4

Alternatives to the Use of Laboratory Dogs

The preceding chapters evaluated the use of laboratory dogs in biomedical research in fields relevant to the mission of the U.S. Department of Veterans Affairs (VA). This chapter explores the landscape of alternatives development in areas of interest to the VA where laboratory dogs are currently used or were used in the recent past—cardiovascular disease (CVD), spinal cord injury (SCI), cancer, and diabetes. Alternatives considered in this chapter include companion dogs, other laboratory species, and non-animal methods.

CURRENT STATUS OF ALTERNATIVES DEVELOPMENT

While laboratory dogs have been used to study a wide range of diseases throughout history, the frequency of their use for research purposes, both in the VA and throughout the United States, has declined over time for a variety of reasons, as described in Chapter 3 and Appendix B. In documents provided by the VA to the committee, justifications for using laboratory dogs in biomedical research largely rely on the availability of historical data and biological similarities between dogs and humans. The committee notes that reliance on historical data and the biological similarities between dogs and humans is not unique to researchers or the scientific review process at the VA—biomedical researchers at institutions throughout the country and world often rely heavily on these factors to justify the use of laboratory dogs or, for that matter, any species. Shifting research to an alternative model (animal or non-animal, including humans) will require successfully addressing those factors that favor the continued use of laboratory dogs.

Four broad categories of alternatives are considered in this chapter. The first is companion dogs volunteered by their owners for clinical trials. The second is laboratory animal models other than the dog, and the third is “new approach methodologies” (NAMs), a diverse array of innovative non-animal approaches. Finally, the fourth category consists of areas where human clinical trials could be used to address research questions currently being studied in laboratory dogs.

As a promising medical discovery is shepherded along the translational pipeline, with testing frequently required in both small (rodent) and large (non-rodent) mammals, it is essential to select

the most scientifically valid approach to study the disease of interest. For animal models, criteria have been proposed to define the ideal model (Schachtschneider et al., 2017a). This model would, for example, mimic a human disease at the molecular and physiological level and display a comparable natural history of the disease, be associated with cell lines for in vitro study, yield reliable and predictable results, provide accurate treatment assessment, and be amenable to imaging.

Although animal models will continue to be part of advancing biomedical research in the near future, they are not always capable of representing the nuances of human pathophysiology and genetic variability critical to efficient translation with impactful clinical outcomes. The desire to improve the translational impact of biomedical research, together with societal pressures to reduce the use of animals for this purpose, has led to significant interest and investment in human-based approaches to biomedical research that do not require the use of animals. These approaches are referred to herein with the broadly descriptive term NAMs, which includes any non-animal technology, methodology, approach, or combination thereof, including in vitro, ex vivo human tissue, computational, and in silico models. While not capable of recapitulating the complexity of intact animal models, NAMs do offer the potential to evaluate specific critical aspects of human biology that are not replicated in animals.

COMPANION DOGS

For multiple reasons, companion dogs (in lieu of laboratory dogs) provide an attractive model in which to study serious medical conditions that affect veterans. In these studies, new diagnostic, preventive, or therapeutic approaches are investigated in dogs with conditions (or genetic predispositions to conditions) of interest. The dogs are “volunteered” by their owners and reside with their owners while under study. The study interventions are carried out in appropriate veterinary clinical settings. Investigators may include licensed veterinary clinicians, Ph.D. biomedical scientists, and physicians focusing on humans, often working as teams. Ethical, legal, and investigative standards comply with federal guidelines for the human owners—if data are collected from them—and also the dogs and the clinical facilities where study interventions are conducted. Veterinarians and animal health businesses have long studied companion dogs as research subjects to enhance the outcomes in the canine patients themselves. Properly controlled and conducted studies also have the potential to provide preclinical data for similar approaches in humans, including veterans. Such use could reduce the use of laboratory dogs while potentially benefiting the companion dogs, proving advantageous on both ethical grounds and in the eyes of the public. As discussed in Chapter 3, companion dogs may also offer certain scientific advantages over highly controlled studies using laboratory dogs. For example, while laboratory animals are generally chosen for their homogeneity, companion dogs display both (well-characterized) genetic heterogeneity and environmental diversity, similar to humans. We note that the genetic and environmental homogeneity of animals bred for laboratory research—notably rodents—can be essential during the early stages of discovery, reducing variables and enabling phenomena to be studied in relative isolation. These studies may be more scientifically valid, easily replicated, and cost-effective as well as more ethically appropriate than initial studies conducted in pet animals. However, evaluations of treatment efficacy of the sort likely to involve dogs may benefit from a more diverse population, and while such studies are more expensive and potentially involve larger group sizes than some studies in laboratory animals, they are certainly less expensive than human clinical trials.

