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European Biopharmaceutical Review
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Use of induced pluripotent stem cells to create innovative,
first-in-class medicines shows much promise. However, before the
benefits can be reaped from the ‘patient in a dish’ approach, a number
of challenges still need to be overcome
It is axiomatic that drug
discovery and drug development efforts have significantly improved the
quality of life in the modern era. However, it is also a near universal
truth that better models which are more reflective of the target biology
or pathology would have the dual effect of significantly increasing the
efficiency of the development process, as well as the safety and
efficacy of new chemical entities. The advent of human embryonic stem
cells (ESCs) and induced pluripotent stem cells (iPSCs) may provide such
models (1,2). The pluripotency and self-renewing capacity of stem cells
offers a nearly limitless supply of biological source material for
generating terminal tissue cells, while the human origin of these cells
in turn may provide a relevant substrate upon which future drug
discovery processes can be built. In addition, iPSC-derived tissues have
the benefit of avoiding many of the ethical concerns of ESCs, while
enabling the incorporation of genetic diversity.
The potential of
stem cell derived tissue cells as models for drug development has been
suggested in recent publications highlighting their recapitulation of in vivo physiology. For example, human stem cell derived cardiomyocytes show
the expected genomic profiling, complement of ion channels, bioenergetic
processes, and contractile activity (3-6). Similarly, iPSC-derived
neurons also present a novel cellular model with in vivo-like
electrophysiology, tau secretion and sensitivity to botulinum neurotoxin
(7,8). This potential for stem cell derived tissue cells as drug
discovery models is further established in their use as a springboard to
Phase 1 clinical trials and as a predictive toxicology tool (9,10).
Although these are a few illustrations of the advances made in the use
of stem cell derived tissue cells, care should be taken not to
‘oversell’ the technology as it is still in relative infancy and
advances in cellular functionality will offer even greater use.
Using Stem Cell Derived Models in Drug Discovery
Improvement
in the quality of healthcare has resulted in a worldwide increase in
life expectancy, but this has also brought about a paradoxical
significant increase in the prevalence of chronic progressive disorders,
particularly of the cardiovascular, metabolic and neural systems. This
statement is backed up by the number of people with diabetes mellitus
worldwide; the number has more than doubled over the past three decades
and is projected to rise to 439 million by 2030.
Despite huge
investments by the pharmaceutical industry to develop new therapeutic
modalities for such disorders, very few new medicines are entering the
market. Drug discovery has proven to be challenging due to the use of
experimental tools which recapitulate subsets of the specific features
of human disease. The advent of patient-specific iPSC technology
represents a revolution in medical science, allowing the study of both
normal and disease pathophysiology in different genetic backgrounds and
their response to drugs very early in the drug discovery process. An
interesting analysis among the first-in-class drugs with new molecular
mechanism of actions (MMOAs) shows that the contribution of phenotypic
screening to the discovery of such molecules exceeded that of
target-based approaches (11). We postulate that the advent of the iPSCs
‘patient in a dish’ – concept, where the aim is to identify multiple
disease-relevant cellular phenotypes – will offer an unprecedented
resource to better investigate disease
biology and ultimately
determine the identification of innovative first-in-class medicines
(12). Here, we will refer the current state of the use of the iPSCs in
drug discovery with respect to cardiac and neural disorders. We will
attempt to report the hurdles, risks and possible rewards that the
pharmaceutical sector will face when implementing such technology.
Implementing iPSC Technology in Pharma
Human
iPSCs have been generated from several tissues using a variety of
approaches. Initial reprogramming efforts used vectors that were
inserted into the genome and then silenced post-iPSC generation. The
alteration of the host genome has given rise to ‘what if’ speculations
of altered function, however such concerns have been unfounded to date.
Nevertheless, given the reported variability among human pluripotent
cell lines with regard to epigenetic information, expression profile and
differentiation properties, their effective implementation in the drug
discovery process relies much more on making the right things more,
rather than doing things right. To that end, newer reprogramming
technologies which avoid insertional mutagenesis or transgene
reactivation should be the preferred tool as the field moves forward.
Such
non-integrating methods will also further enable the transition to
iPSC-based transplantation therapies in the future. Many laboratories
are implementing F-deficient Sendai Viruses and transient transfection
with episomal vectors as preferred reprogramming methods (13). For
similar reasons, the use of reprogramming factors with oncogenic
potential should be avoided. The use of c-Myc could be substituted with
L-Myc and c-Myc mutants, which have shown more efficiency and
specificity to generate human iPSCs.
The generation and
maintenance of iPSC lines is envisioned to be performed in dedicated
core facilities using common reagents and following stringent
standardised operating procedures to better facilitate inter-laboratory
comparisons and compatibility. The concept of using iPSCs from clinical
populations as a ‘patient in a dish’ has to fulfil important
prerequisites, mainly related to the choice of the right controls. This
aspect is even more relevant if we consider that every person has
disease-associated single-nucleotide polymorphisms. Therefore, the
perfect ‘non-diseased/healthy’ control may not exist within the
population, but as explained below, may be able to be fabricated in the
laboratory.
iPSC
technology is particularly amenable to investigating penetrant
monogenetic diseases in which the relationship between genotype and
phenotype is more direct, and because an ad hoc isogenic control is
represented by genetically rescued cell lines. The transcription
activator-like effector nucleases (TALENs) now allows one to genetically
rescue the patient specific monogenetic alterations to generate
genotyped matched lines without the mutation, which may indeed provide
the ‘perfect’ control. Moreover, the TALENs technology – in synergy with
the recent reports for the generation of murine haploid ESCs – provides
a potentially important advance for forward and reverse genetics in
order to target identification and modelling disease pathways in
mammalian cells. For multifactorial disorders, a good control cell line
is represented by cell lines derived from healthy siblings, which may
contribute to decreases in background confounding factors. In the event
that such samples cannot be obtained, an alternative is to compare
specific patient population with extreme ‘fast versus slow progression’,
ideally matching different genders, ethnic and genetic backgrounds.
