From: Robert J. Bradbury (bradbury@www.aeiveos.com)
Date: Mon Sep 06 1999 - 12:38:29 MDT
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:
a) Delivery of the package to the targeted cells
b) Entry of the package into the cells
c) Decloaking the package to release the DNA
d) Transport of the DNA into the nucleus
e) Keeping the DNA in the nucleus (either by integration
into existing DNA or as a separate vector/chromosome)
f) Expression of the gene(s) in the desired cell types
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
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