Germline transmission in transgenic Huntington's disease monkeys

doi:10.1016/j.theriogenology.2015.03.016

Theriogenology

Volume 84, Issue 2, 15 July 2015, Pages 277-285

https://doi.org/10.1016/j.theriogenology.2015.03.016 Get rights and content

Abstract

Transgenic nonhuman primate models are an increasingly popular model for neurologic and neurodegenerative disease because their brain functions and neural anatomies closely resemble those of humans. Transgenic Huntington's disease monkeys (HD monkeys) developed clinical features similar to those seen in HD patients, making the monkeys suitable for a preclinical study of HD. However, until HD monkey colonies can be readily expanded, their use in preclinical studies will be limited. In the present study, we confirmed germline transmission of the mutant huntingtin (mHTT) transgene in both embryonic stem cells generated from three male HD monkey founders (F0) and in second-generation offspring (F1) produced via artificial insemination by using intrauterine insemination technique. A total of five offspring were produced from 15 females that were inseminated by intrauterine insemination using semen collected from the three HD founders (5 of 15, 33%). Thus far, sperm collected from the HD founder (rHD8) has led to two F1 transgenic HD monkeys with germline transmission rate at 100% (2 of 2). mHTT expression was confirmed by quantitative real-time polymerase chain reaction using skin fibroblasts from the F1 HD monkeys and induced pluripotent stem cells established from one of the F1 HD monkeys (rHD8-2). Here, we report the stable germline transmission and expression of the mHTT transgene in HD monkeys, which suggest possible expansion of HD monkey colonies for preclinical and biomedical research studies.

Introduction

The rationale for developing the Huntington's disease (HD) monkey was to create a large preclinical animal model of HD that could pass the mutant huntingtin (mHTT) transgene down through generations, such that offspring with predictable phenotypes could facilitate basic and preclinical research in HD, which remains a terminal disease today [1], [3]. Thus, with such a large animal model, we could develop and test novel treatments using clinical assessment tools and methods similar to those used in humans [1], [3]. A cohort of three male HD monkeys (rHD6, rHD7 and rHD8; rHDs6–8) carrying the mHTT transgene containing exons 1 to 10 of the human HTT gene with an expanded polyglutamine (polyQ) tract (69–72Q) in exon 1 driven by the human HTT promoter were used as founders for the production of F1 HD monkeys [4], [5]. We have been conducting an ongoing longitudinal study to monitor disease onset and progression in the HD monkeys by using a variety of clinical assessment tools, including noninvasive imaging, cognitive behavioral assessments, and molecular profiling studies [4], [5]. Although our model shows great promise as a preclinical large animal model for HD, the practicality of using the HD monkeys in a clinical research setting depends largely on whether the mHTT transgene can be transmitted through the germline, thereby making the cohort of HD monkeys readily expandable and available to researchers. Unlike rodents and small primate species, such as the marmoset, rhesus macaques reach sexual maturity at approximately 4 to 5 years of age, with a subsequent seasonal reproductive cycle peaking between fall and late spring [1], [6]. Because of this, the inheritability of the mHTT transgene has been assessed only recently with F0 HD monkeys reaching pubertal age.

Section snippets

Animal models

Huntington's disease monkeys were generated by lentiviral-mediated transgenesis as previously described [7]. rHD6, rHD7, and rHD8 are male rhesus macaques that carry exons 1 to 10 of the HTT gene with expanded polyQ repeats (66–74Q, 66–72Q, and 71–74Q), under the control of the human HTT promoter [5], [7]. All monkeys received the same treatments and procedures designed for the longitudinal study, including magnetic resonance imaging scans, cognitive behavioral assessments, and scheduled blood

Derivation and genotyping of transgenic HD monkeys' germline and embryonic stem cells

Germline transmission of the mHTT transgene in HD monkeys was assessed primarily by performing PCR genotyping using primers that specifically amplified a region (including the polyQ expansion in exon 1) of the mHTT transgene (Fig. 1A). To confirm the presence of transgene in germ cells, semen was collected from each of the three F0 founders (rHDs6–8). Genotype results confirmed the presence of the mHTT transgene in HD monkey sperm from each of the F0 HD monkeys (Fig. 1B). Polymerase chain

