A the gene, we will use CRISPR to

A technique in gene therapy that allows for the insertion of new genetic material uses the gene gun (4). The gene gun inserts foreign DNA into target cells. It has been used for plants, but has also been studied for its use in humans and its potential in treating various diseases with gene therapy (22). We will use the gene gun for the purpose of introducing a new gene (TgsGP) to the human body which may be helpful in combating the variant-linked FSGS.The first thing that we must do is to isolate the TgsGP gene from the parasite T.b gambiense. The particular protein that is encoded by TgsGP can be found in the parasite’s flagellar pocket, but the gene itself is in the nucleus (1). To extract the gene, we will use CRISPR to cut the gene out. Specifically, the enzyme Cas9 will perform the slicing of the gene (13). In order to get to the nucleus, Cas9 will be encoded with a nuclear localization signal that will direct it to the nucleus. Once inside the nucleus, Cas9 will be able to cut out the TgsGP gene.A study has shown that APOL1 has been confirmed to be expressed in podocytes of the kidney. Podocytes (visceral epithelial cells) are cells found in the kidneys that line the outer surface of the glomerular capillaries and are essential for glomerular filtration (13). After TgsGP is separated from the parasite, podocytes will be extracted from a chlorocebus aethiops (African green monkey) with either the APOL1 G1 or G2 variant. African green monkeys are the only organisms besides humans with the APOL1 gene (21). Once these cells are extracted, we will put them aside for use with a gene gun. The gene gun uses helium to to hit cells with gold nanoparticles covered with the DNA (22). This will allow the particles to penetrate the cells, and the DNA will be released and become infused into the nucleus (6). While this will be a great tool for getting the gene into the podocytes, a disadvantage is that we cannot control exactly where the gene will go in the sequence (22). There are also some disadvantages of using gold particles. Prolonged exposure to the gold particles may be potentially damaging (8). However, there is research that is currently exploring the development of biodegradable particles made of chitosan (a polysaccharide) and poly-gamma-glutamic acid. Studies have shown that these particles are a safe and effective alternative to gold particles (8).For the purpose of this study, we will use the biodegradable nanoparticles with the TgsGP gene, and use the gene gun to inject it into the podocytes. However, we cannot simply take the genes and place them into the target cells. The TgsGP gene that was cut out with the Cas9 must be inserted into a vector, “which can replicate when introduced into a host cell” (20). The vector that we will use are plasmids, as is commonly used. “Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separate from a cell’s chromosomal DNA” (20). In order for the TgsGP gene to be placed in the plasmids, Cas9 will be used again to cut the plasmids. The plasmids will then be attached to biodegradable particles for use with the gene gun. Once the particles are pushed forward with the helium blast, they will enter the podocytes and fuse the new genes into the nucleus (6). With infusion of these new cells into the primate subjects with the APOL G1 or G2 variant, the subjects’ cells will be monitored for any new mutations in the DNA or any effects that the TgsGP has on the cells.