How Gene Therapy Works
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Generally, gene therapy works in four main steps - modification, delivery, activation, and integration. However, before the steps commence, a suitable target gene must be chosen. Not all diseases have a genetic basis, so the disease targeted must have a genetic basis for gene therapy to be effective. Once the disease is chosen, the researcher must find out which genes are involved and mutated in the disease. In addition, the mutation's effects on gene function and the effect of a normal copy of the gene must be known. If these criteria are not met, gene therapy will be useless and possibly dangerous. If these criteria are met, gene therapy can begin.
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The first step in gene therapy is the modification of DNA to the specific target gene. First, a normal copy of the gene is isolated from other cells or ordered from another location, if necessary. The normal copy of the gene and the vector DNA are both cut with restriction enzymes. Two different restriction enzymes can be used to help give directionality when the gene is ligated into the vector. The vector and gene get ligated together by DNA ligase to create a recombinant DNA plasmid containing the gene of interest. This plasmid can now be used to facilitate replication and expression of the gene of interest once inside the body.
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After the creation of a plasmid, it needs to be delivered into cells. There are two main methods of delivery. The first method is in vivo delivery, where the delivery vector containing the plasmid is injected directly into the body. The second method is ex vivo delivery, where the plasmid is inserted into cells in a laboratory setting. Each delivery method has its pros and cons, so the method of delivery is chosen based on each individual patient's needs.
In vivo delivery usually uses viruses, liposomes, or naked DNA to insert the plasmid into cells. Viruses have high specificity and efficiency, but they can elicit an immune response. Liposomes and naked DNA do not elicit an immune response, but are very inefficient.
Ex vivo delivery inserts the plasmid into cells in a laboratory setting, removing the risk of an immune response and increasing efficiency. Unfortunately, removal and reinsertion of organ-specific cells or stem cells can be extremely invasive and strenuous on the patient.
In vivo delivery usually uses viruses, liposomes, or naked DNA to insert the plasmid into cells. Viruses have high specificity and efficiency, but they can elicit an immune response. Liposomes and naked DNA do not elicit an immune response, but are very inefficient.
Ex vivo delivery inserts the plasmid into cells in a laboratory setting, removing the risk of an immune response and increasing efficiency. Unfortunately, removal and reinsertion of organ-specific cells or stem cells can be extremely invasive and strenuous on the patient.
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When delivery is completed, activation of the gene can occur. Through in vivo delivery, the virus will be taken into the cells through receptor binding on the cell surface. If the receptors match, endocytosis will occur and the virus will enter the cells through endosome formation. Through random chance and motion, the endosome may reach the nuclear membrane. If this occurs, the endosome will open up and the virus will insert the plasmid into the nucleus. Through ex vivo delivery, the plasmid will be directly inserted into the nucleus or cytoplasm of the cell. If the plasmid is inserted into the cytoplasm, it will be taken up by the nucleus if contact occurs.
Once inside the nucleus, cell machinery takes over and starts the gene expression pathway. The plasmid DNA is transcribed into mRNA by RNA polymerase II and the complete mRNA is exported to the rough endoplasmic reticulum. At the rough endoplasmic reticulum, the ribosomes and tRNAs read and translate the mRNA into a protein chain. The protein chain will then fold into secondary, tertiary, and quaternary structures. Ultimately, it will result in the protein expression of the gene of interest. Ideally, this protein would remove the condition and cure the disease.
Once inside the nucleus, cell machinery takes over and starts the gene expression pathway. The plasmid DNA is transcribed into mRNA by RNA polymerase II and the complete mRNA is exported to the rough endoplasmic reticulum. At the rough endoplasmic reticulum, the ribosomes and tRNAs read and translate the mRNA into a protein chain. The protein chain will then fold into secondary, tertiary, and quaternary structures. Ultimately, it will result in the protein expression of the gene of interest. Ideally, this protein would remove the condition and cure the disease.
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For the cure to be effective over a long period of time, the normal copy of the gene must integrate into the human genome. The correct cells need to integrate the gene into the correct position within the genome. This step is the most difficult and dangerous because integration is usually random. Additionally, inaccuracy of gene integration can lead to serious or fatal conditions that may be irreversible. If this is accomplished successfully, the gene will replace the mutated copy, leading to full lifetime recovery.