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Double-stranded
RNA triggers potent, specific gene silencing through a process called
RNA interference (RNAi). Greg
Hannon's laboratory has elucidated key biochemical details of the
components of the pathways involved in RNA interference, and is exploiting
these findings to develop molecular tools that can be used for gene
discovery, evaluation of gene function and generation of animal models.
Silencing is accomplished in a two-step process. Double stranded RNAs
are first cleaved to small inhibitory RNA about 22 nucleotides long
by a nuclease called Dicer. These siRNA's are incorporated into a multicomponent
nuclease, RISC, which unwinds the siRNA and uses it to select the complementary
mRNA for cleavage and degradation.
Short hairpin RNAs (shRNAs) were investigated as an alternative to double
stranded RNA in shutting off gene expression, and found to be equally
effective. Expression vectors were constructed that express shRNAs.
When these shRNAs are introduced into cells, they can be stably integrated
and expressed and will turn down expression of their complementary gene.
The shRNAi vectors have proven effective in vitro, and have
been widely used to probe gene function in a variety of experimental
applications.
More recently, shRNA's have been used successfully to down-regulate
gene expression in vivo. Using RNAi directed against a well-characterized
gene (p53), In a collaboration with the Hannon group, Scott Lowe's laboratory
demonstrated that RNAi could stably suppress gene expression in hematopoietic
stem cells, leading to cancer phenotypes upon reconstitution of the
hematopoietic compartment in recepient mice (Figure 3). Furthermore,
shRNAi's directed against different parts of the p53 gene produced distinct
phenotypes, ranging from benign hyperplasia to disseminated malignancy
depending on the degree of p53 suppression intrinsic to each shRNA.
These results suggest that RNAi can be used to produce an 'epi-allelicÅfseries
of hypomorphic mutants for in-depth study of gene function. In addition,
Dr. Hannon, in collaboration with Dr. Thomas Rosenquist, stably integrated
a silencing construct directed against a DNA repair gene (Neil1) into
ES cells, and used these cells to generate transgenic animals. They
demonstrated that decreased expression of this gene was faithfully transmitted
through the germline to produce heritable gene silencing. Together,
these results validate RNAi as an alternative to homologous recombination
for the generation of knock-down (or knock-out) mice. As it circumvents
several of the most difficult and time-consuming steps in homologous
recombination approaches, use of RNAi could allow a much more rapid
assessment of in vivo gene function.
Dr. Hannon's lab has mounted a major project
to generate human and mouse libraries of expressed shRNA's in vectors
that can be introduced into mammalian cells. The goal is to generate
silencing constructs, three per gene, against 30,000 human genes and
a set of 10,000 mouse genes. Once these libraries are established, they
will represent a powerful genetic resource that will allow silencing
of any gene in the genome in a selective manner. These tools have the
potential to greatly simplify gene manipulation and gene discovery for
many biomedical applications, such as validation of gene function, probing
interactions between genes, and establishment of animal models.
Dr. Hannon has devised an approach to use this
library as a tool for cancer gene discovery. In one approach, shRNAs
will be used in a cell-based screen to identify genes that are essential
for the survival of cancer cells, but that are not required for the
survival of normal cells. Such genes represent therapeutic targets for
discovery of drugs that could selectively destroy cancer cells while
leaving normal cells untouched. Since it is clear that not all cancer
cells respond the same way, a number of different cancer lines will
be evaluated in this way to find genes that may be pathway-specific.
In a parallel set of experiments, the same series of silencing constructs
will be used to attempt to modify the response of cancer cells to existing
cancer therapies. The identification of targets for combination therapies
may permit the use of much lower doses of existing therapeutics, maximizing
the chance of obtaining a therapeutic effect while minimizing side effects.
This line of research will complement the work efforts in the laboratories
of Michael Wigler and Robert Lucito to identify cancer genes and correlate
genotype to treatment outcome (see Cancer
Gene Discovery), and will also segue well into studies of genes
and therapies in animal models in the laboratories of Scott Lowe and
Senthil Muthuswamy.
Coupled with discoveries from other labs, including Rob
Martienssen and Shiv
Grewal at Cold Spring Harbor Laboratory, Dr. Hannon's advances with
RNAi research were cited as the "Breakthrough
of the Year" for 2002 by Science Magazine, acknowledging the
rapid progress and impact of this field.
Key Publications:
Dicer: Bernstein E, Caudy AA, Hammond
SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step
of RNA interference. Nature. 2001 Jan 18;409(6818):363-6. abstract
shRNA: Paddison PJ, Caudy AA,
Bernstein E, Hannon GJ, Conklin DS. Short hairpin RNAs (shRNAs) induce
sequence-specific silencing in mammalian cells. Genes Dev. 2002 Apr
15;16(8):948-58. abstract
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Silencing in murine cells: Paddison
PJ, Caudy AA, Hannon GJ. Stable suppression of gene expression by RNAi
in mammalian cells. Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1443-8.
abstract
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release
In vivo p53 silencing: Hemann MT, Fridman
JS, Zilfou JT, Hernando E, Paddison PJ, Cordon-Cardo C, Hannon GJ, Lowe
SW. An epi-allelic series of p53 hypomorphs created by stable RNAi produces
distinct tumor phenotypes in vivo. Nat Genet. 2003 Mar;33(3):396-400.
abstract
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release
In vivo Neil1 silencing: Carmell
MA, Zhang L, Conklin DS, Hannon GJ, Rosenquist TA. Germline transmission
of RNAi in mice. Nat Struct Biol. 2003 Feb;10(2):91-2. abstract
press
releases
RNAi in heterchromatin silencing: Volpe
TA, Kidner C, Hall IM, Teng G, Grewal SI, Martienssen RA. Regulation
of heterochromatic silencing and histone H3 lysine-9 methylation by
RNAi. Science. 2002 Sep 13;297(5588):1833-7. abstract
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