Adeno-Associated Viruses as Gene Therapy Vectors: Difference between revisions
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<br>Adeno-associated viruses (AAV) small viruses that affect humans and other members of the primate species. They belong to the parovirus family and require a helper virus, such as adenovirus, herpes simplex virus, vaccina virus, or CMV, to replicate. For this particular reason they are also part of the dependovirus genus. If no helper virus is present, they are able to incorporate themselves into the host cell’s genome and latently replicate as the host cell replicates. When a helper virus infects the cells that the AAV has latently incorporated itself into, it is able to engage in lytic replication and release itself from the cell. | <br>Adeno-associated viruses (AAV) small viruses that affect humans and other members of the primate species. They belong to the parovirus family and require a helper virus, such as adenovirus, herpes simplex virus, vaccina virus, or CMV, to replicate. For this particular reason they are also part of the dependovirus genus. If no helper virus is present, they are able to incorporate themselves into the host cell’s genome and latently replicate as the host cell replicates. When a helper virus infects the cells that the AAV has latently incorporated itself into, it is able to engage in lytic replication and release itself from the cell. | ||
[[Image:AAV TEM.jpg|thumb| | [[Image:AAV TEM.jpg|thumb|175px|right|Electron micrograph of the Adeno-Associated Virus.]] | ||
<br>The AAV genome is made up of ssDNA, is very small (4.7kbp), and is flanked by two 145 nucleotide-long inverted terminal repeats. It codes for two different types of proteins, the first being Rep, which is responsible for replication and rescue of the virus. The second protein, Cap, is a structural protein that is the chief constituent of the icosahedral capsid that houses the viral genome. Furthermore, the AAV genome contains three promoters, p5, p19, and p40, and two open reading frames. The mRNA transcripts produced in conjunction with each of the promoters is able to code for seven different Rep and Cap proteins due to alternate splicing. AAV’s genome doesn’t code for a DNA polymerase and is consequently dependent on that provided by the host cell. AAV are very simple viruses, but as we will see, they have an extremely wide variety of medicinal applications. | <br>The AAV genome is made up of ssDNA, is very small (4.7kbp), and is flanked by two 145 nucleotide-long inverted terminal repeats. It codes for two different types of proteins, the first being Rep, which is responsible for replication and rescue of the virus. The second protein, Cap, is a structural protein that is the chief constituent of the icosahedral capsid that houses the viral genome. Furthermore, the AAV genome contains three promoters, p5, p19, and p40, and two open reading frames. The mRNA transcripts produced in conjunction with each of the promoters is able to code for seven different Rep and Cap proteins due to alternate splicing. AAV’s genome doesn’t code for a DNA polymerase and is consequently dependent on that provided by the host cell. AAV are very simple viruses, but as we will see, they have an extremely wide variety of medicinal applications. | ||
Revision as of 01:07, 16 April 2009
Adeno-Associated Viruses
Adeno-associated viruses (AAV) small viruses that affect humans and other members of the primate species. They belong to the parovirus family and require a helper virus, such as adenovirus, herpes simplex virus, vaccina virus, or CMV, to replicate. For this particular reason they are also part of the dependovirus genus. If no helper virus is present, they are able to incorporate themselves into the host cell’s genome and latently replicate as the host cell replicates. When a helper virus infects the cells that the AAV has latently incorporated itself into, it is able to engage in lytic replication and release itself from the cell.
The AAV genome is made up of ssDNA, is very small (4.7kbp), and is flanked by two 145 nucleotide-long inverted terminal repeats. It codes for two different types of proteins, the first being Rep, which is responsible for replication and rescue of the virus. The second protein, Cap, is a structural protein that is the chief constituent of the icosahedral capsid that houses the viral genome. Furthermore, the AAV genome contains three promoters, p5, p19, and p40, and two open reading frames. The mRNA transcripts produced in conjunction with each of the promoters is able to code for seven different Rep and Cap proteins due to alternate splicing. AAV’s genome doesn’t code for a DNA polymerase and is consequently dependent on that provided by the host cell. AAV are very simple viruses, but as we will see, they have an extremely wide variety of medicinal applications.
Introduce the topic of your paper. What microorganisms are of interest? Habitat? Applications for medicine and/or environment?
AAV Vector Production (2 Transfection Method)
The first step in producing AAV vectors is finding helper viruses that will aid in replicating the AAV. Many times this helper virus is an adenovirus, which needs a minimum of its E2A, E4, and VA1/II gene regions in order to be effective at replicating the AAV. Then, two plasmids are created, one containing the AAV rep and cap genes plus the adenoviral helper genes prescribed above. The other plasmi containins an expression cassette composed of a CMV promoter, multiple cloning site, and SV40 polyA. This expression cassette on the second plasmid is also flanked by AAV inverted terminal repeats. This concoction is then transfected into a large quantity of 150mm dishes that contain the 293 line of human embryonic kidney cells, which encode the E1 region of the Ad5 genome. After the transfection, the cells are harvested and purified using ultracentrifugation. Another advantage of the AAV system is that large number of AAV can be harvested using these techniques (>10^8vp/ml).
The only portions of the AAV that are absolutely necessary in cis to the vector are the inverted terminal repeats and the therapeutic gene. The packaging and structural genes, rep and cap, are able to be provided in trans. This means that the genome of the AAV can essentially be wiped out to make room for recombinant genes.
Section 2
Include some current research in each topic, with at least one figure showing data.
Section 3
Include some current research in each topic, with at least one figure showing data.
Conclusion
Overall paper length should be 3,000 words, with at least 3 figures.
References
Edited by student of Chad Kurylo for BIOL 238 Microbiology, 2009, Kenyon College.
