Arches. Photo by Daniel Chia
HOPES: Huntington's Outreach Project for Education, at Stanford
May
22
2013

Transgenic HD Monkey Models

Introduction

This article will discuss the advantages and disadvantages of using transgenic monkeys to model Huntington’s disease (HD). Most HD animal research utilizes mouse models of the disease. While there is much that we can learn from mice, animals that are more similar to humans, such as monkeys, could offer more pertinent insights into HD and serve as brand new and promising avenues for HD research. Along these lines, transgenic rhesus macaques carrying a human mutant huntingtin gene have been developed at Yerkes Primate Center in Atlanta, Ga. In addition, transgenic marmosets carrying a fluorescent protein have recently been developed at Keio University, in Japan. Researchers plan to use marmosets to create either a Parkinson’s or HD model in the near future. The new models offer a host of advantages that no other animal model has provided, but they are also constrained by cost, ethical considerations, and time.

Advantages of using a Monkey Model

Monkeys are more genetically similar to humans than rodents. As a result, they have a similar lifespan, metabolism, and physiology to humans. For these reasons, monkeys will probably be better models for monitoring disease progression and the effectiveness of experimental drugs. The transgenic monkeys with the mutant huntingtin gene exhibit an HD phenotype closer to humans and more closely mirror the physical, behavioral, and cognitive symptoms of the disease than any other HD animal model so far.

A variety of established behavioral and cognitive tests are used to assess the monkeys. One such test is the HD primate model scale, modified from the HD scale used for humans. The scale ranges from 0 to 80, in which 80 describes the most severe symptoms and is used to track the number of involuntary movements in the transgenic rhesus macaques. The test shows that the monkeys display chorea and dystonia more clearly than many HD mouse models.

In addition to physical tests, non-invasive fMRI procedures are used to monitor neurodegeneration, and intranuclear huntingtin inclusions, as well as other features of the disease at the neural level. Monkeys have larger brains than rodents, so neural changes can be monitored more accurately. Moreover, a germline of macaque pluripotent stem cells with the mutant huntingtin gene has been developed and is currently being studied by the same researchers. These stem cells can be coaxed into becoming neurons, which could then demonstrate the neural symptoms of the disease. Ethical concerns have prevented the development of a similar human germline, so the transgenic rhesus monkeys provide both an in vivo and in vitro avenue to study the neural progression of HD.

Disadvantages of using a Monkey Model

Many of the advantages of monkey models come hand in hand with their problems. Primate models are more expensive, take more time, and raise more ethical concerns than mouse models. The rhesus macaque for example has one baby at a time, a gestation period of 150 to 160 days, and a long puberty of 3 to 4 years.  The constraints and labor involved in natural and artificial reproduction make the cost of transgenic monkeys significantly higher than transgenic mice. Similarly, monkeys are much larger than mice, so their housing and food costs are more expensive. Because monkeys display HD symptoms several years after they are born, these costs for monkeys are exponentially greater than those for mice. Because macaques and marmosets are similar to humans in terms of emotions, cognition, and behavior, the use of monkeys in lab research raises more ethical concerns and media interest than other animal models. The monkeys are protected by stringent ethical guidelines and procedures determined by the National Institute of Health and the Institutional Animal Care and Use Committee. The use of monkeys in research sparks public interest more than rodents, which can bring both positive and negative attention to transgenic primate research and HD animal model research in general.

Different types of monkeys have their advantages and disadvantages. The rhesus macaque is more closely related to humans than the marmoset but the reproductive traits of the marmoset make it an attractive model in its own right.  The marmoset can have 80 babies over its lifetime, compared to that of 10 offspring for the macaque. The marmoset also has a shorter pregnancy and a faster sexual maturation, making the marmoset model perhaps a more efficient model for studying HD. The marmoset is also smaller making housing and feeding them more manageable economically. However, the marmoset has a smaller brain, making it more difficult to track neurodegeneration on an MRI. Both of these monkey species have far longer pregnancies, pubertal periods, and fewer offspring than a female mouse, who can have up to 10 litters of 3 to 14 mice per year. However, animal models more similar to humans could not only accelerate discoveries in the field, but could potentially reduce the total number of animal models needed in research.

Method of Transgenesis

There are a variety of animal models that are designed through genetic engineering in order to study HD. The only type of monkey model so far is the transgenic model. In this type of model, a transgene is integrated into the animal’s genome. An example of the method used for the transgenic macaques is as follows.  A mutant human huntingtin gene with 84 CAG repeats is inserted into rhesus macaque egg cells through a viral vector. The viral vector is a modified lentivirus, which is a virus commonly used for gene delivery because it infects non-dividing cells. The lentivirus contains the gene encoding for the mutant huntingtin gene as well as a gene encoding green fluorescence protein (GFP) to serve as a marker, so that the scientists can tell whether or not the transgene was integrated into the genome of the monkey. Many of these HD eggs are then artificially fertilized and implanted into female surrogate monkeys. The gene is integrated randomly into the embryo’s genome through reverse transcriptase. Of the HD monkeys that are born, each will vary in terms of the location of the human mutant huntingtin gene on the chromosomes, the number of CAG repeats, and severity of the HD phenotype. Essentially, HD manifests differently in each monkey due to the nature of the transgenic procedure. However, the mutant huntingtin gene is still heritable and dominant despite its variable location in the chromosomes so natural and artificial offspring of transgenic monkeys can also be used to study HD.

