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Novartis
Innovation
Novartis Today:
Friedrich Miescher Institute for
Biomedical Research
Focus on Biomedical Research
The Friedrich Miescher Institute for Biomedical
Research (FMI) located in Basel, Switzerland,
was established in 1970 by a joint decision of
the then-two-separate companies Ciba AG and
J.R. Geigy AG—the predecessors of Novartis.
Since its founding, the FMI has contributed
substantially to a better understanding of the
molecular and cellular basis of disease and
has attained international recognition for its
fundamental biomedical research. Today, the
FMI focuses on neurobiology, quantitative
biology, and the epigenetics*1 of stem cell
development and cell differentiation.
T he F MI w a s nam e d af te r the B a s el
scientist Friedrich Miescher (1844-1895) who
first purified nucleic acids. It is affiliated with
both the University of Basel and the Novartis
Institutes for BioMedical Research (NIBR).
Susan Gasser, Director of FMI
*1 Epigenetics is the study of heritable
changes in gene expression that
do not involve changes to the
underlying DNA sequence.
*2 Proteomics is the comprehensive
study of proteins within an
organism or a cellular system,
focusing on structure and function.
This is an edited version comprised
of several articles by NIBR and FMI.
https://www.nibr.com/
http://www.fmi.ch/
8
Coupling Academic Research and
Biomedical Applications
The FMI is situated at the interface of academic
research and biomedical application. Findings
are published and presented to the scientific
community, contributing to the collective
understanding of human disease. Through
collaborative ef for ts with Novar tis, FMI
scientists also contribute to the development
of both diagnostics and medicines.
The FMI pursues biomedical discoveries
while establishing cutting-edge technology
platforms. FMI scientists make use of the
latest developments in technologies such
as genetic approaches in model organisms,
detailed proteomic*2 and genomic analyses,
microscopy, and structure determination.
Based on an in-depth understanding of the
molecular processes, FMI scientists hope
to uncover new means to combat cancer,
correct degenerative states, and suppress
dise ase s correlate d with physiologic al
dysfunction.
Sus an Gas ser, Direc tor of FMI, s ays:
“Biomedical research aims to describe the
molecular mechanisms at work within living
cells to enable an effective development of
new therapeutics. Now more than ever, it is
clear that biological research has a major
Vol. 3 July 2016
impact on the quality of life of each and
every one of us.”
FMI encourages its scientists to explore
n ove l a r e a s w i t h in t e ll e c t u a ll y d a r in g
approaches. The aim of the Institute is
to continually push back the horizons of
knowledge with original ideas and innovative
techniques. It provides an open, collegial
environment that allows for interdisciplinary
c o ll a b o r a t i o n a n d c r o s s - f e e din g f r o m
one field to another on a daily basis. This
tradition allows FMI to play a leading role in
biomedical research.
Training Young Scientists
According to its founding charter, the FMI
not only seeks to pursue and promote basic
biomedical research but also to provide
young scientists from all over the world with
an opportunity to participate in scientific
research. Currently, FMI laboratories are
home to approximately 100 PhD and MSc
students from about 30 different countries.
Registered at local universities, they are part
of the FMI International PhD Program and
carry out their dissertation studies under the
supervision of FMI group leaders. In addition,
about 90 postdoctoral students from around
the world pursue postgraduate studies at
the FMI. They are exposed to the latest in
molecular and genetic approaches while
being constantly encouraged to examine
biomedical applications.
FMI is internationally recognized as an
excellent training ground for young scientists.
This is testimony not only to the quality of
the research programs and the commitment
to maintaining state-of-the-art platforms but
also to the open, collegial atmosphere.
Issued by Communications Dept., Novartis Pharma K.K.
Toranomon Hills Mori Tower, 23-1, Toranomon
1-chome Minato-ku, Tokyo 105-6333 Japan
NPE00003JG0001 E
2016.07
The Leading Edge:
The Present, Past, and Future of Genomic Medicine
Editing Genes to Potentially Fight Disease
Special Interview
Towards Clinical Application of Genome Editing Technologies
Dr. Kohnosuke Mitani, Professor, Head of the Division of Gene Therapy and Genome Editing,
Research Center for Genomic Medicine, Saitama Medical University
Electrical Brainstorms Traced to Genetic Mutations
Scene:
Stopping Free Radicals at their Source
Novartis Today:
Friedrich Miescher Institute for Biomedical Research
Cover image: Cutting DNA sequence
of HeLa cells using CRISPR.
Image: HeLa cells by William J. Moore,
University of Dundee/Wellcome Images.
Modified by PJ Kaszas.
cancer gene mutations. As described in a
March 2015 paper in Cancer Research 1 ,
NIBR’s research investigator Yi Yang and
others used CRISPR to attach short protein
tags to several genes involved in cancer.
The tagged genes create fused proteins that
wither quickly unless a shield compound
protects them. Changing the amount of the
shield compound mimics how an actual
small molecule drug would inhibit the target
at different doses—without the need to spend
months or years to craft a small-molecule
compound targeted to the gene. CRISPR
enabled the application of the DegronKI approach (originally developed by Tom
Wandless’s laboratory at Stanford University)
to study these genes in a tunable fashion.
Pushing CRISPR into Clinical Applications
CRISPR is inspiring researchers to pursue
new therapeutic programs. In January
2015, NIBR entered into collaboration with
Intellia Therapeutics, a startup biotechnology
company, to sharpen this powerful tool and
investigate new treatments for patients.
Safety is top of mind for the NIBR/Intellia
team members, so the team will start with ex
vivo editing outside the body, which allows
for more control. After human cells have
been modified, the researchers can run them
through a battery of tests to ensure that
they meet stringent requirements before
administering them to patients. “We’re hoping
to treat a number of diseases, beginning with
certain cancers and hematologic disorders,”
says Mickanin. CRISPR also holds potential
for the treatment of certain hematological
disorders, where defective blood cells can be
removed, edited, and then returned to patients.
