Reading these answers I wonder, why doesn't "gene therapy" use self-contained plasmids instead of trying to splice a length into a chromosome?
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2$\begingroup$ IIRC humans have several mechanisms that digest extra-nuclear DNA. Unfortunately I don't have a reference handy, but I'm 95% sure any plasmid would just be destroyed. $\endgroup$– MCMCommented Oct 1, 2015 at 21:05
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$\begingroup$ That makes sense. So "some yeasts" is the exception, having first evolved a mechanism to allow importing of survival traits, possibly trading off resistance to viruses. $\endgroup$– JDługoszCommented Oct 1, 2015 at 21:09
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1$\begingroup$ In mammalian cells, Plasmids are not replicated, which is why in cell culture, you need the extra step of recombining the vector into the genome via homologous recombination. The issue is that this can be done in vitro to cells, but once you try doing it on an organism level there are many problems. One is that Adenovirus usually needs to be used to insert the vector. But adenovirus' capsid can only accommodate about 10kb of DNA. As most homology arms are at least 10kb, each, you cannot do targeted integration. You would need to rely on random integration, and you couldn't predict the effects. $\endgroup$– AMRCommented Oct 2, 2015 at 2:13
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2 Answers
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First, plasmids do not usually integrate into the chromosomes of human cells. In the laboratory, plasmids can be introduced into the cytosol of cells, and plasmid DNA will then be "read" by the host cell and used to make new proteins, for example. But plasmids usually remain separate from the cell's own DNA, and in that sense they are not effective for gene therapy.
Second, our cells are quite good at detecting and destroying plasmids, as well as other foreign DNA. This is a self-defense mechanism, evolved to get rid of viruses and other nasty parasites that might be trying to hijack the cell. In the lab, where we are working with isolated cells in a culture dish, with no immune system, plasmids are typically tolerated. But in a human being, immune cells that have specialized in detecting intruders are very competent at detecting such manipulations, and they will quickly sound the alarm and wreak havoc ... So if injected into the body, plasmids are quickly detected and destroyed.
Research in gene therapy aims to find more efficient ways of "editing" the DNA of human cells, ultimately in a way that can be safely applied in the body. Such a technique does not exist yet. The latest CRISPR-Cas9 gene editing system is a phenomenal tool, and is very efficient at getting into the DNA of human cells; but the CRISPR-Cas9 proteins must themselves be delivered into the cell, for which a virus is typically needed, and this would again cause immune reactions if attempted in a human body.
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I believe we could use plasmid insertion to assist with gene therapy. EBV can maintain a plasmid in the cells it infects and the plasmids can effectively replicate. With a greater understanding of the latency and the implications of inserting genes using EBV, it may be possible for gene therapy to occur via genes on a plasmid. references: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2562867/ http://jvi.asm.org/content/65/1/483.full.pdf
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$\begingroup$ Can you elaborate on «EBV can maintain a plasmid in the cells it infects» in light of Roland’s answer that they are seen as invadors? $\endgroup$– JDługoszCommented Jan 25, 2018 at 4:18
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$\begingroup$ This is a very interesting answer. Whether or not it has any relevence to gene therapy, it is valuable as background information that most people will not be aware of. It could be improved further by better layout and some elaboration. I would split it into three paragraphs: 1. Explain what EBV plasmids are, stressing the eukaryotic replication origin as opposed to the bacterial one the poster may have been thinking of, 2. Suggest how they might be used for gene therapy, 3. list the references. $\endgroup$– DavidCommented Jan 25, 2018 at 14:14