The advantages of transgenesis through the lentivirus vector are that it is very effective and that HD animal models can be produced in high yield. However, through this method the mutant human huntingtin gene could be integrated anywhere in the genome rather than where the gene is normally located. Some knock-in mice have been produced in which the human gene replaces, at least in part, the mouse’s huntingtin gene in the correct location on the chromosome. They should, in theory, be more accurate models than transgenic mice. A knock-in monkey is theoretically feasible, but the laboratory techniques are not yet efficient enough to make this cost-effective.

Current and Future Research

There are currently only two branches of research on transgenic non-human primates at this time: macaques and marmosets. The first successful transgenic monkey was developed in 2001, when Dr. Anthony Chan and his colleagues at Oregon Regional Primate Research Center inserted a GFP gene into an embryo of a rhesus macaque via a retrovirus.  Chan then moved to Emory University where he developed the first HD macaque model in 2008. Five transgenic monkeys were born and four of those expressed HD symptoms. The macaques had integrated human mutant huntingtin genes that varied in CAG repeats and integration location. Two of these monkeys died within the same day of birth, probably because they had longer CAG repeats. This feature is known to cause a quicker onset of symptoms in humans, and thus expressed a more severe phenotype –motor impairment and difficulty breathing– than the others.  They both also had multiple mutant huntingtin integration sites, which may have lead to the overexpression of the huntingtin gene as well. One monkey is still living today. Its huntingtin protein had only 29 CAG repeats, within the range of a normal huntingtin gene, so it will probably not develop the disease. The other two monkeys had 83 and 84 CAG repeats and lived long enough to be studied by the researchers. All the monkeys were studied based on their physical behaviors of dystonia, chorea, difficulty swallowing and difficulty breathing. These symptoms manifested in varying degrees in all monkeys except the monkey with 29 CAG repeats.

The lab now has a second generation of monkeys that are currently being studied. They are from the same germline of one of the monkeys from the first generation, meaning they are genetically related. Though the research is unpublished, the new group of monkeys model HD even better, reflecting a delayed onset of the disease and milder phenotypes. The macaques’ brains will be studied through fMRI, which is non-invasive. The macaques could potentially be used to study promising HD medications.

In 2009, Japanese researchers developed the first transgenic marmoset model expressing GFP. Five monkeys made up the first generation of transgenic marmosets. The marmosets glow under a specific wavelength of light because, like the macaques, they carry the GFP gene derived from jellyfish DNA. The researchers hope to use the same techniques to integrate genes coding for Parkinson’s, but are also considering HD. Because the marmosets reproduce more frequently and in larger numbers, transgenic marmosets could even further accelerate HD research.

HD is easier to study than some other diseases because it involves a mutation in one gene, where as Parkinson’s, ALS, and Alzheimer’s have more complex genetic origins. So HD is, in many ways, a gateway for all neurodegenerative research.  As more labs start working with transgenic non-human primate models, advances in understanding the progression, physiology, and neurobiology of HD are sure to follow. Because HD monkeys are also an intermediate between mouse and humans, they serve as a new avenue for drug research, which could reduce the time it takes to bring a new drug to market. The new monkey models put HD research on the cusp of major breakthroughs for both understanding and treating the disease.

Further Reading

Anthony W.S. Chan, Pei-Haun Cheng, Adam Neumann and Jin-Jing Yang (2010) Reprogramming Huntington Monkey Skin Cells into Pluripotent Stem Cells. Cellular Reprogramming (In Press). Dense and unnecessary to read.

Anthony W.S. Chan (2009). Transgenic primate research paves the path to a better animal model: are we a step closer to curing inherited human genetic disorders? J Mol Cell Biol 1(1):13-14. PMID:19671628. Great article that discusses the advantages of a monkey model and is easy to read.

Anthony W.S. Chan and Shang-Husn Yang (2009). Generation of Transgenic Monkeys with Human Inherited Genetic Disease. Methods 2009 May 23 [Epub ahead of print] PMID: 19467335. Scientific research and difficult read but helpful for understanding setting up a germline.

Bachevalier, Stuart M.  Zola, Shihua Li, Xiao-Jiang Li and Anthony WS Chan (2008) Toward a Transgenic Model of Huntington’s Disease in the Nonhuman Primate. Nature 453(7197): 921-924. PMID:18488016. Scientific and dense but excellent description of methods used for transgenesis.

B.Snyder, A. M. Chiu, D. Prockop and Anthony W.S. Chan (2010) Human Multipotent Stromal Cells (MSCs) Increase Neurogenesis and Decrease Atrophy of the Striatum in a Transgenic Mouse Model for Huntington’s Disease. Dense and unnecessary to read.

Chan, A. W. Personal Interview.  4 Jan. 2013.

Cyranoski, David. “Marmoset Model Takes Centre Stage.” Nature.com. Nature Publishing Group, 27 May 2009. Web. Good summary article of more difficult published research.

Nature 459, 523-527 (28 May 2009) | doi:10.1038/nature08090; Received 27 September 2008; Accepted 30 April 2009. Difficult but worthwhile read on the marmoset models.