As noted in Chapter 3, many conditions that the VA was investigating prior to the initiation of this study, including cardiac arrhythmia, cardiomyopathy, SCI, cancer, and narcolepsy, are experienced as spontaneous conditions in the companion dog population. Other topics of research at the

VA that are endemic to dogs include infectious diseases, chronic pain, senile dementia, and obesity. Additionally, military working dogs experience physical trauma, and there is general clinical suspicion that psychological trauma may also occur, with dogs going on to develop canine posttraumatic stress disorder (Broach, 2018).

There are limits to the utility of companion dogs as research subjects. One limit is biological and results from the significant differences between humans and dogs with regard to disease prevalence, natural history, and/or pathophysiology. For example, while dogs display cardiac arrhythmias, cardiomyopathy, and heart failure, they do not experience atherosclerosis at anywhere close to the human rate. Diabetes mellitus (DM) occurs at a rate of roughly 0.2 percent in companion dogs, with type 1 more prevalent than type 2 (Banfield Pet Hospital, 2016). Although dogs contract some infectious diseases in common with humans (e.g., rabies, Lyme disease, leishmaniasis) (CFSPH, 2011; Petersen, 2019), the manifestations can be different; for example, the most common serious sequela of Lyme disease in dogs is kidney disease, but neurologic disease is more common in humans. Furthermore, many significant human infectious diseases do not affect dogs.

Although many types of human cancer are seen in companion dogs, the use of companion dog models could be limited by certain factors. Recruiting sufficient numbers of companion dogs with a particular cancer for a randomized controlled trial in a timely manner could be a challenge, for example. In cancer and other disease areas, the variability inherent in companion dog clinical cases may require greater treatment group sizes than if laboratory dogs are used. In companion dogs (depending on the scientific questions that the researcher wants to address), controlling environmental factors, such as outdoor exposure, diet, and environmental stressors, all of which may be confounders, may also be a challenge. The use of companion dogs also limits the ability to collect certain experimental endpoints that can be obtained from laboratory dogs, such as histopathology, gross pathology, and necropsy.

Companion dogs experience natural SCI at a high rate in the thoracic and lumbar regions, but cervical injuries—the most common type in humans and an area of research at the VA—are rarely seen in companion dogs. If a dog with a high spinal injury suffered respiratory paralysis or severe compromise, sustaining it through prolonged ventilator support would not be consistent with humane and ethical practice; this also applies to the long-term study of cervical SCI in laboratory animals. Therefore, companion dogs can be useful for studying SCI and its response to treatment but not for research on quadriplegia or long-term respiratory support.

While studying a disease of interest in companion dogs is more attractive than working on laboratory animals on both ethical and public perception grounds, it requires certain preconditions that have yet to be met for many disorders. First, the disease must have been sufficiently well studied in dogs to establish its relevance for humans. Second, it requires that researchers studying the human condition collaborate with clinician–scientist partners competent in companion dog studies. Both conditions have been met in the fields of cancer and chronic pain, as discussed further below, and there is the potential to satisfy them for other areas of interest to the VA.

In addition to biological and scientific constraints, another factor limiting the expansion of research to companion dogs is administrative. With notable exceptions (such as for cancer and SCI, as discussed previously), there has been little investment in developing the infrastructure needed to enable biomedical researchers to identify those human diseases that are amenable to being studied in dogs and to find high-quality clinical trials with which they might collaborate. For many diseases, even where the science supports use of a companion and a working dog model, there is no existing network for conducting clinical trials in dogs. This is not an insurmountable issue; indeed, a relatively minor investment in administrative infrastructure and network building could yield significant value.

