Download Preclinical models: Needed in translation?

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Preclinical Models: Needed in Translation? A Pro/Con Debate
Thomas Philips PhD,1 Jeffrey D. Rothstein MD, PhD,1 and Mahmoud A. Pouladi PhD2*
1
Johns Hopkins University, Brain Science Institute, Baltimore, Maryland, USA
Translational Laboratory in Genetic Medicine (TLGM), Agency for Science Technology and Research (A*STAR), and Department of Medicine,
Yong Loo Lin School of Medicine, National University of Singapore (NUS), Singapore
2
ABSTRACT:
The discovery of the causative
mutations and many of the predisposing risk factors for
neurodegenerative disorders such as Amyotrophic Lateral Sclerosis, Alzheimer’s, Parkinson’s, and Huntington’s disease (HD), has led to the development of a
large number of genetic animal models of disease. In
the case of HD, for example, over 20 different transgenic rodent models have been generated. These models have been of immense value in providing novel
insights into mechanisms of disease, with the promise
of accelerating the development of therapies that can
For many neurodegenerative diseases, including
Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s, Parkinson’s, and Huntington’s disease (HD), the causative
mutations and many of the predisposing genetic risk
factors have been known for many years. In the case of
HD, for example, the mutation in HTT was identified
more than 20 years ago, enabling the development of
genetic animal models and leading to a wealth of
knowledge and novel insights into mechanisms of disease. Yet, despite the availability and intensive use of
such preclinical models, a treatment that can delay the
onset of disease or slow its rate of progression is yet to
be developed for HD or any of the other neurodegenerative diseases. This leads one to raise the question: Are
preclinical models needed in translational research?
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*Correspondence to: Mahmoud A. Pouladi, Translational Laboratory in
Genetic Medicine (TLGM), Agency for Science Technology and Research
and National University of Singapore, 8A Biomedical Grove, Immunos L5,
Singapore 138648. E-mail: [email protected]
Funding agencies: M.A.P. is supported by grants from the Agency for
Science Technology and Research (A*STAR), and the National University
of Singapore (NUS).
Relevant conflicts of interest/financial disclosures: Nothing to report.
Full financial disclosures and author roles may be found in the online version of this article.
Received: 5 August 2014; Accepted: 11 August 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com). DOI: 10.1002/mds.26010
delay the onset or slow the progression of the disease.
Yet, despite extensive use of such models, no effective
treatment for HD has been developed. Here, we discuss the value of animal models, highlighting their
strengths and shortcomings in the context of translaC 2014 International Parkinson
tional research for HD. V
and Movement Disorder Society
K e y W o r d s : Animal models; Huntington’s; HTT;
transgenic; translation
CON: The Case Against Preclinical
Models in HD
Many different animal models for studying HD have
been generated, ranging from nonhuman primates,
mice, Caenorhabditis elegans, and Drosophila melanogaster and zebrafish. Many of these animal models
develop a phenotype reminiscent of some aspects of disease as seen in humans, including the loss of gaminobutyric acid–ergic medium spiny neurons (MSN
neurons) in the striatum (as reviewed in Pouladi et al.1).
In fact, interference with specific disease pathways in
these models has led to improvement of the disease phenotype and an improvement of our understanding of
some basic biological aspects of neurodegeneration in
HD. None of these models, especially the mice models
of HD, have so far led to the development of an effective treatment that can halt or slow the disease from
progressing in HD patients. We describe several characteristics of preclinical models of HD that often limit
their translational value for studying HD in humans.