Discussion

Development of a transgenic nonhuman primate model has proved a challenging task that requires the efficient and innovative use of genetic techniques; not only this, the innate physiological parameters of the primate itself pose a major obstacle because the rhesus macaque does not reach reproductive maturity until 4 to 5 years of age with seasonal breeding cycles [1], [4], [8]. Although genetically modified primate models such as the marmoset have certain physiological advantages, such as their

Acknowledgments

All animal procedures and experiments were performed in a BSL-2 facility at the Yerkes National Primate Research Center (YNPRC) and were approved by the Emory University's Institutional Animal Care and Use Committee. The Yerkes National Primate Research Center is a fully Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facility. All animals in the colony are managed in accordance with the applicable United States Department of Agriculture (USDA's)

References (29)

A.W. Chan et al.
Generation of transgenic monkeys with human inherited genetic disease
Methods
(2009)
A.P. Goursaud et al.
Social attachment in juvenile monkeys with neonatal lesion of the hippocampus, amygdala and orbital frontal cortex
Behav Brain Res
(2007)
R.L. Carter et al.
Reversal of cellular phenotypes in neural cells derived from Huntington's disease monkey–induced pluripotent stem cells
Stem Cell Rep
(2014)
Y. Chen et al.
Cell-based therapies for Huntington's disease
Drug Discov Today
(2014)
H. Okano et al.
The common marmoset as a novel animal model system for biomedical and neuroscience research applications
Semin Fetal Neonatal Med
(2012)
H. Liu et al.
TALEN-mediated gene mutagenesis in rhesus and cynomolgus monkeys
Cell Stem Cell
(2014)
Y. Niu et al.
Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos
Cell
(2014)
F. Squitieri et al.
Juvenile Huntington's disease: does a dosage-effect pathogenic mechanism differ from the classical adult disease?
Mech Ageing Dev
(2006)
A.W. Chan
Progress and prospects for genetic modification of nonhuman primate models in biomedical research
ILAR J
(2013)
M.A. Pouladi et al.
Choosing an animal model for the study of Huntington's disease
Nat Rev Neurosci
(2013)

A. Morton et al.

Large genetic animal models of Huntington's disease

J Huntingtons Dis

(2013)

A.W. Chan et al.

A two years longitudinal study of a transgenic Huntington disease monkey

BMC Neurosci

(2014)

J. Kocerha et al.

Longitudinal transcriptomic dysregulation in the peripheral blood of transgenic Huntington's disease monkeys

BMC Neurosci

(2013)

S.H. Yang et al.

Towards a transgenic model of Huntington's disease in a non-human primate

Nature

(2008)

Cited by (30)

