Download Gene discovery and validation technologies

BIOTECHNOLOGY
Gene discovery
and validation
technologies
A novel technology platform makes it possible to assess how the blocking of
specific genes can alter disease progression.
Dr Klaus Giese, Atugen AG
T
The real
work of
genomics ...
has only
just begun
he human genome project will, for the first
time, provide a complete nucleotide
sequence for every human gene. This
information will have immediate application in the
diagnostic field but excitement over its medical
impact will be limited by the lack of functional
data. Unravelling the function of genes will take a
much larger effort and time, and will require new
strategies. In particular, technologies are required
to rapidly select and validate putative drug targets
in order to understand disease initiation and
progression. The real work of genomics, therefore,
has only just begun.
Atugen AG has a fully integrated functional
genomics programme to rapidly identify gene
function and is, therefore, in a pivotal position to
address this future task. The company designs
small nuclease-resistant oligonucleotides (termed
GeneBlocsTM) and ribozymes against target genes to
selectively inhibit their expression and establish
their role in disease. The approach is applicable
in a high-throughput mode, and can be used
in dose-response studies at all stages of animal
development. Moreover, the function of
GeneBlocsTM can be studied in several animal
models which are predictive of human disease.
This article provides an overview of Atugen’s
technologies and the company’s approach to
finding novel genes involved in disease initiation
and progression.
Tools and technologies
Atugen has exclusively licensed a target validation
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and discovery technology based primarily on
ribozymes, GeneBlocsTM, and delivery vehicles,
developed at Ribozyme Pharmaceuticals Inc, USA.
Since the company’s inception, the technology
has been further developed and applied to animal
models of human diseases. The result is a
technology which can be used in a highthroughput mode to discover and validate putative
drug targets in vitro and in vivo.
Ribozymes are catalytic RNA molecules that
inhibit gene expression in a highly specific
manner by binding to and cleaving the target
mRNA, thereby preventing translation into the
protein. Atugen is using ribozymes expressed from
vectors for discovery purposes only, as described
in detail below.
GeneBlocs TM are improved antisense
molecules with outstanding properties, such as
nuclease resistance, high binding affinity and
specificity to target RNA. In contrast to other
commercially available antisense molecules,
GeneBlocsTM cause very little toxicity in
mammalian cells. This is a major advantage,
allowing the expression of target genes to be
specifically inhibited for several days without
causing oligonucleotide-mediated side effects.
Delivery vehicles The third important tool is
Atugen’s proprietary delivery vehicles, which are
used to deliver GeneBlocsTM to mammalian cells.
Some of the important characteristics of these
proprietary formulations are serum compatibility
and the ability to achieve sustained delivery over
several days. The company is using these tools Innovations in Pharmaceutical Technology
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Figure 1.
The highly efficient delivery of GeneBlocsTM to mammalian cells. A) Mammalian cells were
transfected with a fluorescently-labelled GeneBlocTM and the degree of intracellular delivery
analysed by flow cytometry. B) GeneBlocTM screen against a specific target mRNA. Eight
different GeneBlocsTM were designed and tested for inhibition of the target mRNA expression.
Quantification of mRNA was done by multiplex quantitative reverse transcriptase polymerase
chain reaction. 1 and 2, untransfected and control GeneBlocTM-transfected cells, respectively;
3-10, GeneBlocsTM directed against the target mRNA.
either alone or in combination with other
technologies - to discover new targets and validate
gene function.
Target validation
The result is a
technology
which can be
used in a
highthroughput
mode to
discover and
validate
putative drug
targets in
vitro and
in vivo
78
The Atugen delivery system can achieve
transfection efficiencies of over 90% (Figure 1A).
After an initial step of identifying and optimising
the appropriate formulation for a cell type from
our extensive list of delivery vehicles, a small set of
GeneBlocsTM is designed against putative targets.
To successfully complete this step for the gene
of interest, nucleotide sequence information as
short as only an expressed sequence tag is required.