Caveats of Small Animal Models to Study HD
Small animal models such as zebrafish, Drosophila,
and C. elegans do have the advantage of being relatively cheap to maintain and being a great tool for
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high-throughput genetic or drug screenings. However,
small animal models have the disadvantage of not being
very biologically related to humans in terms of their
phylogenetic differences and complexity of their nervous system, particularly of relevance for HD, in which
patients’ cognitive and psychiatric functions are highly
affected. In addition, some of the small animal models
lack a corresponding disease-causing genetic homolog,
for example, the C. elegans models for HD, in which
human mutant Huntingtin protein (mHTT) is inserted
in the worm genome to model disease.2 In both these
small but also the larger animal models discussed later,
many caveats are associated with insertion of exogenous genes in a host genome (reviewed in O’Sullivan
et al.3). Other caveats are associated with the design of
small animal models. For example, HD is caused by a
dominant mutation in a single gene encoding huntingtin
(HTT). Because the HTT gene is a particularly large
gene (approximately 170 kbp), developing HD animal
models has been very difficult. Therefore, many HD
models only express a (small) part of the mHTT gene,
with excluded but potentially pathological regions of
the mHTT gene not represented in the phenotype being
observed.2,4 C. elegans and Drosophila models of HD
usually express either truncated or full-length mHTT
and often express mHTT from complementary DNA
constructs, potentially failing to recapitulate pathological events related to other regions of the mHTT gene
such as mHTT introns.2,5,6
Many caveats are associated with the phenotype
that small animal models of neurodegeneration
develop. These often do not recapitulate the unique
spectrum of the disease phenotype as seen in patients
with neurodegenerative disease. In HD, specific hallmarks of the disease such as dysphagia and dysarthria
are clearly very difficult to model in such small animal
models. Drosophila models clearly recapitulate motor
dysfunction as seen in reduced climbing behavior (negative geotaxis),6 but this is phenotypically very similar
to what is seen in Drosophila models of other, but
very different, neurodegenerative disease such as
ALS.7 Distinctive motor symptoms such as the rigidity
and chorea seen in HD patients, as well as the cognitive and psychiatric features characterizing the HD
phenotype are very hard to model using small animal
models. Therefore, these studies using small animal
models might rather lead to enhanced insight into less
subtle disease mechanisms that are relevant for a
broader range of neurodegenerative diseases.
Caveats of Rodent and Large Animal Models
to Study HD
Similarly to the small HD models, one has to be very
careful about the biological differences between rodent
models and humans. These might confound proper
translation to the human clinic. For example, given the
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differences in brain mass (3,0003 larger in humans as
compared with mice) and number of brain neurons (86
billion in humans versus 90 million in mice), and the
much larger complexity of brain structure in humans,
one should not be surprised that accurately modeling
human disease in rodent HD models is very difficult.
Therefore, although particular aspects of human HD
are well modeled in rodents (see later discussion), other
aspects of human HD such as facial movements, oculomotor deficiencies, dysarthria, and dysphagia are very
difficult to model in rodent models of HD.
More than 20 different transgenic rodent models
expressing the human or mouse HTT gene have been
generated (reviewed in Pouladi et al.1). These rodents
express either truncated or full-length fragments of
mHTT with varying CAG repeat length. Although these
rodent models develop symptoms of cognitive loss and
motor neuron dysfunction, some major caveats are associated with the development of these rodent models.
Adequate gene dosage is essential during the development of animal models of neurodegeneration. High
gene dosage in transgenic animals, or long repeat
lengths in models of expansion disorder diseases such
as HD, often lead to a fast progressive disease phenotype because of the ubiquitous expression of a high
copy number or long repeat length of a transgene.
This could be motivated by the financial aspects of
animal research, which is usually a significant financial
burden for a research facility, as well as the publication pressure in a usually highly competitive environment. This fast progressive disease phenotype often
precludes the analysis of specific pathology related to
human HD. Conversely, using these models, one
might try to explore whether alterations in expression
of genes affecting aging might modify the phenotype.
In HD, mice overexpressing truncated mHTT often
develop very-early-onset, fast progressive fatal disease
that is more reminiscent of a generalized degenerative
phenotype rather than specifically recapitulating HD
symptoms as seen in patients.4 Therefore, in HD
rodent models, the generalized expression of a CAG
repeat expansion in cell types only mildly involved in
human HD could have significant impact on the presentation of the phenotype in these rodents, potentiating fast progression and early demise. For example,
mHTT expression in mouse motor cortex or other
brain regions, whose corresponding brain regions in
human HD are more mildly affected, might contribute
to a very fast progressive motor phenotype, which is
usually not seen in HD patients.