Image*: SPL, PPS
*CRISPR-Cas9 gene editing
complex. The Cas9 protein is
shown in blue. The guide RNA
is red, double-strands of the
target DNA are yellow, and the
non-target DNA strand is pink.
Craig Mickanin, Director of
TDT BioArchive, NIBR.
Photo by PJ Kaszas, NIBR
2
The Leading Edge:
Editing Genes to
Potentially Fight Disease
The Rise of CRISPR, a New Editing Tool
Imagine trying to find and correct a single
typo in a novel that is 2.3 million pages
long—more than 3,000 times the length
of Moby Dick—without computer software.
Scientists face an equivalent task when
editing the human genome, which contains
approximately 3 billion pairs of DNA “letters.”
With previous tools, it was difficult to efficiently
hone in on a particular segment of DNA and
make necessary changes.
An emerging tool known as the CRISPRCas9 system (or "CRISPR") makes the job
easier. This tool—and the technology behind
it—allow scientists to make precise cuts or
patch in new DNA. Think of CRISPR as a pair
of molecular scissors capable of snipping
DNA. The tiny shears are combined with an
RNA-based targeting molecule that scans
the genome for specific sequences and
makes a controlled cut at a single site.“The
key advantage of CRISPR is that it relies on
RNA to recognize specific DNA sequences
and direct the cutting machinery,” explains
Craig Mickanin, who leads a technologybased group within the Developmental &
Molecular Pathways department at the
Novartis Institutes for BioMedical Research
(NIBR). “We can quickly and easily design
RNA guide sequences. Then the cutting is
done with Cas9, a protein.”
CRISPR Fueling Cancer Drug Discovery
NIBR has adopted CRISPR to research
potential cell and gene therapies and to
identify drug targets. Researchers are using
CRISPR, for example, to quickly and precisely
investigate thousands of genes related
to cancer as potential drug targets. “The
technology improves the efficiency of cancer
drug target selection, aiding decisions on
which ones should advance to drug discovery
projects,” says Rob McDonald, an expert in
target identification for Oncology at NIBR.
Seeking answers about the roots of many
cancers, NIBR scientists have started using
CRISPR to study a large collection of cancer
cell lines known as the Cancer Cell Line
Encyclopedia (CCLE), developed jointly at NIBR
and the Broad Institute of MIT and Harvard.
The researchers have also combined
CRISPR with other molecular tools to study
“Editing Genes to Potentially
Fight Disease” is an edited
version of NIBR articles.
http://www.nibr.com/
Possible Synergies with Immunotherapy
The team is exploring how CRISPR can
enhance a program that Novartis runs
in collaboration with the University of
Pennsylvania to reengineer T cells and
unleash them on cancer in patients. These
chimeric antigen receptor (CAR) T cells—
“ninjas by design”—recognize a marker
that is unique to the surface of cancer cells
1. Zhou, Q. et al., Cancer Res. 2015; 75(10): 1949-58.
and launch an attack. In early phase clinical
trials, the investigational treatment is proving
most effective in certain blood cancers. “The
first clinical validation of gene editing using
CRISPR may be the one that reinforces
therapies for blood cancers with CART cells.”
“There are a number of challenges that
we face with the CART program going
forward, and we view gene editing as one of
the possible solutions,” says Phil Gotwals,
who leads a CART group at NIBR. It also
might offer improved ways to turn off cell
activity if patients have overly strong immune
reactions, or to add other immunotherapy
weaponry, Yang speculates.
CRISPR’s Advantages and Drawbacks
Compared with an earlier genome editing
method called TALEN, the CRISPR system
enables researchers to edit genes much more
efficiently and 200 times less expensively.
TALEN costs about $4,000 per gene and takes
months longer to perform than using CRISPR.
Additionally, CRISPR offers two huge
improvements over RNA interference (RNAi),
a gene-silencing method. The first is that
CRISPR can achieve complete protein
loss, as compared with only partial protein
reduction via RNAi. The second comes from
the improved specificity of CRISPR. While
the field is still learning about the drawbacks
of CRISPR, RNA interference research has
historically been plagued by off-target
effects that complicate the interpretation of
experiments.
Of cou r se, as wi t h al l g en et i c t o o l s ,
“there’s a lot of room for improvement with
CRISPR,” Yang notes. Among its drawbacks,
CRISPR doesn’t work with all genes, and it
sometimes attaches to off-target DNA. Labs
around the world—including at NIBR—are
rapidly developing alternative versions of
CRISPR that better address these problems,
cleanly activate gene expression or repress
genes without cutting them, or bring other
advantages. Beyond work in cells and animal
models, “the race is on and we need to see
who will be the first to push CRISPR into
clinical applications.”
3
cancer gene mutations. As described in a
March 2015 paper in Cancer Research 1 ,
NIBR’s research investigator Yi Yang and
others used CRISPR to attach short protein
tags to several genes involved in cancer.
The tagged genes create fused proteins that
wither quickly unless a shield compound
protects them. Changing the amount of the
shield compound mimics how an actual
small molecule drug would inhibit the target
at different doses—without the need to spend
months or years to craft a small-molecule
compound targeted to the gene. CRISPR
enabled the application of the DegronKI approach (originally developed by Tom
Wandless’s laboratory at Stanford University)
to study these genes in a tunable fashion.
Pushing CRISPR into Clinical Applications
CRISPR is inspiring researchers to pursue
new therapeutic programs. In January
2015, NIBR entered into collaboration with
Intellia Therapeutics, a startup biotechnology
company, to sharpen this powerful tool and
investigate new treatments for patients.
Safety is top of mind for the NIBR/Intellia
team members, so the team will start with ex
vivo editing outside the body, which allows
for more control. After human cells have
been modified, the researchers can run them
through a battery of tests to ensure that
they meet stringent requirements before
administering them to patients. “We’re hoping
to treat a number of diseases, beginning with
certain cancers and hematologic disorders,”
says Mickanin. CRISPR also holds potential
for the treatment of certain hematological
disorders, where defective blood cells can be
removed, edited, and then returned to patients.
