Hi Mike,
AAV--"site specific integration
Wild type AAV can infect a cell and specifically integrates its DNA into a specific site in chromosome 19 of the host cell...which does not disrupt an active region of the host DNA. Although AAV vectors usually don't integrate its DNA into chromosome 19, usually the integration is random, and not site specific into the host genome.
I recently glanced @ the ARIA rapamycin paper, and thought that their system is interesting...I'm wondering what is the size of each promoter component. From what I think I can understand, is that there are two different AAV or Ad vectors that must infect the same cell. If only one infects a particular cell, then nothing is supposed to happen with or without rapamycin...but I think there is some still some expression (although they did say that they had a second generation that is better). The results of the shortened length of expression for the Ad vector in immunocompetent mice was expected, since the Ad vector seems to be an E1 E3 deleted version that has leaky expression of the wild type Ad proteins, which creates a cytotoxic T cell immune response that attacks the transduced cells...shortening the length of expression. I think if they would of used a different vector system for comparison would have been more interesting(eg. retrovirus, lentivirus, helper-dependent Ad, etc.)<imo>. If they really wanted to they probably could of put both components into the same Ad vector, but I think they developed the 2 part system to try and evade the packaging size limitation of AAV, maybe by splitting the coding sequence into two parts, and only when both parts are present the gene is expressed. Getting the gene into the cell is only one of the obstacles, another important obstacle is regulating expression of the gene over a period of time, and they have a very interesting system that could be very beneficial.
efficient
For efficient use in vivo, direct injection into the host, as opposed to ex vivo (harvesting cells from the body...transducing them in tissue culture...reimplanting the transduced cells back into the host), one usually needs to have a titer of @ least ~100,000,000 infections particles/ml (naked DNA, or liposomes are inefficient...they usually get trapped inside endosomes, and get degraded...viruses usually have mechanisms to escape the endosome and the degradation pathway).
working with monkeys
From what I understand, for the FDA to approve of a gene therapy protocol to be approved for human studies, there needs to be tests on @ least 2 different animal species(if I'm wrong...please correct me : )
Best of luck, Where'd He Go? |
