Large Animal Models are Critical for Rationally Advancing Regenerative Therapies

Keywords::

• GDNF
• gene therapy
• large animal model
• MPTP
• nonhuman primate
• Parkinson‘s disease
• stem/precursor cell
• transplantation
• Type 1 diabetes
• regenerative medicine

Enthusiasm for therapies based on the transplantation of exogenous cells or the transfer of genes by viral vectors has burgeoned over the past 30 years, accompanied by a predictable exhortation to launch clinical trials as soon as possible. Most data regarding safety, efficacy and mechanisms of these therapies have been derived from studies in rodents alone. While such ‘small-animal‘ systems offer invaluable insights into fundamental biological questions, it is often misleading and perilous to unquestionably equate the higher order motor, sensory and cognitive processes that characterize human disease with that gleaned from a mouse or rat. Indeed, the literature is littered with clinical trials that failed and, in some cases, led to unforeseen adverse outcomes because the field leap-frogged over the requisite large-animal model. Large animals often provide an essential bridge between insights into fundamental biology and pathophysiology gleaned from simple systems and the realities of treating a human disease. Often, this is especially true for neurological disorders where not only differences in size and scale pertain, but also in neuroanatomical connections and organization, cognitive capacities, signaling pathways, genetic redundancy or the disease etiology.

While the gene therapy field has increased their use of nonhuman primates prior to the application of viral vectors in clinical trials, the cellular therapy field – represented most conspicuously of late by the stem cell field – has only recently begun to properly address this requirement. Monkeys and the minipig may prove to be excellent preclinical models owing to their similar comparative anatomy, pharmacokinetics and physiological and metabolic interactions. These models have proven to be extremely useful for studying endocrinological diseases, such as Type 1 diabetes, and neurological disorders, including Parkinson‘s disease (PD), spinal cord injury (SCI) and multiple sclerosis (MS). Prudence would argue that clinical trials for diseases in which differences between rodent and human transcend size and scale should require clear and definitive proof-of-concept in at least one relevant large-animal model in order to safeguard patients. This need, of course, is counterbalanced by considerations of the substantial cost, time and ethical circumspection that typically accompanies such research. In this editorial, we will attempt to help researchers reason through the potential need and advisability of using a large-animal model for their particular biological question.

There are numerous differences between rodents and humans that make the unqualified translation of rodent data inadequate. The most relevant differences start at the genomic level and are manifested as differences in pharmacological, biochemical, developmental, behavioral and functional responses to perturbations and interventions [1]. Anatomical and cytoarchitectural differences are particularly profound in the brain, where innervation of specific regions is critical to addressing a neurological deficit (Figure 1). In PD for example, it has been estimated that for each volume of tissue innervated by a rat dopaminergic (DA) neuron, a monkey neuron must innervate 20-times that volume, while humans would need a staggering volume of 200 units to mimic similar reinnervation of nigrastriatal projections [2]. In addition, differences in monoamine biochemistry and developmental life span make extrapolating rodent data to human therapies risky and insufficient [3–7]. Behavioral differences between rodents and humans are also of great significance [8–10]. It is likely that only a large animal model can adequately mirror the complexities of studying and restoring bipedal gait, balance, tremor, hand preference, fine motor coordination, spontaneous blink rate and cognitive deficits [11–16].

Figure 1. Coronal images of Nissl stained sections from Mus musculus, Chlorocebus aethiops, Macaca mulatta, and MRI of human cortex [135].

The minipig

For research into PD, the nonhuman primate model is ideal, although, the technical, financial and organizational complexities of monkey colony maintenance may require the use of an alternative, somewhat more accessible, species, such as the Gottingen miniature pig [17]. The ‘minipig‘, developed in the early 1960s [18,19], presents many advantages over the rodent as a model for human diseases. Its organ systems are organized similar to that of the human and it displays similar physiological and pathophysiological responses [20]. Similarities in pancreas size, position and shape [21], gastrointestinal tract structure and function [20], and pharmacokinetic responses make the minipig an especially useful model for Type 1 diabetes [22]. The kinetics and dynamics after subcutaneous delivery of insulin, [23,24] as well as aspects of metabolism [25] and glucose tolerance [26,27], closely resemble those of humans. Furthermore, several stable models for chemical induction of Type 1 diabetes have been established in minipigs using pharmacological insult to ??cells with streptozotocin (STZ) [28–31] or alloxan [32]. Recently, the transplantation of both human and porcine islets of Langerhans in Type 1 diabetes has been studied in diabetic pigs, as well as in dogs, monkeys and patients [33–41].

Complications in xenograft survival related to differences in the HLA and SLA systems between humans and pigs, respectively, are of concern, but may be partially overcome by using a more recent variation of the minipig – the ‘NIH minipig‘. First described by Sachs in 1976 for transplantation studies [42,43], the NIH minipig, homozygous for known alleles of the major histocompatibility complex (MHC) [44], has been shown to be effective for modeling immunological and genetic aspects of heart, lung and spleen transplant tolerance [45,46,22] and is sufficiently bred to accept bone marrow and possibly hematopoetic stem cell transplants [47,48], as well as skin and heart grafts [22].