Transgenic animal models expressing more modest
levels of the transgene, similar to the levels of the
endogenous gene, or mouse models of repeat expansion disorders with limited repeat length, might circumvent this issue but often lead to a phenotype that
is only partial or very mild1,8 and might take a long
time to develop. This might be more relevant for
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modeling disease as seen in patients, because HD
onset usually occurs in mid-adult life and might take
many years or decades to progress until significant
burden in the patient is noticed. Very often in such
instances the animal model might not live long enough
to develop the whole plethora of the neurodegenerative phenotype as seen in patients. These rodent versions of HD often show very simplistic clinical
representations of the disease and lack the many relevant nuances (clinical and pathological) seen in human
HD. For example, HD patients develop significant loss
of MSN neurons in the striatum. Conversely, a rat
model of HD, the BACHD rat, develops many alterations in the striatal compartment but shows no loss of
striatal neurons.9 In fact, many rodent models for HD
only present certain aspects of disease, and effective
therapeutic drug trials using HD rodents are of low
translational value because their implications for many
other disease-related mechanisms could not be
explored in these models. When only partial aspects of
disease are being targeted, that a clinical trial will fail
in the earlier stages might not be surprising, because
drug effects on other disease-relevant pathways have
not been taken into account in the preclinical model.
Nonhuman primate models might give the best correlation to human disease. The earliest generated nonhuman primate models for HD were generated by
administration of neurotoxins that selectively ablated
striatal neuronal subpopulations.10 These models do
not recapitulate the phenotype seen in HD patients,
who develop progressive widespread cortical, subcortical, and even peripheral nervous system degeneration,
in contrast to the acute localized lesions seen in the
neurotoxin models. Transgenic large animal models
for HD have also been developed. Unfortunately, none
of these models is a true HD animal model. Transgenic mHTT macaque monkeys develop a very aggressive phenotype, with early death and unusual
symptoms that are not seen in early stages of human
HD.11 Transgenic sheep and minipigs did not develop
overt disease symptoms, however, although this could
be because of the limited mHTT CAG repeat size
these models expressed.12,13
Even if one is successful in developing a large animal model to study HD, still many inherent caveats
will be associated with these studies. Large animal
studies, especially those using nonhuman primate
models most relevant to study human disease, are
extremely expensive to maintain, and the neurodegenerative disease in these animals takes many years to
develop. In fact, HD onset in humans is usually midadult life, and so disease progression might need to be
studied during several decades. Even if they were to
develop disease, given the genetic variability of primates (like humans), this would likely necessitate a large
number of animals—similar to human trials—to see a
positive and reproducible clinical response. This makes
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their use for drug screening suboptimal, and studies
using nonhuman primate models usually take many
years if not decades to complete.
Alternatives to Preclinical Animal Models:
Value of iPS Cells in Studying HD
Rather than using animal models to study HD disease
pathogenesis and treatment opportunities, one could
consider the use of patient-derived induced pluripotent
stem (iPS) cells. The development of iPS cells with a
patient-specific disease signature could be explored
both for understanding disease pathogenesis as well as
for testing drugs for therapeutical efficacy. The iPS cells
could be differentiated into different central nervous
system cell types such as neurons, astrocytes, and oligodendrocytes. Protocols have recently been established
for specifically generating MSN neurons from iPS
cells.14 The concept that neuronal death is non–cell
autonomous, that is, not only neurons but also specific
glial cell types and other neuronal cell types contribute
to the disease pathogenesis, is well established.15 For
example, in HD mouse models, astrocyte GLT-1 glutamate transporters are significantly reduced, which contributes to glutamate-mediated excitotoxic cell death of
the nearby g-aminobutyric acid–ergic neurons most susceptible in HD.16 The ability to model all contributing
cell types in a dish, by analyzing a patient-specific disease signature with effects of co-morbidity and disease
modifier genes incorporated as well, is most relevant for
the specific HD patient under study.
Of particular interest is that in all HD patients, disease is caused in a monogenetic fashion by a CAG
repeat expansion of varying length. Unlike in other
neurodegenerative diseases in which most cases are
sporadic with the underlying cause still at large, specific targeting of the HD disease–causing gene using
RNAse H active antisense oligonucleotides or small
interfering RNAs targeting single nucleotide polymorphisms associated with the expanded allele is of interest to all HD patients. A recent study has shown that
transient infusion of antisense oligonucleotides into
the cerebrospinal fluid effectively targeted the mHTT
gene and reversed the phenotype in HD models.17 In
addition, infused nonhuman primate models showed a
clear reduction in wild-type HTT protein expression
in many brain regions.17 The development of iPSderived neuronal and non-neuronal cells from individual HD patients could at least in part bypass the need
for these preclinical therapeutic trials in HD animal
models by providing us a great tool to assess the feasibility and therapeutic dosage of antisense treatment
specifically designed for the patient under study. In
addition, this antisense therapy also could be directed
against many potential disease modifier genes in highthroughput screenings. In fact, many different genes
have been reported to aggravate or alleviate HD
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disease symptoms.18 Conversely, studying these cells
in a dish, extracted from their usual microenvironment, might lead to significant loss of their translational value. Therefore, whether specific HD disease–
related phenotypes can be effectively mimicked in the
iPS-derived cell culture system remains to be seen.