Current State of the Art for Companion Dog Clinical Trials

Veterinary clinical trials have a long history (Boothe and Slater, 1995), and the past decade has seen an increased interest in using companion dogs for these studies (Davies et al., 2017; Dean, 2017; Giuffrida, 2016; Rice et al., 2008). International guidelines for carrying out these trials have been discussed, with a focus on regulatory drug approval studies (Alvarez and Fortsch, 2005), although no international regulations are currently in effect (Nature, 2016).

Among the studies performed on companion dogs, the most longstanding and well organized are those investigating treatments for cancer (Paoloni and Khanna, 2008). While juvenile dogs usually die from trauma, congenital disease, and infections, cancer is the leading cause of death in adults (Fleming et al., 2011). Dogs spontaneously develop cancers at rates similar to those in humans, and companion dog models have been successfully used in preclinical trials of cancer therapies (Cornelius, 2018; MacNeill et al., 2018).

The National Cancer Institute (NCI) supports a Comparative Oncology Trials Consortium (COTC) using companion dogs to study cancer (NCI CCR, n.d.a). COTC studies have demonstrated an exemplary level of collaboration between canine-focused and human-focused clinicians and resulted in multiple publications (NCI CCR, n.d.b).

A consortium to investigate spinal cord injuries, the Canine Spinal Cord Injury Consortium, is also well developed, with promising results from a recent trial that tested cell-based therapy in companion dogs with SCI (Jeffery, 2019; Moore et al., 2017) and another that tested intraspinal injection of chondroitinase (Hu et al., 2018; Jeffery, 2019). Studies of novel approaches to relieving pain caused by chronic disease, which is particularly difficult to study humanely in a laboratory setting, have also yielded promising results (Cimino Brown, 2017; Iadarola et al., 2018).

The committee’s task included the consideration of future areas of research likely to be relevant to veterans. These future topics may include, for example, injuries sustained in battle. Dogs endure battleground injuries, and a recent article in Military Medicine called for the establishment of a military working dog trauma registry to collect these data to improve casualty care (Orman et al., 2018). There is also interest in the diagnosis and treatment of canine PTSD, which, due to the nature of their work, military working dogs are prone to develop (Broach, 2018).¹

Indeed, the VA is to be recognized for its pioneering research using companion dogs, including a current evaluation of immunocytokine therapy for melanoma (VA ORD, n.d.). If media coverage is any indication, then public interest in companion dog research is high (NCI CCR, n.d.b). Another indicator of such interest occurred on December 19, 2019, when the U.S. Senate unanimously passed Senate Resolution 462, designating January 2020 as “National One Health Awareness Month” to promote collaboration among public, animal, and environmental health scientists (Feinstein, 2019).

Optimization of VA Research to Use Companion Dogs

The VA has an opportunity to become a premier biomedical research entity engaging formally with veterinary expertise, both to enhance the experience of laboratory dogs and to conduct clinical trials in companion dogs, using companion dog studies to replace laboratory dog research wherever possible. Accomplishing this goal will require the following:

1. Engaging with experts in canine medicine and research to optimize both clinical methods and research goals.

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¹ While clinical criteria have been established by the American College of Veterinary Behaviorists (https://www.cliniciansbrief.com/article/acvb-avsab-capsules [accessed June 16, 2020]), as of this writing, there are no published scientific studies of canine PTSD.

The study of comparative species medicine and biomedical research underpin the fundamental argument that studying non-humans will inform us about humans. Veterinarians dedicate their careers to this work, and the application of veterinary expertise to more than simply assuring humane treatment and regulatory compliance would strengthen the quality of the VA’s work. While board-certified veterinary clinical specialists could (and should) ensure that the most modern techniques are applied to all procedures, the VA can aim higher, with the broad evolutionary perspective of “One Health” serving as a guide to new avenues of inquiry.

