Adeno Associated Virus Research
The specific research I conducted at my biotechnology internship was about the use of Adeno-Associated Virus (AAV) to treat and kill cancer cells in humans. Although my internship concluded before the completion of the project, the CEO of the company published a paper detailing the findings from this research project. Because my research was quite recent, there are no good illustrations depicting the process so I complied and edited images to create a simple, yet effective means of visualizing the research. Further details can be found in the published paper.
First, a host plasmid was obtained for use in the company's AAV system. The AAV system packages the plasmid into the AAV, so a specific plasmid must be used. In addition, scientific research was carried out to determine which genes in various bacteria could be used to create toxins to kill cancer cells. The challenge was finding toxins that would stay within the cancer cells and that would not spread to other cells even after those cells died. Once toxic genes that met the criteria were found, those genes were isolated from their natural host or ordered from another company, if possible. Both plasmid and toxic gene would get cut with the same restriction enzymes, leading to specific directional ligation. The plasmid and toxic gene would ligate together and the toxic gene would be inserted into the plasmid DNA. The resulting toxic plasmid would produce the toxin if the plasmid was transcribed and translated.
After the toxic gene was inserted into the plasmid, the plasmid is then packaged into the AAV. However, before packaging, an AAV strain needs to be chosen. Each AAV strain targets a different set of cells. For example, AAV2 targets skeletal muscle cells, vascular smooth muscle cells, and neurons. On the other hand, AAV8 targets hepatocytes. The specific strain of AAV needs to be chosen based on which cells are going to be targeted. AAV has this specificity because each strain has a separate set of receptors, which allow AAV to target certain cells while leaving other cells alone. When packaging is complete, the virus is injected into the bloodstream in high titer. It is injected in high titer because the amount of AAV that reaches the target cells is based on random chance. High titer allows for high probability of AAV infection in those cells. As the AAV travels through the body, it will bind to cells where the receptors match. The cells that bind AAV will then take in the AAV through endosome formation.
Within the cell, the endosome carrying the AAV will bind to the nucleus. Again, the number of endosomes that reach the nucleus is also based on random chance, which is why using high titer AAV is important. Once the endosome reaches the nucleus, it will bind to the nuclear membrane and the endosome will open up. The AAV carried within will become exposed to the nuclear membrane. The AAV will then insert the toxic plasmid into the nucleus, where gene expression can begin.
Inside the nucleus, the cell machinery can transcribe the plasmid and create mRNA. RNA polymerase II will synthesize mRNA after transcription of the plasmid. Because the toxic gene was cloned into the plasmid at the beginning, RNA polymerase II also transcribes the toxic gene into mRNA. The mRNA is transported out of the nucleus and into the cytoplasm for protein synthesis. In the cytoplasm, the mRNA is translated across the membrane of the endoplasmic reticulum. Ribosomes and tRNAs facilitate the creation of amino acid chains from the mRNA. Once the translation is complete, the amino acid chain will fold up in the correct conformation, leading to an active protein. In this case, the protein will be a toxin which will end up killing the cancer cells that it is produced in. In the end, the cancer cells targeted by the AAV will die from the toxins resulting in a reduction in the size of the tumor.