sion.22 That animal models allow the examination of
the neuroprotective potential of cellular and molecular
targets implicated in HD provides a degree of reassurance before the initiation of potentially complicated
and likely costly clinical trials, further emphasizing the
importance of animal models in translational studies.
PRO: The Case for Preclinical
Models in HD
High Construct and Face Validity
of Animal Models of HD
In spite of the various caveats about the HD models,
preclinical animal, and in particular rodent, models of
HD have played an indisputably important role in identifying disease processes and elucidating cellular pathways disrupted in Huntington’s disease.1 Indeed, much
of our current understanding of the pathogenic mechanisms implicated in HD has been shaped by studies of
animal models of HD.19 In addition to their utility in
discovering disrupted cellular processes, animal models
have been used to evaluate the impact of normalizing
the identified cellular and molecular deficits on diseaserelated phenotypes,20,21 shedding light on their potential as targets for therapeutic intervention.
Despite developments that have led to the value of
animal models in translational research being debated,
animal models of HD will remain important in bridging the translational gap from basic discoveries to trials in patients with HD for a number of reasons.
Because rare disorders such as HD have limited
patient populations that are available to participate in
the trial of potential therapies, approaches that help
build confidence and provide evidence, even if only in
proof-of-concept form, that supports a given therapeutic strategy are of immense importance. Such data provide an additional criterion by which to prioritize
experimental therapies and may help in the nomination of promising candidates for clinical evaluation. In
this regard, animal models provide an opportunity to
test the therapeutic potential of candidate compounds
before initiating clinical trials in the limited pool of
patients with HD that may compromise their ability
to participate in subsequent trials.
Value of Proof-of-Concept Studies of
Neuroprotection
Furthermore, although treatments that target symptoms are generally the first line of therapies developed
for many neurodegenerative disorders, including HD,
the ultimate goal of translational research is not only to
provide temporary relief of symptoms but also to delay
the onset and slow the progression of disease.22 For
these slow progressing diseases, the development of
such neuroprotective therapies is challenging not only
from a clinical trial design point of view, but also economically because of the longer duration of trials
needed to ascertain treatment effects on disease progres-
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Unlike many forms of neurodegenerative diseases,
HD is extraordinary in its monogenic etiology and its
dominant and highly penetrant presentation.23 These
features entail not only a clearly defined patient population, but also animal models of high apparent validity.
Three broad criteria are often used to judge a given
model’s relevance: construct validity, face validity, and
predictive validity.1 Many animal models of HD are considered to be of high construct validity given that they
closely reproduce the single genetic mutation that underlies HD in humans, namely CAG trinucleotide repeat
expanded HTT allele(s).1 Similarly, animal models of HD
exhibit high face validity, recapitulating many phenotypic
features of HD, including progressive motor, cognitive,
and psychiatric-like disturbances coupled with HD-like
neuropathological and molecular changes.1 In terms of
predictive validity, which is gauged based on how closely
response to treatment in the animal model parallel, or
predict, improvements in patients, this aspect is yet to be
proven, because no disease-modifying therapies have been
developed. Indeed, failure of a therapy shown to be effective in humans to improve disease phenotypes in animal
models would be cause for pause. With respect to the
only treatment currently approved for HD,24,25 which is
mostly of symptomatic benefit, evidence indicates that tetrabenazine improves disease phenotypes in an animal
model of HD.26 Furthermore, the outcome of a given
clinical trial is influenced by a number of factors, which
include not only the role of the target being engaged in
the disease process, but also issues of dosage, frequency,
disease stage, and specific outcomes being assessed. This
can be readily appreciated when considering that for
many clinically approved therapies, multiple clinical trials
of a given treatment were often necessary to arrive at the
right combination of dose, disease stage, and outcome
measure. Thus, for treatment approaches showing robust
and reproducible benefits in animal models, caution is
warranted when interpreting discordance between animal
and human trials in which the disease stage or outcome
measures may have been different.