“One Health” highlights the intellectual and practical benefits of recognizing the linkage between human health and veterinary medicine. To enable a more holistic approach to human, animal, and environmental health, many veterinary colleges have relationships with parallel health colleges. The Association of American Veterinary Medical Colleges (AAVMC) provides leadership and liaison to all veterinary schools accredited by the American Veterinary Medical Association (AVMA) Council on Education, the accrediting body for all colleges and schools of veterinary medicine in the United States. The AAVMC, with its mission to advance the quality of academic veterinary medicine, serves to convene those schools that support improving the quality of research conducted on domestic animals. Additionally, the AAVMC liaises with AVMA’s American Board of Veterinary Specialties, providing a link to the mostly highly trained and credentialed specialists.

Of particular relevance to the VA at present is a formal memorandum of understanding among the cardiology colleges, which engages veterinary experts and non-veterinarians involved in biomedical research related to cardiovascular conditions and diseases shared across multiple species. Thus, the AAVMC is positioned to provide expertise to the VA, should it choose to proactively seek veterinary input to develop and refine studies that may benefit the species of animal under study as well as the primary intended beneficiaries, veterans.

2. Collaborating with researchers conducting clinical trials in companion dogs to identify or develop trials to benefit veterans.

Animals in general, and dogs specifically, often experience disorders similar to those being researched by the VA. As a result, studies aimed at improving the understanding, diagnosis, treatment, and prevention of these disorders in dogs can benefit humans as well. There is a need for a standardization of methods to ensure that clinical trials performed on companion dogs produce high-quality results (Boothe and Slater, 1995; Davies et al., 2017; Dean, 2017; Giuffrida, 2016; Rice et al., 2008). Nonetheless, clinical trials have become a focus of academic veterinary centers internationally (Alvarez and Fortsch, 2005), with an increasing commitment to conducting high quality, multi-center trials. Such trials are subject to ethical and legal regulation to safeguard both owners and patients. The most longstanding and well-organized area of interest is cancer, with NCI supporting the COTC, as described earlier (NCI CCR, n.d.a). Several modes of treatment, especially immunological approaches, have moved forward into human clinical use as a result of such trials (Dow, 2020; Gardner et al., 2016; NCI CCR, n.d.b; Prouteau and Andre, 2019; Tarone et al., 2019). As described in Chapter 3, a consortium to investigate SCIs is also well developed (Moore, 2019; Moore et al., 2017).

As described above, studies of novel approaches to pain relief from chronic disease have yielded promising results, providing evidence that results obtained in companion dogs may reliably predict efficacy in humans (Cimino Brown, 2017; Lascelles et al., 2018). The genetic, environmental, and lifestyle variation seen in companion animals may offer a better model of the human condition—and therefore of much human disease—than the highly controlled genetics and environment that are standard for laboratory animals (Lascelles et al., 2018).

3. Participating in efforts to develop a registry connecting human research needs with companion dog clinical trials.

Biomedical researchers and clinicians need to know the status of clinical trials in their field. Most desirable would be a fully funded registry to help researchers in human disease quickly determine whether a relevant companion dog trial exists or could be developed. AVMA has an online clinical trial registry, albeit one focused on linking pet owners and primary care veterinarians with clinical trials (AVMA, n.d.). To fill in the missing link—that is, connecting biomedical researchers focused on human disease with companion dog clinical trials—perhaps the best route would be through an existing consortium of veterinary schools and medical schools supported by the National Center for Advancing Translational Sciences institute of the National Institutes of Health (NIH). This consortium, the Clinical and Translational Science Award One Health Alliance, currently consists of 15 schools and has received funding to enhance its presence and impact. Its website is designed to educate and link researchers who are interested in such studies (COHA, n.d.).

The committee envisions the VA becoming a pioneer among biomedical research institutions in fostering collaborative work to benefit both veterans and canine patients, thereby addressing many of the current ethical difficulties inherent in using laboratory dogs. It is notable that companion dog studies receive enormous positive public interest and, most importantly, have the potential to accelerate the translation of biomedical research to improvements in human health.