The GeneBlocsTM are then tested for their
potential to inhibit gene expression; this is
determined by a multiplex Taqman assay
(quantitative reverse transcriptase polymerase
chain reaction, Figure 1B). The high percentage
of GeneBlocsTM which inhibit gene expression at
a level of 70% or greater attests to the improved
target RNA binding properties of Atugen’s
antisense molecules and the power of the
proprietary site-selection programme which
predicts suitable binding sites in the target RNA.
At least two independent GeneBlocsTM are
selected for each target for further biological or
phenotypic assays.
The GeneBlocTM technology is applied at two
distinct steps in the overall drug development
process (Figure 2). In a first step, the best targets
are selected from a large pool of candidates for
subsequent screening of small molecules. For
this task - termed Target Validation - potent
GeneBlocsTM are screened for that significantly
reduce the expression of the target mRNA in
mammalian cells. The achieved mRNA knockdown is correlated with changes in specific
biochemical pathways and/or phenotypic assays
to either validate or invalidate the target. By
providing functional information to nucleotide
sequences, crucial data is added - making the
patent applications of customers more viable.
In a second step, Atugen helps to optimise new
chemical entities in the preclinical development
stage. This approach is of particular importance
when putative drug targets belong to gene families
- such as members of protein kinases, protein
phosphatases and transcription factors - which
share a common DNA binding domain.
GeneBlocsTM can be viewed as small molecule-like
inhibitors with higher specificity and can therefore
be used to select the safest and most efficacious
small molecule lead - for example, by comparing
the gene expression changes induced by the small
molecule and the GeneBlocTM.
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Figure 2.
Atugen can make a significant contribution to the overall drug
discovery process. See text for details.
Figure 3.
High-throughput screening assay. GeneBlocsTM are screened in this assay using phenotypic
read-outs. Up to 40 genes can be analysed in parallel using a 384 well-plate format. Top and
bottom panels, inhibition of gene expression analysed by cell counting using light microscopy,
or by using a fluorescently-labelled marker, respectively.
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Figure 4.
Schematic diagram of the step-wise analysis of molecular changes. Atugen uses GeneBlocsTM
to induce a process of molecular changes by inhibiting the expression of specific genes. The
timely order of the molecular changes can be analysed at the protein and DNA level.
GeneBlocsTM and microarrays
The high specificity of GeneBlocsTM for target
RNAs makes them powerful tools to unravel the
sequential order of gene expression cascades. For
this programme, RNA isolated from GeneBlocsTM
and control-treated cells is hybridised to DNA
microarrays. This powerful combination of
technologies - specific mRNA knock-down
combined with gene expression profiling - enables
the dissection of gene expression cascades which
would otherwise be impossible to investigate
using other technologies. The instant transient
knock-down of gene expression induced after
GeneBlocTM treatment does not allow for any
compensatory changes which the cell might
undergo using conventional techniques, such as
stable cell lines or classical knock-outs.
High-throughput screening assays
A major bottleneck in the use of today’s gene
validation technologies is the inability to screen
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large numbers of candidate drug targets in a
timely fashion. A solution to this problem is
provided through use of a high-throughput
screening system with phenotypic endpoints as a
read-out; this mode of screening for novel drug targets is only possible because GeneBlocsTM
have significantly reduced toxicity compared with
other commercially available reagents. The low
level of toxicity not only reduces the occurrence
of false positives but also allows long-term
inhibition of gene expression - which is imperative
for studying the role that certain genes play in
pathology. An example of a high-throughput
screen based on a 384 well-plate format and
capable of analysing up to 40 genes in parallel is
shown in Figure 3. This screening assay has been
combined with an imaging system to follow more
than one biochemical or biological change at a
time; the imaging system can measure up to five
different fluorescent-based phenotypic endpoints
simultaneously including proliferation, apoptosis,
mitochondrial activity and calcium flux.