Image*: SPL, PPS
*CRISPR-Cas9 gene editing
complex. The Cas9 protein is
shown in blue. The guide RNA
is red, double-strands of the
target DNA are yellow, and the
non-target DNA strand is pink.
Craig Mickanin, Director of
TDT BioArchive, NIBR.
Photo by PJ Kaszas, NIBR
2
The Leading Edge:
Editing Genes to
Potentially Fight Disease
The Rise of CRISPR, a New Editing Tool
Imagine trying to find and correct a single
typo in a novel that is 2.3 million pages
long—more than 3,000 times the length
of Moby Dick—without computer software.
Scientists face an equivalent task when
editing the human genome, which contains
approximately 3 billion pairs of DNA “letters.”
With previous tools, it was difficult to efficiently
hone in on a particular segment of DNA and
make necessary changes.
An emerging tool known as the CRISPRCas9 system (or "CRISPR") makes the job
easier. This tool—and the technology behind
it—allow scientists to make precise cuts or
patch in new DNA. Think of CRISPR as a pair
of molecular scissors capable of snipping
DNA. The tiny shears are combined with an
RNA-based targeting molecule that scans
the genome for specific sequences and
makes a controlled cut at a single site.“The
key advantage of CRISPR is that it relies on
RNA to recognize specific DNA sequences
and direct the cutting machinery,” explains
Craig Mickanin, who leads a technologybased group within the Developmental &
Molecular Pathways department at the
Novartis Institutes for BioMedical Research
(NIBR). “We can quickly and easily design
RNA guide sequences. Then the cutting is
done with Cas9, a protein.”
CRISPR Fueling Cancer Drug Discovery
NIBR has adopted CRISPR to research
potential cell and gene therapies and to
identify drug targets. Researchers are using
CRISPR, for example, to quickly and precisely
investigate thousands of genes related
to cancer as potential drug targets. “The
technology improves the efficiency of cancer
drug target selection, aiding decisions on
which ones should advance to drug discovery
projects,” says Rob McDonald, an expert in
target identification for Oncology at NIBR.
Seeking answers about the roots of many
cancers, NIBR scientists have started using
CRISPR to study a large collection of cancer
cell lines known as the Cancer Cell Line
Encyclopedia (CCLE), developed jointly at NIBR
and the Broad Institute of MIT and Harvard.
The researchers have also combined
CRISPR with other molecular tools to study
“Editing Genes to Potentially
Fight Disease” is an edited
version of NIBR articles.
http://www.nibr.com/
Possible Synergies with Immunotherapy
The team is exploring how CRISPR can
enhance a program that Novartis runs
in collaboration with the University of
Pennsylvania to reengineer T cells and
unleash them on cancer in patients. These
chimeric antigen receptor (CAR) T cells—
“ninjas by design”—recognize a marker
that is unique to the surface of cancer cells
1. Zhou, Q. et al., Cancer Res. 2015; 75(10): 1949-58.
and launch an attack. In early phase clinical
trials, the investigational treatment is proving
most effective in certain blood cancers. “The
first clinical validation of gene editing using
CRISPR may be the one that reinforces
therapies for blood cancers with CART cells.”
“There are a number of challenges that
we face with the CART program going
forward, and we view gene editing as one of
the possible solutions,” says Phil Gotwals,
who leads a CART group at NIBR. It also
might offer improved ways to turn off cell
activity if patients have overly strong immune
reactions, or to add other immunotherapy
weaponry, Yang speculates.
CRISPR’s Advantages and Drawbacks
Compared with an earlier genome editing
method called TALEN, the CRISPR system
enables researchers to edit genes much more
efficiently and 200 times less expensively.
TALEN costs about $4,000 per gene and takes
months longer to perform than using CRISPR.
Additionally, CRISPR offers two huge
improvements over RNA interference (RNAi),
a gene-silencing method. The first is that
CRISPR can achieve complete protein
loss, as compared with only partial protein
reduction via RNAi. The second comes from
the improved specificity of CRISPR. While
the field is still learning about the drawbacks
of CRISPR, RNA interference research has
historically been plagued by off-target
effects that complicate the interpretation of
experiments.
Of cou r se, as wi t h al l g en et i c t o o l s ,
“there’s a lot of room for improvement with
CRISPR,” Yang notes. Among its drawbacks,
CRISPR doesn’t work with all genes, and it
sometimes attaches to off-target DNA. Labs
around the world—including at NIBR—are
rapidly developing alternative versions of
CRISPR that better address these problems,
cleanly activate gene expression or repress
genes without cutting them, or bring other
advantages. Beyond work in cells and animal
models, “the race is on and we need to see
who will be the first to push CRISPR into
clinical applications.”
3
Figure 1: Genome Editing Method
The Leading Edge: Special Interview
Target chromosome
Mutation
Towards Clinical
Application of Genome
Editing Technologies
Conventional method
(natural mechanism of cells)
Gene Knockout and Knockin
Recently, a new technique called “genome
editing” has been developed. Unlike
conventional gene therapy, in which a
therapeutic cDNA is transferred into a cell,
genome editing utilizes DNA repair machinery.
Genome editing is generally classified based
on whether it involves the “gene knockout”
or the “gene knockin” approach. The former
cleaves the chromosomal DNA using artificial
nucleases such as TALEN and CRISPR-Cas9,
which can recognize and cut a specific DNA
sequence, leading to gene disruption through
an error-prone DNA repair pathway. The latter
accurately inserts an artificial DNA fragment
(donor DNA) into the targeted site through
homologous recombination to allow the gene
to acquire a specific function [Figure 1].