Conclusion 4-1: The use of companion dogs in biomedical research aimed at benefiting both dogs and humans is a preferred alternative to the use of laboratory dogs. Companion dogs experience many of the same naturally occurring diseases as humans and stand to benefit from the results of the research in which they participate. Established areas of clinical companion dog research with relevance to preclinical studies in veterans include cancer and (thoracic) spinal cord injury. Other disorders of interest to the U.S. Department of Veterans Affairs likely to benefit from development of a companion dog model include chronic pain, diabetes, cardiovascular disease, and senile dementia, including Alzheimer’s disease. The utility of companion dogs may increase if other biomedical research areas wherein their use is scientifically valid could be identified and if there is an infrastructure in place to facilitate the conduct of studies that use companion dogs.

Conclusion 4-2: A significant barrier to conducting clinical studies in companion dogs is a lack of administrative infrastructure to connect U.S. Department of Veterans Affairs (VA) investigators to the veterinary researchers who conduct such trials. The regulatory infrastructure to address ethical and legal issues for clinical trials in dogs is already established, but the mechanism for using these studies to supplement, complement, or accelerate collaborations with investigators who are interested in conducting human clinical trials does not exist. With validation of the utility and relevance of the naturally occurring canine disease or disorder for the study of the human equivalent disease or disorder a network for developing a companion animal clinical trials registry could be created. The VA could move forward with supporting new collaborations to establish the relevance of dog studies to humans, both for conditions of likely future interest to the VA (e.g., posttraumatic stress disorder, natural infectious disease, Alzheimer’s disease, and obesity) and for areas currently under study.

OTHER ANIMAL MODELS

The committee explored alternative animal models and non-animal alternatives in those fields where dogs have recently been (or are currently) used at the VA. Accordingly, the committee con-

sidered non-dog animal models currently used or in development for research on CVD, SCI, cancer, and diabetes. The models discussed below represent a small subset of what is available; readers seeking a more comprehensive overview are directed to recent review articles cited in the text.

Cardiovascular Disease

Although animals are used to study a wide array of CVDs, atherosclerosis and arrhythmias present especially critical domains for animal studies, with VA dog research focused on elucidating the mechanisms of heart failure and arrhythmia. Atherosclerosis (fatty deposits within the arteries) is the most common cause of heart failure but does not often present until later in life, despite the fact that atherosclerotic lesions commonly begin to form during the teenage years (PDAY Research Group, 1993). Human studies of the chronologic development of atherosclerosis typically rely on indirect means of assessment (e.g., ultrasound imaging) (Saxena et al., 2019) or a direct assessment of arterial tissue collected during autopsy; thus, no true longitudinal studies are possible in humans. In contrast, animal models have enabled the direct study of the progression of the disease (Daugherty et al., 2017). Over time, atherosclerosis can progress to ischemia and arrhythmia, with arrhythmia often serving as the fatal event for diseases of the heart (Santangeli et al., 2017; Srinivasan and Schilling, 2018). Understanding atherosclerosis and arrhythmia would go a long way toward elucidating many of the heart pathologies that affect veterans.

Rodents

For decades, the majority of experimental studies of atherosclerosis have been conducted in mice. The mouse’s short life span, genetic makeup (including similarities in genes associated with increased risk in humans), ease of breeding, and low cost have made it a favored model (Getz and Reardon, 2012). The tractability of the murine genome is also advantageous; because mice are naturally resistant to atherosclerosis, genetic manipulation of their lipid metabolism is necessary (Meir and Leitersdorf, 2004). ApoE-/- mice, generated in 1992 (Piedrahita et al., 1992), develop total plasma cholesterol levels three- to eight-fold higher than their wild-type counterparts on a regular “chow” diet, with even higher levels on a Western diet (Nakashima et al., 1994; Plump and Breslow, 1995), and they demonstrate advanced atherosclerotic lesions by 8 to 10 months of age (Reddick et al., 1994). Another valuable model for development of atherosclerosis is the low-density lipoprotein (LDL)-receptor-deficient mouse (Ishibashi et al., 1993). Both mouse models have made significant contributions to our understanding of atherosclerosis (reviewed in Getz and Reardon, 2016a,b).