GeneBlocsTM
can be viewed
as small
molecule-like
inhibitors
with higher
specificity and
can therefore
be used to
select the safest
and most
efficacious
small molecule
lead ...
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BIOTECHNOLOGY
Ribozyme target discovery technology
A ribozyme-based system has been developed at
Atugen to discover and validate target genes in a
one-step process; this technology represents a
new and fast way to identify genes involved in
normal or disease processes. The system produces a
combinatorial ribozyme library, encoding all
possible ribozymes, and cloned in an expression
vector for transfection of target cells. The selection
of ribozymes is based on specific phenotypes; after
identification of the ribozyme, the corresponding
target gene is identified through bioinformatics providing a one-step process to identify and
validate target genes.
Target discovery
The stepwise
analysis of
signalling
pathways
will enable
identification of
the crucial
markers of
disease and the
development of
diagnostic and
therapeutic
molecules
Figure 5.
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Using this new approach, novel genes involved in
human disease can be discovered. In contrast to
the current approach taken by the pharmaceutical
industry - which historically has been to compare
normal and pathological tissues - Atugen is able to
unravel the unknown links that underlie the long
transition from, for example, a low to a high
metastatic tumour tissue (Figure 4). The stepwise
analysis of signalling pathways will enable
identification of the crucial markers of disease and
the development of diagnostic and therapeutic
molecules - diagnostic markers to allow early
detection of disease, as well as its progression, and
therapeutic products to intercept the pathogenic
process. As mentioned above, GeneBlocsTM can
subsequently be used to model better drugs against
specific targets.
Atugen’s first priority is to target tumour
suppressor genes, which are believed to play an
important role in preventing cancer and metastatic
events. An example of such an approach is shown
Analysis of biochemical pathways using GeneBlocTM
technology. A) Signals activating the PI-3K pathway are
controlled by the tumour suppressor, PTEN. B) Immunoblot
analysis of cell lysates after GeneBlocTM treatment. Cells
were transfected with either a control or a PTEN-specific
GeneBlocTM. p110, subunit of PI-3K.
in Figure 5; in this experiment, the expression of a
specific tumour suppressor, termed PTEN, is
inhibited and the effect on cellular signalling
changes measured. Activation of the survival
protein Akt (PKB) in the absence of PTEN protein
rapidly and elegantly verifies the tumour
suppressor function, which previously could only
be shown by means of time-consuming animal
knock-out experiments. RNA and protein extracts
from GeneBlocTM-treated cells are currently
analysed for changes at the RNA and protein level.
In vivo gene validation and discovery
Atugen operates an animal facility, which allows
it to move quickly from cell-based assays to
animal models of human disease; this enables
putative target genes to be studied in their
relevant environment. The biological effects of
GeneBlocsTM have been demonstrated in several
animal models; effectiveness in these animal
models not only verifies the cell-based assay data
but also indicates the potential usefulness of
GeneBlocsTM as drugs. In addition, we offer
traditional transgenic and “knock-out” animals as
part of our commercial TVD (target validation and
discovery) programmes.
Conclusion
Atugen has developed a technology platform to
bridge the gap between nucleotide sequence
information and the function of a gene. The
technologies make it possible to assess how
the blocking of specific genes can alter disease
progression. Moreover, the technologies allow
multigenic diseases to be studied by inhibiting
several different genes at the same time using
specific sets of GeneBlocsTM. This represents a
major advantage over other technologies, which
can only study one gene at a time.
Klaus Giese is a Vice President of Research at Atugen
AG. Dr Giese received his PhD in Biochemistry
from the Free University of Berlin in 1990. As a
postdoctoral fellow at the University of California,
San Francisco, he focused on the regulation of
lymphocyte-specific gene expression. In 1994, he
joined Chiron Corporation where he worked on the
discovery and development of new pharmaceuticals
for the treatment of human disease. As a Vice
President of Research, he is responsible for the
scientific programme at both Atugen’s headquarters in
Berlin (Germany) and its subsidiary in Boulder
(Colorado, USA).
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