Particularly for such diseases of unknown genetic origins, it must be
clear which cells in a tissue are the major contributor of the disease
pathophysiology, although even this may be unknown still for some
diseases. In addition to selecting the correct patient population and
appropriate controls, a suitable differentiation protocol(s) must be
chosen for the cell type(s) of interest to enable suitable maturation
and detection of the cellular disease pattern. For diseases with low
penetrance and late-onset phenotypes, the iPSC-derived cellular models
may be treated with stressors mimicking the disease specific
micro-environmental factors (see Figure 1).
Several studies have
reported a proof of concept (PoC) that human iPSCs can be used to model
diseases ‘in a dish’ recapitulating specific disease traits.
Cardiovascular diseases were among the first where successful PoCs have
been generated. Prominent examples are long QT syndromes (LQTSs) that
are caused by mutations in ion channel genes. Moretti and colleagues not
only reported that the duration of the action potential was markedly
prolonged in the myocyte cells derived from patients, but for the first
time introduced the concept of rescuing the phenotype from
pharmacological intervention (14). Indeed pretreatment with a
nonselective beta-blocker substantially blunted the effect of a
chronotropic agent (isoproterenol) in myocytes from patients with
long-QT syndrome type 1.
More recently, PoCs have been reported
for inherited cardiomyopathies associated with a mutation in a
sarcomeric protein (15). Indeed, the R173W point mutation in the cardiac
muscle troponin T could recapitulate some morphological and functional
characteristics of dilated cardiomyopathy (DCM) in vitro, such as
altered sarcomere disarray, impaired Ca2+ kinetics, and decreased
contractility. They have also shown in this study that overexpression of
Serca2a markedly improves the contractility of DCM iPSC-derived
cardiomyocytes. These results are intriguing, especially considering
that a Sereca2a gene therapy treatment is under evaluation in clinical
trials for heart failure. Identifying small molecules that can resolve
the deficiency in contractile force production in DCM-cardiomyocytes may
present a possible new avenue to treat end stage heart failure disease.
In
the neuroscience context, many different examples have been achieved as
well. For example, patient-specific iPSCs lines for modelling
Parkinson’s disease (PD) have been described. In particular,
iPSC-derived dopaminergic neurons bearing mutations in Leu-rich repeat
kinase 2 (LRRK2), a gene causing a significant age-associated cumulative
risk for a form of PD, showed an increased response to oxidative
stressors (16,17). More recently, Israel and colleagues probed sporadic
and familial Alzheimer’s disease (AD) using iPSCs and reported
phenotypes relevant to AD, even though it can take decades for overt
disease to manifest in patients (18).
One of the two patients
with sporadic AD exhibited significantly higher levels of the
pathological markers amyloid-b (1-40), phospho-tau (Thr 231) and active
glycogen synthase kinase-3b (aGSK-3b); as did two patients with familial
AD, both caused by a duplication of the amyloid-b precursor protein
gene (APP).
The use of large-scale screening using familial
dysautonomia iPSCs enabled the identification of compounds that rescue
inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase
complex-associated protein (IKBKAP) expression (19). Target
identification for the identified phenotypic hit SKF-86466 implicated
the discovery of the adrenergic receptor pathway in regulating IKBKAP
expression. By providing cellular systems that closely resemble human
cell biology and pathophysiology in vitro, the iPSC technology will realise the possibility of conducting ‘in vitro Phase 3 studies’. In particular in this visionary proposal, a shared
consensus from the EMA, the FDA and the pharmaceutical industry will be
essential to create clear guidelines, standard SOPs, and well-validated
iPSC line collections with different ethnic and genetic backgrounds. In
conclusion, the ‘patient in a dish’ approach definitely retains all the
potentials to trigger a new era in drug discovery, but numerous
challenges still need to be overcome before its clinical translation is
complete.
Summary
Drug discovery and development
is a dynamic process that has yielded great successes and yet faces
continual challenges. ESC and iPSC technologies and related methods
provide one avenue through which some of these challenges can be
overcome. These technologies have brought relevant human targets and
systems into the drug development pipeline earlier than ever before,
enabling mechanistic investigations into human biology and
pathophysiology and predictive toxicology from healthy and clinical
populations. Continued effort at improving the ESC and iPSC tissue cell
models will only strengthen the foundation upon which these early
successes are built.
References
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K, Tanabe K, Ohnuki M et al, Induction of pluripotent stem cells from
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J, Vodyanik MA, Smuga-Otto K et al, Induced pluripotent stem cell lines
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JE, Ravon M et al, Determination of the human cardiomyocyte mRNA and
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electrophysiological properties of action potentials and ionic currents,
Am J Physiol Heart Circ Physiol 301: H2,006-H2,017, 2011
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HN, Byers B, Cord B et al, LRRK2 mutant iPSC-derived DA neurons
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G, Ramirez CN, Kim H et al, Large-scale screening using familial
dysautonomia induced pluripotent stem cells identifies compounds that
rescue IKBKAP expression, Nat Biotechnol, Letter 25 November 2012
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