Gene knockout is relatively easy to achieve
in various cell types. Ongoing clinical studies
aimed at treating AIDS involve the ex vivo
generation of CD4-positive T cells with a
CCR5 gene knockout. Another clinical study
related to cancer immunological gene therapy
has started, using chimeric antigen receptionexpressing T cells (CAR-T) with the T cell
receptor α-chain being knocked out. Such
cells then can be used for patients in a human
leucocyte antigen (HLA)-independent manner.
The gene knockin technique would be
applicable to gene repair therapy, including
that for dominantly inherited diseases.
However, it requires donor DNA and it
is more inefficient than gene knockout
technology. Gene knockin was successful for
only about 30% of human hematopoietic
stem/progenitor cells—a far less efficient
4
Gene knockin
(gene repair)
Cleaving target chromosome
with artificial nucleases
Homologous recombination
Gene knockin
(gene repair)
Gene knockout
Dramatically improved efficiency with artificial nuclease development. Figure by Kohnosuke Mitani.
Dr. Mitani, genome editing expert
problems, such as the immunogenicity of Cas9
derived from bacteria and the accumulation
of off-target mutations due to the sustained
expression of Cas9 over a period of years.
However, spontaneous mutations occur in
one out of every 1010 bases of genome DNA
per cell division. Furthermore, each of us
is known to possesses up to 200 loss-offunction mutations and also about 20 diseasecausing mutations. Thus, we face difficulties in
distinguishing normal variants from off-target
mutations. Neither genome editing nor any
other therapies are completely safe. Whether
a therapy is performed is thus determined
based on its risks and benefits. Therefore, the
establishment of methods and standards to
evaluate safety and specificity is an urgent task.
figure than that for conventional gene
therapy, which achieves strong expression of
therapeutic genes in the majority of cells. For
in vivo gene repair in the liver, gene knockin
has only been successful in less than 10% of
the cells in mouse studies.
Importance of Safety Evaluation Standards
Genome editing poses many challenges. These
include issues regarding efficacy, safety, target
cells, and diseases, as well as ethical and social
concerns. Off-target mutations that occur in nontargeted sequences are problematic in terms
of safety. For example, Cas9 tends to cleave
DNA sequences that are similar to the target
sequence. In addition, in the cell population
subjected to genome editing, both gene repair
and gene knockout (repair failure) may occur in
individual cells or in each of two alleles, causing
a mosaic pattern resulting from gene repair,
gene knockout, and unaltered alleles.
There is still no method to comprehensively
detect the off-target mutation sites and the
frequency thereof in the targeted cells. Most
research performed with the aim of applying
genome editing to clinical application has
introduced large quantities of Cas9 or donor
DNA into cells to improve efficiency. Off-target
mutation, however, has not been extensively
analyzed. Our research showed that off-target
incorporation of donor DNA occurs more
frequently than estimated—a fact overlooked
in most research reports.
Genome editing in vivo also faces other
Introduction of breaks at target
chromosome with artificial nucleases
Normal DNA sequence
(donor DNA)
Recombination between
homologous sequences
Dr. Kohnosuke Mitani
Professor, Head of the Division of Gene Therapy and
Genome Editing, Research Center for Genomic Medicine,
Saitama Medical University
Artificial nucleases
(Interviewed on May 23, 2016 at
Saitama Medical University)
Somatic Genome Editing Issues
In 2015, the American and Japanese Societies
of Gene and Cell Therapy and the international
Human Gene-Editing Initiative issued separate
statements and proposed that in ethical terms,
clinical use of gene editing in somatic cells
can be appropriately and rigorously evaluated
within the existing and evolving regulatory
framework for gene therapy.
Gene knockout is now at a level that makes
clinical application possible. However, it should
be applied based on comprehensive judgments
on risk-benefit, which include whether the
target cells are dividing or non-dividing cells,
whether the purpose is gene disruption or
gene repair, and whether the patients are
children or adults. At present, gene knockout
would likely be utilized for the treatment
of cancer or infections. Ex vivo gene repair
targeting hematopoietic stem cells would also
be feasible. It is essential to compare genome
editing and conventional gene therapy in terms
of efficacy and safety, and the comparison
should be used as a benchmark for making
decisions on when to use genome editing
instead of conventional gene addition therapy.
Germline Genome Editing Issues
Genome editing in human germline cells
faces technical, biological, and ethical
issues. In addition, long-term follow-ups
regarding efficacy and safety are impossible.
It is problematic that genome editing may
be applied to genetic enhancements. My
greatest concern is that the CRISPR-Cas9
system may be used at private clinics,
because the techniques are quite simple.
Even if such treatment were to be regulated
in one country, there is a possibility that
couples with unborn babies that will surely
develop hereditary diseases may seek germ
cell genome editing overseas.
Genome editing is making rapid progress,
and its application to clinical treatment is
anticipated. However, safety and ethical
problems still need to be solved and social
consensus is required for its application.
Scientists have a responsibility to provide
accurate information to the public for
practical implementation of genome editing.
5
Figure 1: Genome Editing Method
The Leading Edge: Special Interview
Target chromosome
Mutation
Towards Clinical
Application of Genome
Editing Technologies
Conventional method
(natural mechanism of cells)
Gene Knockout and Knockin
Recently, a new technique called “genome
editing” has been developed. Unlike
conventional gene therapy, in which a
therapeutic cDNA is transferred into a cell,
genome editing utilizes DNA repair machinery.
Genome editing is generally classified based
on whether it involves the “gene knockout”
or the “gene knockin” approach. The former
cleaves the chromosomal DNA using artificial
nucleases such as TALEN and CRISPR-Cas9,
which can recognize and cut a specific DNA
sequence, leading to gene disruption through
an error-prone DNA repair pathway. The latter
accurately inserts an artificial DNA fragment
(donor DNA) into the targeted site through
homologous recombination to allow the gene
to acquire a specific function [Figure 1].