The minipig has a relatively large brain, approximately ten-times the mass of the marmoset [49,50], making it of recent interest in neurological disease studies. The size of the brain makes it especially appealing for imaging and stereotactic manipulation. There is a growing list of neurological diseases that have been modeled in the minipig: experimental allergic encephalomyelitis (EAE) [51], mimicking the autoimmune, demyelinating disease MS; Huntington‘s disease (HD) CAG triplet repeats [52]; and 1?methyl?4?phenyl?1,2,3,6?tetrahydropyridine (MPTP)-induced Parkinsonism [53]. Such models enable a more representative analysis of cell transplantation therapies for these disorders. In fact, porcine xenografts have already been tested in Phase I trials in both HD and PD patients [54,55], as well as in a variety of other disorders [56,57], validating the use of the minipig as a bridge between rodent and human clinical trials.

The minipig has also been used to study implants of progenitor cells into the ischemic and contused spinal cord (M Marsala; correspondence). Indeed, it is work in the spinal cord and the difficulties in attempting to adapt procedures developed in rodent to the pig that highlight the absolute need for large animal studies as a precursor to human trials for such disorders [58].

Lessons from Parkinson‘s disease

PD is characterized by degeneration of DA neurons in the substantia nigra pars compacta and subsequent loss of striatal DA release. Cell-based strategies for replacement of damaged DA neurons have been a promising strategy since the early 1970s, when Olsen and colleagues performed their initial transplantation experiments in rat brains [59–63]. Several subsequent studies in rats provided extended evidence for fetal cell transplants repairing DA deficiency [64,65]; however, despite numerous attempts by many groups, these results could never be repeated in primates [1]. Coupled with mounting ethical considerations, researchers turned to adrenal allografts as a new source of tissue; however, these also failed and subsequent clinical trials were abandoned [66–68]. New hope came again after 1985 when human fetal neural cells were derived and successfully implanted into the rat and monkey brain [69–76]; however ethical opponents argued that the large amount of fetal tissue required made these procedures for routine regenerative therapy unfeasible and, therefore, unfundable.

Based on the varying methods published in the mid-1970s, several groups began preliminary efficacy and safety clinical studies of fetal ventral mesencephalic grafts in PD patients. Initial studies in Sweden [77], England [78] and Mexico [79] produced variable results, elevating the ethical debate and political controversy, eventually leading to a funding moratorium in the USA [80,81]. Subsequent studies appeared promising, although the overall consensus was that the results were inconclusive owing to variability within small studies, limited short-term functional improvements and overall difficulty in comparing independent procedures [1]. To investigate this problem and improve the ability to compare studies, a panel of experts published a recommended protocol for future investigations, termed Core Assessment Program for Intracerebral Transplantation (CAPIT) [82].

In the same year, the first randomized controlled clinical trial, headed by Curt Freed and Stanley Fahn, was initiated studying 40 PD patients over 7 years, half of whom underwent bilateral stereotactic transplantation of human embryonic mesencephalic tissue into the putamen [83]. Although initial results appeared promising, it was determined that no benefit in primary outcome measure had been attained after 12 months post operation. However, a small subset of patients under 60 years old did show small double-blind improvements on a validated PD rating scale. More importantly, predictions made from animal models failed to predict the relatively high occurrence of dystonia and dyskinesia associated with long-term survival [84]. These gross abnormalities occurred in five out of 33 patients that lived 3 years post transplant, of whom, all five were aged less than 60 years and had displayed improvements in PD score after 12 months post surgery.

The human trials appeared to have a solid rationale based on a decade of rodent studies. Hundreds of investigators had reported survival and functional recovery from fetal ventral mesencephalic-derived neural tissue transplanted into the striatum of rats [85]. The grafts were shown to express tyrosine hydroxylase (TH), increase DA concentrations, project long outgrowths, form synaptic connections and respond to afferent stimulation [86–90]. However, the rat models of PD overestimated the likely success of fetal precursor transplants and failed to anticipate the dyskinetic side effects in human trials [84]. Furthermore, although large animal models were tested, it is possible that they were not followed for a long enough time, simply owing to the vast difference between rodents and primates (of which human is an example) was not appreciated. Also, while the Freed and colleagues study made extremely valuable contributions to our understanding of PD, small design flaws, such as method of tissue preparation, limited target area and lack of immunosuppression, may have inherently impeded the overall success of the study [1]. Many of these details may have been worked out in large animals before reaching the clinic, potentially predicting adverse effects. Unfortunately, cell replacement therapy received negative media coverage [91–94], creating an impression of defeat in the public‘s mind and even creating dissention among PD investigators [84,92,95–102]. Much of this may have been avoided had a higher priority been placed on more extensive primate studies with larger numbers and longer follow-up.

Going forward in Parkinson‘s disease: a case-in-point for choosing the correct model

Having detailed the pitfalls into which the PD field slipped by relying upon nonrepresentative data for its first cell-based clinical trial; how might it recover its momentum, and can this serve as a lesson for future trials for other diseases?

