuracy, rather 95% of the time ( Vector Toolbox par.2). While integrating retroviral DNA into the host cell s genome poses a risk for disrupting other crucial genes, there is less of a risk for AAV delivery system integration as the virus will only integrate its DNA on chromosome 19 ( Vector Toolbox par.3). Unlike retroviruses and adenoviruses, adeno-associated viruses will not trigger an immune response and are defined as one of the more safer viral vectors, because they are harmless to the human patient (Flotte et al. 11). Researchers are still perfecting the gene delivery system technique for

6 Patibandla 6 adeno-associated viruses and aiming to take advantage of its harmless nature and safer integration strategy. The last family of viruses that has proven to be very promising for gene therapy delivery systems is the herpes simplex viruses. Herpes simplex viruses are popularly known for being the infectious agents responsible for oral and genital herpes ( Vector Toolbox par. 1). The herpes simplex virus is responsible for small painful cold sores around the oral and genital areas and is known for introducing its DNA into the genome without integrating it into the chromosomes. Instead of integrating their DNA into the host cell s chromosomes like retroviruses and adeno-associated viruses, herpes simplex viruses insert their DNA into the host cell s nucleus as a separate circular molecule ( Vector Toolbox par.3). As discussed before, the DNA of adenoviruses will not be passed down when the infected cell replicates because the DNA does not integrate with the host cell s chromosomes. However, the DNA of herpes simplex viruses will be passed down from the infected cell to its daughter cell, maintaining the herpes simplex virus infection. Herpes simplex viruses usually infect and target cells in the nervous system, which are quiescent and never replicate and divide. Research has shown that the herpes simplex viruses are useful for gene therapy in nervous systems, as they remain latent in our body systems and continue to express the targeted genes in the targeted cells. Like retroviruses and adenoviruses, herpes simplex viruses are capable of triggering an immune response, which can be quieted by manipulating the specific antigens on the recombinant virus. Herpes simplex viruses are garnering a lot of attention for their ability to exert permanent influence on their target cells without a dangerous integration system, showing promise for a more efficient viral vector delivery system.

7 Patibandla 7 All four viral vectors, retroviruses, adenoviruses, adeno-associated viruses and herpes simplex viruses, are limited by the amount of recombinant DNA that they can carry. Viral DNA is very rigid and cannot be expanded or modified to add additional recombinant DNA. Researchers therefore need to consider the size of the recombinant DNA they want to insert in the viral vector. The maximum length of RNA that can be inserted into the retrovirus is 8000 base pairs, while the maximum length of DNA that can be inserted into the adenovirus is 7500 base pairs ( Vector Toolbox par.4). The maximum length of DNA than can be inserted into the adeno-associated virus is around 5000 base pairs and the maximum length of DNA that can be inserted into the herpes simplex virus is 20,000 base pairs ( Vector Toolbox par.4). Looking at these different size requirements, researchers have to determine which viral vectors to use according to type of cell, side effects and size. In order to proceed successfully with gene therapy, researchers must consider the different viral vectors that are needed to carry our selected genes into the target cells. The four most common viral vectors that are being studied currently are retroviruses, adenoviruses, adenoassociated viruses, and herpes simplex viruses. Each virus has its own specific molecular characteristics, benefits and limitations that must be evaluated before being used for experimentation. As one of the core bases for gene therapy, viral vectors are a promising subject for research. Even though the race to win and master gene therapy began a long time ago, we haven t truly reached the end of the race. Viral vectors, in the form of these four discussed viruses, are still mostly enigmas that need to solved. We are so much closer to winning with every single experiment, trial and study. Maybe not in this day and age, but soon, will the fantastical idea of gene therapy become a solid, concrete reality throughout our daily lives.

8 Patibandla 8 Works Cited Flotte, T. R., and K. I. Berns, eds. Adeno-Associated Viral Vectors For Gene Therapy. Vol. 31. Amsterdam: Elsevier B.V., Print. Quesenberry, Peter J., Gary S. Stein, Bernard Forget, and Sherman Weissman, eds. Stem Cell Biology and Gene Therapy. Canada: John Wiley & Sons, Inc., Print. Gene Therapy Vectors. News-Medical.net, Web. 24 July <http://www.newsmedical.net/health/gene-therapy-vectors.aspx>. Human Genome Project Information: Gene Therapy. U.S. Department of Energy Genome Program's Biological and Environmental Research Information System (BERIS, 24 Aug Web. 24 July <http://web.ornl.gov/sci/techresources/human_genome/medicine/genetherapy.shtml>. Learn. Genetics Genetics Science Learning Center. University of Utah & NIH, Web. 24 July <http://learn.genetics.utah.edu/content/tech/genetherapy/gttools/>.

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