Gene knockout is relatively easy to achieve
in various cell types. Ongoing clinical studies
aimed at treating AIDS involve the ex vivo
generation of CD4-positive T cells with a
CCR5 gene knockout. Another clinical study
related to cancer immunological gene therapy
has started, using chimeric antigen receptionexpressing T cells (CAR-T) with the T cell
receptor α-chain being knocked out. Such
cells then can be used for patients in a human
leucocyte antigen (HLA)-independent manner.
The gene knockin technique would be
applicable to gene repair therapy, including
that for dominantly inherited diseases.
However, it requires donor DNA and it
is more inefficient than gene knockout
technology. Gene knockin was successful for
only about 30% of human hematopoietic
stem/progenitor cells—a far less efficient
4
Gene knockin
(gene repair)
Cleaving target chromosome
with artificial nucleases
Homologous recombination
Gene knockin
(gene repair)
Gene knockout
Dramatically improved efficiency with artificial nuclease development. Figure by Kohnosuke Mitani.
Dr. Mitani, genome editing expert
problems, such as the immunogenicity of Cas9
derived from bacteria and the accumulation
of off-target mutations due to the sustained
expression of Cas9 over a period of years.
However, spontaneous mutations occur in
one out of every 1010 bases of genome DNA
per cell division. Furthermore, each of us
is known to possesses up to 200 loss-offunction mutations and also about 20 diseasecausing mutations. Thus, we face difficulties in
distinguishing normal variants from off-target
mutations. Neither genome editing nor any
other therapies are completely safe. Whether
a therapy is performed is thus determined
based on its risks and benefits. Therefore, the
establishment of methods and standards to
evaluate safety and specificity is an urgent task.
figure than that for conventional gene
therapy, which achieves strong expression of
therapeutic genes in the majority of cells. For
in vivo gene repair in the liver, gene knockin
has only been successful in less than 10% of
the cells in mouse studies.
Importance of Safety Evaluation Standards
Genome editing poses many challenges. These
include issues regarding efficacy, safety, target
cells, and diseases, as well as ethical and social
concerns. Off-target mutations that occur in nontargeted sequences are problematic in terms
of safety. For example, Cas9 tends to cleave
DNA sequences that are similar to the target
sequence. In addition, in the cell population
subjected to genome editing, both gene repair
and gene knockout (repair failure) may occur in
individual cells or in each of two alleles, causing
a mosaic pattern resulting from gene repair,
gene knockout, and unaltered alleles.
There is still no method to comprehensively
detect the off-target mutation sites and the
frequency thereof in the targeted cells. Most
research performed with the aim of applying
genome editing to clinical application has
introduced large quantities of Cas9 or donor
DNA into cells to improve efficiency. Off-target
mutation, however, has not been extensively
analyzed. Our research showed that off-target
incorporation of donor DNA occurs more
frequently than estimated—a fact overlooked
in most research reports.
Genome editing in vivo also faces other
Introduction of breaks at target
chromosome with artificial nucleases
Normal DNA sequence
(donor DNA)
Recombination between
homologous sequences
Dr. Kohnosuke Mitani
Professor, Head of the Division of Gene Therapy and
Genome Editing, Research Center for Genomic Medicine,
Saitama Medical University
Artificial nucleases
(Interviewed on May 23, 2016 at
Saitama Medical University)
Somatic Genome Editing Issues
In 2015, the American and Japanese Societies
of Gene and Cell Therapy and the international
Human Gene-Editing Initiative issued separate
statements and proposed that in ethical terms,
clinical use of gene editing in somatic cells
can be appropriately and rigorously evaluated
within the existing and evolving regulatory
framework for gene therapy.
Gene knockout is now at a level that makes
clinical application possible. However, it should
be applied based on comprehensive judgments
on risk-benefit, which include whether the
target cells are dividing or non-dividing cells,
whether the purpose is gene disruption or
gene repair, and whether the patients are
children or adults. At present, gene knockout
would likely be utilized for the treatment
of cancer or infections. Ex vivo gene repair
targeting hematopoietic stem cells would also
be feasible. It is essential to compare genome
editing and conventional gene therapy in terms
of efficacy and safety, and the comparison
should be used as a benchmark for making
decisions on when to use genome editing
instead of conventional gene addition therapy.
Germline Genome Editing Issues
Genome editing in human germline cells
faces technical, biological, and ethical
issues. In addition, long-term follow-ups
regarding efficacy and safety are impossible.
It is problematic that genome editing may
be applied to genetic enhancements. My
greatest concern is that the CRISPR-Cas9
system may be used at private clinics,
because the techniques are quite simple.
Even if such treatment were to be regulated
in one country, there is a possibility that
couples with unborn babies that will surely
develop hereditary diseases may seek germ
cell genome editing overseas.
Genome editing is making rapid progress,
and its application to clinical treatment is
anticipated. However, safety and ethical
problems still need to be solved and social
consensus is required for its application.
Scientists have a responsibility to provide
accurate information to the public for
practical implementation of genome editing.
5
*An mTOR mutation identified
from FCD patients introduced
into rat neurons. The neurons
are enlarged, similar to what
is seen in brain tissue from the
patients.
The Leading Edge:
Electrical Brainstorms
Traced to Genetic
Mutations
Electrical signals pulse through the gray
matter of your brain, allowing you to read
and understand this sentence. The cerebral
cortex—home to your gray matter—is packed
with more than 20 billion neurons, which are
organized into circuits1. The results can be dire
when the circuits don’t form properly.
Take children with a disease called focal
cortical dysplasia (FCD)2. Born with an enlarged,
disorganized area of the cortex, these patients
often experience seizures, which are brainstorms
of uncontrolled electrical activity that can lead
to developmental delays and disabilities.
Collaborators from Seattle Children’s
Research Institute, the Novartis Institutes
for BioMedical Research (NIBR), and other
organizations recently traced cases of FCD
to genetic mutations3. Specifically, the team
identified mutations in a molecular pathway
called mTOR (mammalian target of rapamycin),
which plays an essential role in regulating cell
growth. The discovery bolsters a growing body
of evidence4 that such diseases can be genetic
and suggests new treatment approaches.