As aforementioned, most of the early transplantation studies in PD were performed in rodents that had received a unilateral injection of the dopaminergic toxin 6?hydroxydopamine (6?OHDA) into the medial forebrain bundle or nearby [103,104]. Although transplantation appeared to promote recovery from the resultant PD-like symptoms, these behaviors actually bore little resemblance to human PD. If therapies were to be tested solely on such a rodent model, the results would likely have poor predictive value for actual patients. Conversely, through serendipity in the 1960s and 70s, it was discovered that the complex I inhibitor, MPTP, created pathology quite similar to human idiopathic PD in humans [105–107] and monkeys [70,108–112], especially the vervet [113,114]. MPTP depletes substantia nigra TH?immunoreactive and striatal DA to levels similar to PD, and produces characteristic Parkinsonian symptomatology, including akinesia, rigidity, postural alterations and tremor [9,10]. Overexpressing ??synuclein in conjunction with MPTP may create Lewy bodies [115,116]. Hence, systemically administered MPTP in African green monkeys has become acknowledged as the most authentic model for actual human idiopathic PD and the model in which the safety and efficacy of viral vector- and cell-based therapies in humans can most reliably be judged [1,8–10,117]. Indeed, the potential use of lentiviral and adeno-associated virus-based vectors for delivering neurotrophic factors in PD patients has been, and is currently being, tested in monkies [118–120]. Such studies, in turn, set the stage for some Phase I clinical trials [121–131]. It is disheartening that, to date, very few investigators in the cell therapy field (including the stem cell field) have taken advantage of this model for work in PD, despite widespread recognition of its power. This reticence is, most probably, due to the huge expense entailed in performing such complex studies with a sufficiently large sample size and with suitably objective and predictive readouts over a long enough period of observation. Nevertheless, that expense is vastly smaller than the cost to the healthcare system, science and patient well-being when an ineffective or injurious clinical intervention becomes misguidedly launched. Funding agencies and scientists must begin to change their thinking and view adequately powered large animal studies for some diseases not as a luxury, but as a necessity.

Another source of hesitancy in the use of large animals, particularly monkeys, is not solely financial, but also ethical. There is no question that experimentation on ‘higher order‘ animals provokes, more than any other type of scientific study, circumspection regarding the appropriateness of animal experimentation. Although we have outlined its scientific justification, there is no question that investigators must be acutely sensitive (as they should be for all animal work) in using the minimal number of monkeys required to statistically overcome type 1 and 2 error rates [1], to treating the animals humanely and to only using animals in the most judicious manner and only for exquisitely well-conceived and rigorously designed experiments.

Another level of ethical consideration that has recently emerged as a consequence of the burgeoning human stem cell field, is whether integrating human cells into the brains of nonhuman primates (particularly prenatally) somehow ‘humanizes‘ them. In other words, might a human–primate chimeric brain develop consciousness and, hence, merit consideration beyond that of just an experimental laboratory animal [131–134]. While an interesting metaphysical consideration, chimerism in our hands, even under the most optimal transplantation conditions, yields only a relatively small human cell contribution to an overwhelmingly primate CNS structure. Furthermore, we have never observed anything but typical primate behaviors, emotions and skills in our animals.

Summary & conclusions

Appropriately assessing the safety and efficacy of many future interventions in regenerative medicine will require large animal preclinical testing to avoid the failures of previous studies in patients. Such recognition requires scientists, investors, funding agencies and the public to recalibrate their thinking. Simpler rodent models, while useful for unraveling certain biological conundrums, often fall short in their predictive value for human disease or their appropriateness for devising effective delivery methods. A greater number of multicenter collaborations may be required to insure adequate funding and expertise for large animal studies. Such a method requires a less egocentric approach to scientific accomplishment. The ultimate cost of not including the rationale use of large animal models in preclinical studies – the price of unsuccessful trials, injury to patients and failure to bring potentially groundbreaking therapies to the bedside – far outweighs the short-term expense of the studies themselves.

Acknowledgements

We thank DE Redmond and JH Kordower for helpful suggestions. DR Wakeman is supported by the National Institute of Health (NIH)/National Institute of General Medical Sciences (NIGMS) funded UCSD Genetics Training Program (T32 GM08666) and is a recipient of an American Society for Neural Therapy and Repair Travel Fellowship (R13NSO55615; DR Wakeman and AM Crain are supported by an NIH Stem Cell Center Grant (1 P20 GM075059–01) and a grant from the American Parkinson‘s Disease Association.

DR Wakeman, AM Crain and EY Snyder acknowledge no conflicts of interest.

Additional information

Funding

We thank DE Redmond and JH Kordower for helpful suggestions. DR Wakeman is supported by the National Institute of Health (NIH)/National Institute of General Medical Sciences (NIGMS) funded UCSD Genetics Training Program (T32 GM08666) and is a recipient of an American Society for Neural Therapy and Repair Travel Fellowship (R13NSO55615; DR Wakeman and AM Crain are supported by an NIH Stem Cell Center Grant (1 P20 GM075059–01) and a grant from the American Parkinson‘s Disease Association. DR Wakeman, AM Crain and EY Snyder acknowledge no conflicts of interest.