On Mon, 6 Sep 1999 GBurch1@aol.com wrote:
>
> A probably naive question: Are these mechanisms exploitable to do wholesale
> somatic cell genetic engineering, i.e. can a retrovirus be tinkered with to
> be a vector to carry specified genome "edits" into lots and lots of
> (targeted?) somatic cells?
>
Somatic Gene Engineering is complex, it requires:
Dowdy's TAT-fusion protein method may be a great way to solve (a-b). They cite that most of the cells in culture take up their "package" (which does not include any DNA/RNA). There are multiple other methods under development to solve the delivery problem.
All of the problems have been solved to varying degrees, (d) & (e) are the most difficult to solve well because there are not natural mechanisms for this for large pieces of DNA.
Retroviruses are not the best vectors because they only integrate in DNA in dividing cells. That may be fine for cancer treatments but for general purpose somatic gene therapy it is insufficient. Adeno-associated-virus (AAV) seems to be the prefered carrier at this point because it preferentially integrates in a specific site on the q arm of chromosome 19. The fact that the virus can do this, would seem to imply that with sufficient engineering, we could probably target genes to specific locations. [Emphasis on *sufficient* since it probably requires the X-ray crystal structures of the molecules that do this fancy footwork and some advanced molecular modeling by some very clever people on how to change the proteins so they do the job on different DNA sequences.]
AAV (a parvovirus) and is a very small virus (~5000 bases) and can only carry very few genes. Retroviruses are similarly small. Herpesvirus and Cytomegalovirus (the cause of mononucleosis) on the other hand are much more complex (~200-300K bases) and can carry many more genes (50+). *All* viruses have solved the problem of how do I get my genes into a cell, usually into the nucleus (a-e) above. The bible of virology, "Fields Virology", is a two volume 2300+ page door stop. There are over 20 different families of viruses from which to select the gene engineering toolkit. For example, you would like to engineer a cell line (factory) that produces large quantities of cytomegalovirus-like enveloped vectors with 50+ anti-aging/intelligence/... genes that incorporate the site-specific integration capabilities found in AAV. This is a very complex field and we have probably just scratched the surface.
The knowledge for (f) becomes clearer when we have isolated all of the transcription factors in the genome and determined their DNA binding sequences (3-5 years probably).
So in answer to the question, no, a retrovirus would not be able to do what you want. However, we have existance proofs that suggest what you want to do is highly likely to be achievable.
As some of you are aware there was a difference of opinion between myself, Greg Stock and John Campbell regarding the feasibility of somatic gene therapy at Extro4. I've got a stack of papers that I need to go through on topic and will be writing a "Why Somatic Gene Therapy Will Happen" background paper to address some of Greg & John's concerns. The shortest answer possible is - Its the only thing that can really "cure" cancer. I would also argue that somatic gene therapy is much more important to many more people, and has fewer ethical mines, than germ line engineering is or does. Because of its greater advantages to more people (who would be willing to pay for it), its development is likely to proceed faster, even if it "were" more difficult, which is debatable.
The question is not if, but when.
Robert