Clues in Patients’ Brain Tissues
By 2012, the research team at Seattle
Children’s Hospital had gathered tantalizing
clues by studying the brain tissue from patients
who underwent epilepsy surgery. Biochemical
tests indicated that the mTOR pathway was
overactive in many of the samples. DNA
sequencing revealed mutations 5 in key
components of the pathway, but only in patients
with diffuse brain overgrowth. The mutations
didn’t show up in any patients with FCD.
Two clinical geneticists at Seattle Children’s
Hospital suspected that the mutations were
simply hidden due to a quirk of biology. They
wondered if there was a small population of
neurons with mTOR mutations in FCD patients.
6
Image*: David Furness, Wellcome Images
Image*: Jonathan Biag, Novartis
Perhaps the population was so small that the
mutations weren’t registering with standard
DNA sequencing techniques.
Uncovering Hidden Mutations
NIBR’s next-generation sequencing group in
Oncology had the tools to test the hypothesis,
and together with the Seattle Children’s
Research Institute, it embarked on this
research. The cancer sequencing lab specializes
in finding mutations that only occur in a small
fraction of cells. The team analyzed samples
from eight patients with FCD and their parents
by tuning their software to catch mutations
that occur in less than 5 percent of the cells.
They identified mTOR pathway mutations—
including genetic lesions identical to those seen
in cancer patients—at a low level in four of the
FCD patients.
In parallel, NIBR scientists set out to
determine exactly how the mutations affect
brain cells and introduced such mutations
into rat neurons, which proceeded to grow
very large. The researchers also tested mTOR
pathway activity in the neurons and confirmed
that it had become elevated. When the team
applied an mTOR inhibitor to the mutant
neurons, the cells shrank to a healthy size,
pointing toward a potential therapeutic strategy.
“This pathway is extremely well known in the
cancer space, but now it is coming up as an
important target in neuroscience,” says Leon
Murphy, who led the validation effort at NIBR.
“It might be possible to repurpose cancer
drugs for these diseases based on preclinical
data and potentially provide patients with more
options at some point.”
1. Pelvig, D.P. et al., Neurobiol Aging. 2008; 29 (11): 1754-62.
2. Kabat, J., Król P., Pol J Radiol. 2012; 77 (2): 35-43.
3. Mirzaa, G.M. et al., JAMA Neurol. 2016 May 9. doi: 10.1001/jamaneurol. 2016. 0363.
4. Blümcke, I., Sarnat, H.B., Curr Opin Neurol. 2016 Jun; 29 (3): 0. doi: 10.1097/WCO. 0000000000000303.
5. Rivière, J.B. et al., Nat Genet. 2012; 44 (8): 934-40.
*Scanning electron microscopy
shows mitochondria (blue
areas), where free radicals are
created.
“Electrical Brainstorms Traced
to Genetic Mutations” and
“Stopping Free Radicals at their
Source” are edited versions of
NIBR articles.
http://www.nibr.com/
Scene:
Stopping Free Radicals
at their Source
Identifying Chemical Compounds that Block
the Production of Free Radicals in Cells
Free radicals stand accused of aiding or
abetting just about every form of human
disease. These chemically reactive molecules,
flooding through cells under stress, generally
contain oxygen, and there has long been hope
that anti-oxidants such as Vitamin C can help
to protect against their damaging effects.
However, in clinical trials, antioxidants have
yielded disappointing results.
Now, however, scientists at the Genomics
Institute of the Novartis Research Foundation
(GNF) in San Diego, California and the Buck
Institute for Research on Aging in Novato,
California have identified chemical compounds
that can block the production of certain free
radicals in cells without changing the energy
metabolism of such cells1. These compounds
point toward potential new therapies.
“These compounds can be used as a
scalpel in research to slow down or prevent
specific signaling pathways in conditions
such as Alzheimer’s disease,” says Martin
Brand, professor at the Buck Institute. “They
also can be used to help design more druglike molecules.” Ed Ainscow at GNF explains
that therapies based on such compounds
would take a very different approach than
antioxidants. “Instead of increasing the
degradation of free radicals, we want to
decrease their production.” While mitochondria
primarily act as the cell’s powerhouse, they
spin off free radicals as normal byproducts,
a n d t h i s p ro d u c t i o n m a y ra m p u p t o
damaging levels as the cell is stressed.
One mitochondrial site called IIIQ is
thought to be a leading source of free
radicals, especially when cells are starved of
oxygen. Brand and his colleagues developed
a test assay to find compounds that could not
only suppress free radicals at the IIIQ site but
also maintain the site’s energy production.
The scientists screened the 635,000 small
molecules in mitochondria isolated from
muscle. In a second round of tests, the
researchers examined how three promising
compounds performed to protect cells from
various forms of stress.
Targeting Diseases Connected to Free Radicals
Scientists looked at whether the compounds
could help to safeguard insulin-producing
pancreatic beta cells, which are vulnerable to a
lack of oxygen—a weakness that has plagued
pancreatic cell transplants for people with type 1
diabetes. “Some of the atrophy and cell death
you see in transplanted cells is due to excess
free radical production,” Ainscow says. When
researchers stressed animal pancreatic cells
with another antibiotic that normally boosts free
radical production, the compounds helped to
reduce production of the molecules and keep
the cells healthy. This positive result suggests
that similar chemicals could help with other
kinds of transplants as well. The compounds
also might target increased free radical
production in numerous diseases. Examples
include neurodegenerative illnesses, chronic
inflammation, and macular degeneration.
Furthermore, rapidly growing tumors often
have a core of cells that receive low levels
of oxygen, which slows tumor growth. Free
radicals can send out signals to increase the
blood supply to these cells, boosting their
oxygen supply. “If we can interfere with this
signaling and stop these solid tumors from
growing, the tumors then can be hit by other
therapies,” Brand suggests.