Availability of Versatile Animal-Based
Experimental Tools to Dissect Disease
Processes and Mechanism of Action of
Therapeutic Candidates
Safe, specific, and brain-permeable pharmacological
compounds are available for a relatively limited
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number of molecular targets, and in most cases,
screening for and development of novel compounds to
engage molecular targets of interest, a nontrivial
undertaking, is required. The ability to assess the
impact of specifically enhancing or decreasing the
activity of a given target before the initiation of potentially costly and lengthy drug screening and development efforts is of great value. Since the generation of
the first transgenic rodent model over three decades
ago,27 thousands of genetically modified animal lines
have been established that make such studies possible.
By cross-breeding to animal models of HD, these lines
have allowed for the interrogation of the effect of
increased or decreased expression, and hence activity,
of a given gene of interest on HD-related phenotypes.
Furthermore, the availability of transgenic animal
technologies, such as inducible and tissue-specific Cre
recombinase lines,28 allows the dissection of timedependent and tissue-specific cellular processes
involved in HD. These versatile transgenic animalbased tools have enabled a better understanding of the
pathogenesis of HD and will continue to play an
important role in providing novel insights into HD.2931
Transgenic animal lines also may be of translational
value in examining the specificity and the mechanism
of action of therapeutic candidates of interest.
Alternative Approaches Are Not as Well
Developed
An exciting recent development that promises to
revolutionize the study of human disease, and in particular brain disorders, is the discovery that somatic
and terminally differentiated cells may be induced to a
pluripotent stem cell state that may subsequently be
differentiated into any cell type of interest.32 For brain
disorders such as HD, a major challenge has been the
inability to directly study the cell type most affected in
the disease, namely striatal medium spiny neurons, in
a human context. The ability to generate iPS cells
from readily accessible patient tissues (eg, fibroblasts
or peripheral blood monocytes), coupled with the
development of genome editing tools, such as the transcription activator–like effector nucleases and the
RNA-based clustered regularly interspaced short palindromic repeats–Cas9 system,33 which enable the correction of the HD mutation, provides an opportunity
to study pathogenic processes and to evaluate potential therapies in isogenic stem cell–derived MSNs of
human origin. This potential has indeed been demonstrated in a number of recent studies.34-37
The value of stem cell–derived human neurons in
the study of HD cannot be overestimated; however, a
number of factors may limit their utility, at least in
the short-term. First, whereas in animals isolating and
studying virtually any cell type of interest is possible,
the ability to do the same using human stem cells
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depends largely on the availability of the appropriate
directed differentiation protocols necessary to derive
the cell type of interest. Furthermore, with respect to
MSNs, the most highly validated protocols available
to derive them from human stem cells are relatively
inefficient, with only a small fraction (10-15%) of
the differentiated neurons exhibiting the desired MSN
identity at the conclusion of the differentiation procedure.14,38 Second, in their simplest and most widely
adopted configuration, studies of human stem cell–
derived neurons neglect to recreate the complex milieu
that includes other central nervous system cell types,
such as astrocytes, microglia, and oligodendrocytes,
which may be key to recapitulating disease phenotypes. The importance of non–cell autonomous effects
in HD disease processes is indeed supported by a number of recent animal studies.29,31,39-41 By failing to
model many of the environmental cues in stem cell–
based neuronal models, important non–cell autonomous disease processes may be missed. Finally, agedependent progression of whole system, complex phenotypes, in particular cognitive, motor, and psychiatric
features, which can be readily assessed in animal models of HD, simply cannot be examined in stem cell–
derived neuronal systems. Thus, much room for
improvement in stem cell–based modeling of HD
remains, and the exciting advances in our ability to
study HD disease processes in neurons and cell types
of human origin will serve to complement studies in
animal models of HD.
Conclusion
Numerous factors argue for the continued use of
preclinical animal models of HD in translational
research, including the fact that the patient population
available to participate in clinical trials is limited; the
costs of failure for trials seeking to document neuroprotection are high; the available animal models show
a high level of validity because of HD’s dominant,
monogenic, and highly penetrant nature; and finally,
the available alternative experimental systems are less
developed.
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