The marmoset as a model for investigating the neural basis of social cognition in health and disease
2022, Neuroscience and Biobehavioral Reviews
Citation Excerpt :
Being phylogenetically closer to humans, non-human primates can resolve this issue. While macaque disease models were generated using lentiviral transgenesis (Yang et al., 2008; Moran et al., 2015; Niu et al., 2015; Liu et al., 2016), success of germline transmission is low (Moran et al., 2015; Liu et al., 2016). Moreover, even though macaques are phylogenetically closer to humans than marmosets (Finstermeier et al., 2013), for long-term trans-generational studies, because of longer gestation periods, reproductive seasonality, susceptibility to zoonotic diseases, and low fecundity of macaques, marmosets still stand as advantageous disease models (Elmore and Eberle, 2008).
Social-cognitive processes facilitate the use of environmental cues to understand others, and to be understood by others. Animal models provide vital insights into the neural underpinning of social behaviours. To understand social cognition at even deeper behavioural, cognitive, neural, and molecular levels, we need to develop more representative study models, which allow testing of novel hypotheses using human-relevant cognitive tasks. Due to their cooperative breeding system and relatively small size, common marmosets (Callithrix jacchus) offer a promising translational model for such endeavours. In addition to having social behavioural patterns and group dynamics analogous to those of humans, marmosets have cortical brain areas relevant for the mechanistic analysis of human social cognition, albeit in simplified form. Thus, they are likely suitable animal models for deciphering the physiological processes, connectivity and molecular mechanisms supporting advanced cognitive functions. Here, we review findings emerging from marmoset social and behavioural studies, which have already provided significant insights into executive, motivational, social, and emotional dysfunction associated with neurological and psychiatric disorders.
The three sisters of fate: Genetics, pathophysiology and outcomes of animal models of neurodegenerative diseases
2022, Neuroscience and Biobehavioral Reviews
Citation Excerpt :
In addition, preliminary behavioural observations showed some evidence of chorea and dystonia (Yang et al., 2008). To date, few studies have been done using these NHP HD model, however germline transmission of the mutant HD transgene has been confirmed (Moran et al., 2015). A number of toxin-induced lesion models that have been developed for HD are described in the sub-sections below (summarized in Table 3B).
Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) are neurodegenerative disorders characterized by progressive structural and functional loss of specific neuronal populations, protein aggregation, an insidious adult onset, and chronic progression. Modeling AD, PD, and HD in animal models is useful for studying the relationship between neuronal dysfunction and abnormal behaviours. Animal models are also excellent tools to test therapeutic approaches. Numerous genetic and toxin-induced models have been generated to replicate these neurodegenerative disorders. These differ in the genetic manipulation employed or the toxin used and the brain region lesioned, and in the extent to which they mimic the neuropathological and behavioral deficits seen in the corresponding human condition. Each model exhibits unique advantages and drawbacks. Here we present a comprehensive overview of the numerous AD, PD, and HD animal models currently available, with a focus on their utilities and limitations. Differences among models might underlie some of the discrepancies encountered in the literature and should be taken into consideration when designing new studies and testing putative therapies.
Generation of nonhuman primate retinitis pigmentosa model by in situ knockout of RHO in rhesus macaque retina
2021, Science Bulletin
Retinitis pigmentosa (RP) is a form of inherited retinal degenerative diseases that ultimately involves the macula, which is present in primates but not in the rodents. Therefore, creating nonhuman primate (NHP) models of RP is of critical importance to study its mechanism of pathogenesis and to evaluate potential therapeutic options in the future. Here we applied adeno-associated virus (AAV)-delivered CRISPR/SaCas9 technology to knockout the RHO gene in the retinae of the adult rhesus macaque (Macaca mulatta) to investigate the hypothesis whether non-germline mutation of the RHO gene is sufficient to recapitulate RP. Through a series of studies, we were able to demonstrate successful somatic editing of the RHO gene and reduced RHO protein expression. More importantly, the mutant macaque retinae displayed clinical RP phenotypes, including photoreceptor degeneration, retinal thinning, abnormal rod subcellular structures, and reduced photoresponse. Therefore, we suggest somatic editing of the RHO gene is able to phenocopy RP, and the reduced time span in generating NHP mutant accelerates RP research and expands the utility of NHP model for human disease study.
Longitudinal characterization of cognitive and motor deficits in an excitotoxic lesion model of striatal dysfunction in non-human primates
2019, Neurobiology of Disease
Citation Excerpt :
Post-mortem analysis revealed neuronal dysfunction and astrogliosis alongside the presence of neuritic and nuclear ubiquitinated aggregates up to 30 weeks following viral vector injections. In parallel, an outstanding effort was deployed by Chan and colleagues to develop a transgenic macaque model of HD that reproduces the pathological landmarks of the disease, including reduced striatal volume, inflammation (Raper et al., 2016), and shows variable motor deficits, irritability, and subtle fine cognitive alterations in striatal function (Chan et al., 2015; Chan et al., 2014; Korecka et al., 2016; Moran et al., 2015; Yang et al., 2008). This work has advanced important knowledge on the disease itself, however, the small number of animals generated so far and the mild phenotype observed at 5 years of age might not respond to the needs of the HD community for pre-clinical therapeutic efficacy studies.