1. Orr, A.L. et al., Nat Chem Biol. 2015; 11(11): 834-6.
7
*An mTOR mutation identified
from FCD patients introduced
into rat neurons. The neurons
are enlarged, similar to what
is seen in brain tissue from the
patients.
The Leading Edge:
Electrical Brainstorms
Traced to Genetic
Mutations
Electrical signals pulse through the gray
matter of your brain, allowing you to read
and understand this sentence. The cerebral
cortex—home to your gray matter—is packed
with more than 20 billion neurons, which are
organized into circuits1. The results can be dire
when the circuits don’t form properly.
Take children with a disease called focal
cortical dysplasia (FCD)2. Born with an enlarged,
disorganized area of the cortex, these patients
often experience seizures, which are brainstorms
of uncontrolled electrical activity that can lead
to developmental delays and disabilities.
Collaborators from Seattle Children’s
Research Institute, the Novartis Institutes
for BioMedical Research (NIBR), and other
organizations recently traced cases of FCD
to genetic mutations3. Specifically, the team
identified mutations in a molecular pathway
called mTOR (mammalian target of rapamycin),
which plays an essential role in regulating cell
growth. The discovery bolsters a growing body
of evidence4 that such diseases can be genetic
and suggests new treatment approaches.
Clues in Patients’ Brain Tissues
By 2012, the research team at Seattle
Children’s Hospital had gathered tantalizing
clues by studying the brain tissue from patients
who underwent epilepsy surgery. Biochemical
tests indicated that the mTOR pathway was
overactive in many of the samples. DNA
sequencing revealed mutations 5 in key
components of the pathway, but only in patients
with diffuse brain overgrowth. The mutations
didn’t show up in any patients with FCD.
Two clinical geneticists at Seattle Children’s
Hospital suspected that the mutations were
simply hidden due to a quirk of biology. They
wondered if there was a small population of
neurons with mTOR mutations in FCD patients.
6
Image*: David Furness, Wellcome Images
Image*: Jonathan Biag, Novartis
Perhaps the population was so small that the
mutations weren’t registering with standard
DNA sequencing techniques.
Uncovering Hidden Mutations
NIBR’s next-generation sequencing group in
Oncology had the tools to test the hypothesis,
and together with the Seattle Children’s
Research Institute, it embarked on this
research. The cancer sequencing lab specializes
in finding mutations that only occur in a small
fraction of cells. The team analyzed samples
from eight patients with FCD and their parents
by tuning their software to catch mutations
that occur in less than 5 percent of the cells.
They identified mTOR pathway mutations—
including genetic lesions identical to those seen
in cancer patients—at a low level in four of the
FCD patients.
In parallel, NIBR scientists set out to
determine exactly how the mutations affect
brain cells and introduced such mutations
into rat neurons, which proceeded to grow
very large. The researchers also tested mTOR
pathway activity in the neurons and confirmed
that it had become elevated. When the team
applied an mTOR inhibitor to the mutant
neurons, the cells shrank to a healthy size,
pointing toward a potential therapeutic strategy.
“This pathway is extremely well known in the
cancer space, but now it is coming up as an
important target in neuroscience,” says Leon
Murphy, who led the validation effort at NIBR.
“It might be possible to repurpose cancer
drugs for these diseases based on preclinical
data and potentially provide patients with more
options at some point.”
1. Pelvig, D.P. et al., Neurobiol Aging. 2008; 29 (11): 1754-62.
2. Kabat, J., Król P., Pol J Radiol. 2012; 77 (2): 35-43.
3. Mirzaa, G.M. et al., JAMA Neurol. 2016 May 9. doi: 10.1001/jamaneurol. 2016. 0363.
4. Blümcke, I., Sarnat, H.B., Curr Opin Neurol. 2016 Jun; 29 (3): 0. doi: 10.1097/WCO. 0000000000000303.
5. Rivière, J.B. et al., Nat Genet. 2012; 44 (8): 934-40.
*Scanning electron microscopy
shows mitochondria (blue
areas), where free radicals are
created.
“Electrical Brainstorms Traced
to Genetic Mutations” and
“Stopping Free Radicals at their
Source” are edited versions of
NIBR articles.
http://www.nibr.com/
Scene:
Stopping Free Radicals
at their Source
Identifying Chemical Compounds that Block
the Production of Free Radicals in Cells
Free radicals stand accused of aiding or
abetting just about every form of human
disease. These chemically reactive molecules,
flooding through cells under stress, generally
contain oxygen, and there has long been hope
that anti-oxidants such as Vitamin C can help
to protect against their damaging effects.
However, in clinical trials, antioxidants have
yielded disappointing results.
Now, however, scientists at the Genomics
Institute of the Novartis Research Foundation
(GNF) in San Diego, California and the Buck
Institute for Research on Aging in Novato,
California have identified chemical compounds
that can block the production of certain free
radicals in cells without changing the energy
metabolism of such cells1. These compounds
point toward potential new therapies.
“These compounds can be used as a
scalpel in research to slow down or prevent
specific signaling pathways in conditions
such as Alzheimer’s disease,” says Martin
Brand, professor at the Buck Institute. “They
also can be used to help design more druglike molecules.” Ed Ainscow at GNF explains
that therapies based on such compounds
would take a very different approach than
antioxidants. “Instead of increasing the
degradation of free radicals, we want to
decrease their production.” While mitochondria
primarily act as the cell’s powerhouse, they
spin off free radicals as normal byproducts,
a n d t h i s p ro d u c t i o n m a y ra m p u p t o
damaging levels as the cell is stressed.
One mitochondrial site called IIIQ is
thought to be a leading source of free
radicals, especially when cells are starved of
oxygen. Brand and his colleagues developed
a test assay to find compounds that could not
only suppress free radicals at the IIIQ site but
also maintain the site’s energy production.
