Mitochondrial Gene Therapy Via Nested Viral Vectors

Gene therapy is the mitigation of an undesirable phenotype by way of modifying the genetic information. For many years this held enormous promise, however, not much has happened in the clinic.

The reasons for this centre around delivery of the new genetic information. The best way of getting DNA into cells is by virus. Viruses are really fantastic at this job. The viruses that scientists tend to pick for this task fall into two main categories: those which infect and then express their DNA cargo directly, such as adenoviruses. Or those which infect and subsequently incorporate selected information into the host genome, such as lentivirus, which is a kind of retro virus.

Each have their own problems, adenovirus expression tends to be temporary, and repeated doses are far from a cheap/simple solution. Lentiviruses tend to incorporate themselves into the genome in an indiscriminate manner, where they may interrupt an important gene... doing more harm than good. Specifically, they may cause cancer.

Mitochondria have their own genome. They used to be bacteria in the dark history of evolution, before they were captured, and they now live as obligate intra cellular symbiotes. Most of their genome has been stolen away and now resides in the dusty library of the nucleus (carefully robbing them of their independence, naturally). However, a number of genes remain. Almost exclusively, these code for the proteins of the electron transport chain (ETC). A clever series of proteins that makes eukaryotes a whole lot more efficient. However, there are a number of defects associated with these proteins leading to very real human diseases.

The example I will use is Leber's hereditary optic neuropathy <link>. This disease is caused by mutations of the mitochondrial genome, all of which have been mapped, which prevent the normal operation of mitochondrial complex 1.

Now, there are viruses that infect bacteria; bacteriophage. There are even bacteriophage which infect bacteria and subsequently incorporate themselves into the genome <link>. This is great, it means that it may be possible to infect mitochondria with bacteriophage. Then, if you've been a good little molecular biologist, you can get your phage to incorporate itself into the mitochondrial genome and start expressing a FUNCTIONAL copy of the affected mitochondrial complex 1 sub unit.

Here's the first problem: The phage will not be able to infect or enter a eukaryotic cell. It will never reach the mitochondria. Worse, in a clinical setting it would just tool around in the bloodstream before getting destroyed by the immune system.

Here's the solution: create an adenovirus (more accurately an adenovirus associated vector; AAV) which encodes the phage genome. The phage will then be directly manufactured and constrained within individual human cells. The phage genomes will fit; AAV inserts go to about 5.5kb, phage genomes are rarely bigger than 4.5, and we may not need all of it.

Then, your AAV infects human cells, it's DNA starts being transcribed and replicated, the Phage then self- assembles inside the host cell, the functional phage infects the mitochondria, incorporates into the genome and then SOME of the complex 1 subunits begin to function normally.

There should be no problems with causing cancer: while there are MANY mitochondrial proteins associated with control of cell death etc. They are all encoded for by nuclear genes. There should be no problems with the AAV, you can give pretty massive doses of AAV which are replication-deficient. The phage should only ever be present INSIDE cells, so there should be no immune problems.

Potential problems: 1. There may be no phage capable of infecting mitochondria; it's been a while since they were free- living, and so their outer membrane may have become a little too specialized (would make a cool paper to infect mitos w/a micro-injected phage though).

2. The phage may not self-assemble in a eukaryotic cell: phage proteins may not be correctly processed and get stuck in the ER/fold incorrectly etc.

3. There may not be a way of controlling where the phage incorperates its DNA. You may fix complex1, but destroy complex 2. This may be mitigated by the multiple copies of mitochondrial DNA.... you should end up with a full complement of functional proteins, even if they're from diverse DNA molecules.