As research progresses in the understanding of the molecular and cellular mechanisms underlying neurodegenerative diseases like Huntington's disease (HD) and expands towards preclinical work for the development of new therapies, highly relevant animal models are increasingly needed to test new hypotheses and to validate new therapeutic approaches. In this light, we characterized an excitotoxic lesion model of striatal dysfunction in non-human primates (NHPs) using cognitive and motor behaviour assessment as well as functional imaging and post-mortem anatomical analyses.
NHPs received intra-striatal stereotaxic injections of quinolinic acid bilaterally in the caudate nucleus and unilaterally in the left sensorimotor putamen. Post-operative MRI scans showed atrophy of the caudate nucleus and a large ventricular enlargement in all 6 NHPs that correlated with post-mortem measurements. Behavioral analysis showed deficits in 2 analogues of the Wisconsin card sorting test (perseverative behavior) and in an executive task, while no deficits were observed in a visual recognition or an episodic memory task at 6 months following surgery. Spontaneous locomotor activity was decreased after lesion and the incidence of apomorphine-induced dyskinesias was significantly increased at 3 and 6 months following lesion. Positron emission tomography scans obtained at end-point showed a major deficit in glucose metabolism and D2 receptor density limited to the lesioned striatum of all NHPs compared to controls. Post-mortem analyses revealed a significant loss of medium-sized spiny neurons in the striatum, a loss of neurons and fibers in the globus pallidus, a unilateral decrease in dopaminergic neurons of the substantia nigra and a loss of neurons in the motor and dorsolateral prefrontal cortex.
Overall, we show that this robust NHP model presents specific behavioral (learning, execution and retention of cognitive tests) and metabolic functional deficits that, to the best of our knowledge, are currently not mimicked in any available large animal model of striatal dysfunction. Moreover, we used non-invasive, translational techniques like behavior and imaging to quantify such deficits and found that they correlate to a significant cell loss in the striatum and its main input and output structures. This model can thus significantly contribute to the pre-clinical longitudinal evaluation of the ability of new therapeutic cell, gene or pharmacotherapy approaches in restoring the functionality of the striatal circuitry.
Investigation of brain science and neurological/psychiatric disorders using genetically modified non-human primates
2018, Current Opinion in Neurobiology
Citation Excerpt :
Five transgenic macaques carrying mutant HTT were born, and the monkeys displayed difficulty in movement coordination and involuntary movement. While the first report showed only the phenotypes of the founder animals, the follow-up paper confirmed the germline transmission of the transgene in 2015 [44]. Furthermore, a recent study on autism model transgenic macaques was reported [45].
Although mice have been the most frequently used experimental animals in many research fields due to well-established gene manipulation techniques, recent evidence has revealed that rodent models do not always recapitulate pathophysiology of human neurological and psychiatric diseases due to the differences between humans and rodents. The recent developments in gene manipulation of non-human primate have been attracting much attention in the biomedical research field, because non-human primates have more applicable brain structure and function than rodents. In this review, we summarize recent progress on genetically-modified non-human primates including transgenic and knockout animals using genome editing technology.
A transgenic monkey model of huntington’s disease
2018, Conn's Handbook of Models for Human Aging
No matter how well an animal, cellular or computation simulation model were developed; no model system can capture the full spectrum of clinical features and progression of human diseases. Therefore, the development of the best animal model possible with maximum potential to advance our knowledge on diseases such as Huntington's disease (HD) may better fit the mission. Transgenic HD monkeys that developed clinical features as the animals age are similar to those observed in human patients. Although HD monkeys hold great potential as a preclinical large animal model, HD monkey stem cells provide a unique platform for investigating the cellular pathogenesis and therapy discovery that is limited in HD monkeys. Here we present a fact check on the HD monkey model and HD monkey neural stem/progenitor cell model and their roles in advancing our knowledge in HD onset and development and their potential roles in future preclinical therapy development.

View all citing articles on Scopus

View full text

Research articleGermline transmission in transgenic Huntington's disease monkeys

Abstract

Introduction

Section snippets

Animal models

Derivation and genotyping of transgenic HD monkeys' germline and embryonic stem cells

Discussion

Acknowledgments

Methods

Behav Brain Res

Stem Cell Rep

Drug Discov Today

Semin Fetal Neonatal Med

Cell Stem Cell

Cell

Mech Ageing Dev

Progress and prospects for genetic modification of nonhuman primate models in biomedical research

ILAR J

Choosing an animal model for the study of Huntington's disease

Nat Rev Neurosci

Large genetic animal models of Huntington's disease

J Huntingtons Dis

A two years longitudinal study of a transgenic Huntington disease monkey

BMC Neurosci

Longitudinal transcriptomic dysregulation in the peripheral blood of transgenic Huntington's disease monkeys

BMC Neurosci

Towards a transgenic model of Huntington's disease in a non-human primate

Nature

Research article
Germline transmission in transgenic Huntington's disease monkeys