The scientists screened the 635,000 small
molecules in mitochondria isolated from
muscle. In a second round of tests, the
researchers examined how three promising
compounds performed to protect cells from
various forms of stress.
Targeting Diseases Connected to Free Radicals
Scientists looked at whether the compounds
could help to safeguard insulin-producing
pancreatic beta cells, which are vulnerable to a
lack of oxygen—a weakness that has plagued
pancreatic cell transplants for people with type 1
diabetes. “Some of the atrophy and cell death
you see in transplanted cells is due to excess
free radical production,” Ainscow says. When
researchers stressed animal pancreatic cells
with another antibiotic that normally boosts free
radical production, the compounds helped to
reduce production of the molecules and keep
the cells healthy. This positive result suggests
that similar chemicals could help with other
kinds of transplants as well. The compounds
also might target increased free radical
production in numerous diseases. Examples
include neurodegenerative illnesses, chronic
inflammation, and macular degeneration.
Furthermore, rapidly growing tumors often
have a core of cells that receive low levels
of oxygen, which slows tumor growth. Free
radicals can send out signals to increase the
blood supply to these cells, boosting their
oxygen supply. “If we can interfere with this
signaling and stop these solid tumors from
growing, the tumors then can be hit by other
therapies,” Brand suggests.
1. Orr, A.L. et al., Nat Chem Biol. 2015; 11(11): 834-6.
7
Novartis
Innovation
Novartis Today:
Friedrich Miescher Institute for
Biomedical Research
Focus on Biomedical Research
The Friedrich Miescher Institute for Biomedical
Research (FMI) located in Basel, Switzerland,
was established in 1970 by a joint decision of
the then-two-separate companies Ciba AG and
J.R. Geigy AG—the predecessors of Novartis.
Since its founding, the FMI has contributed
substantially to a better understanding of the
molecular and cellular basis of disease and
has attained international recognition for its
fundamental biomedical research. Today, the
FMI focuses on neurobiology, quantitative
biology, and the epigenetics*1 of stem cell
development and cell differentiation.
T he F MI w a s nam e d af te r the B a s el
scientist Friedrich Miescher (1844-1895) who
first purified nucleic acids. It is affiliated with
both the University of Basel and the Novartis
Institutes for BioMedical Research (NIBR).
Susan Gasser, Director of FMI
*1 Epigenetics is the study of heritable
changes in gene expression that
do not involve changes to the
underlying DNA sequence.
*2 Proteomics is the comprehensive
study of proteins within an
organism or a cellular system,
focusing on structure and function.
This is an edited version comprised
of several articles by NIBR and FMI.
https://www.nibr.com/
http://www.fmi.ch/
8
Coupling Academic Research and
Biomedical Applications
The FMI is situated at the interface of academic
research and biomedical application. Findings
are published and presented to the scientific
community, contributing to the collective
understanding of human disease. Through
collaborative ef for ts with Novar tis, FMI
scientists also contribute to the development
of both diagnostics and medicines.
The FMI pursues biomedical discoveries
while establishing cutting-edge technology
platforms. FMI scientists make use of the
latest developments in technologies such
as genetic approaches in model organisms,
detailed proteomic*2 and genomic analyses,
microscopy, and structure determination.
Based on an in-depth understanding of the
molecular processes, FMI scientists hope
to uncover new means to combat cancer,
correct degenerative states, and suppress
dise ase s correlate d with physiologic al
dysfunction.
Sus an Gas ser, Direc tor of FMI, s ays:
“Biomedical research aims to describe the
molecular mechanisms at work within living
cells to enable an effective development of
new therapeutics. Now more than ever, it is
clear that biological research has a major
Vol. 3 July 2016
impact on the quality of life of each and
every one of us.”
FMI encourages its scientists to explore
n ove l a r e a s w i t h in t e ll e c t u a ll y d a r in g
approaches. The aim of the Institute is
to continually push back the horizons of
knowledge with original ideas and innovative
techniques. It provides an open, collegial
environment that allows for interdisciplinary
c o ll a b o r a t i o n a n d c r o s s - f e e din g f r o m
one field to another on a daily basis. This
tradition allows FMI to play a leading role in
biomedical research.
Training Young Scientists
According to its founding charter, the FMI
not only seeks to pursue and promote basic
biomedical research but also to provide
young scientists from all over the world with
an opportunity to participate in scientific
research. Currently, FMI laboratories are
home to approximately 100 PhD and MSc
students from about 30 different countries.
Registered at local universities, they are part
of the FMI International PhD Program and
carry out their dissertation studies under the
supervision of FMI group leaders. In addition,
about 90 postdoctoral students from around
the world pursue postgraduate studies at
the FMI. They are exposed to the latest in
molecular and genetic approaches while
being constantly encouraged to examine
biomedical applications.
FMI is internationally recognized as an
excellent training ground for young scientists.
This is testimony not only to the quality of
the research programs and the commitment
to maintaining state-of-the-art platforms but
also to the open, collegial atmosphere.
Issued by Communications Dept., Novartis Pharma K.K.
Toranomon Hills Mori Tower, 23-1, Toranomon
1-chome Minato-ku, Tokyo 105-6333 Japan
NPE00003JG0001 E
2016.07
The Leading Edge:
The Present, Past, and Future of Genomic Medicine
Editing Genes to Potentially Fight Disease
Special Interview
Towards Clinical Application of Genome Editing Technologies
Dr. Kohnosuke Mitani, Professor, Head of the Division of Gene Therapy and Genome Editing,
Research Center for Genomic Medicine, Saitama Medical University
Electrical Brainstorms Traced to Genetic Mutations
Scene:
Stopping Free Radicals at their Source
Novartis Today:
Friedrich Miescher Institute for Biomedical Research
Cover image: Cutting DNA sequence
of HeLa cells using CRISPR.
Image: HeLa cells by William J. Moore,
University of Dundee/Wellcome Images.
Modified by PJ Kaszas.