There remain some critical differences between mouse models of atherosclerosis and humans; these include the identity of the primary lipoprotein, the diversity of lipoprotein, and the failure of mice to express cholesteryl ester transfer protein (CETP), a target of interest for reducing cardiovascular risk (Davidson et al., 2009; Kosmas et al., 2016; Tanigawa et al., 2007). Mice differ from humans in the distribution of lesions, the frequency of plaque ruptures, plaque size and composition, biological processes, and mechanics (Schwartz et al., 2007). Efforts are under way to develop a murine model that mimics the plaque instability seen in humans (van der Heiden et al., 2016).

Rabbits

Before the availability of genetically modified mice, New Zealand white rabbits (NZWRs) were the primary model for experimental atherosclerosis research (Shim et al., 2016). Rabbit models helped reveal the significant role of plasma cholesterol in the etiology of atherosclerosis as

well as the importance of differently sized lipoproteins (Nordestgaard and Zilversmit, 1988). Unlike mice, rabbits transport significant fractions of cholesterol via LDL (Fan and Watanabe, 2000; Lee et al., 2017); in this regard, their lipid metabolism resembles that of humans. With regard to their expression of CETP, a possible target for reduction of cardiovascular risk, rabbits resemble humans more closely than do either mice (Lee et al., 2017; Wang et al., 2017) or dogs (Guyard-Dangremont et al., 1998; Tsutsumi et al., 2001). Other advantages of rabbit models include their relatively larger size compared with mice, which affords more tissue for analysis and enables the implantation of stents (Daugherty et al., 2017). Like mice, rabbits do not exhibit spontaneous plaque rupture. However, due to their larger size, rupture can be more readily induced (van der Heiden et al., 2016). Nonetheless, rabbits have key differences from humans, both in the location and composition of their lesions, and the small size of the animals’ arteries limits the ability to use catheter-based or non-invasive imaging.

NZWRs fed a high-fat/high-cholesterol diet are the most commonly used rabbit model in atherosclerosis. In certain genetic models or with the use of mechanical manipulations, lesions will form in coronary arteries (Fan and Watanabe, 2000; Shiomi et al., 2003; van der Heiden et al., 2016). Like mice, NZWR models differ from humans in their primary circulating lipoprotein. The use of NZWRs is further complicated by their low concentration of plasma apoA-II, high levels of which are associated with susceptibility to atherosclerosis (Castellani et al., 2001), as well as the long period of high-cholesterol feeding required to induce atherosclerosis, which results in jaundice and fatty livers (Daugherty et al., 2017). Another popular model, the Watanabe hereditary hypercholesterolemic (WHHL) rabbit, lacks LDL receptors, resulting in increased plasma LDL and the eventual development of atherosclerotic markers in the coronary arteries and aorta (Buja et al., 1983). On an enriched diet, this model develops lesions similar to those observed in human familial hypercholesterolemia (FH) (Atkinson et al., 1989; Phelan et al., 1985). The recently developed ApoE knockout (Ji et al., 2015) and lipoprotein receptor (Ldlr) knockout (Lu et al., 2018) rabbit models will likely prove valuable in future studies of atherosclerosis.

Pigs

Compared to mice and rabbits, pigs share more characteristics with humans with respect to physiology and lipoprotein profile, the location and mechanics of lesion development, and the opportunity for non-invasive measurement of arteries, while providing ample tissue for analysis (Lee et al., 2017). The pig genome is more similar to humans than is the mouse genome (Wernersson et al., 2005). Nonetheless, a number of significant differences remain. For example, pig plasma, like that of mice, has very low levels of CETP activity (Guyard-Dangremont et al., 1998).

When normal pigs are fed a high-cholesterol diet, they develop hypercholesterolemia and atherosclerotic lesions similar to those in humans (Granada et al., 2009). The domestic crossbred farm pig (Sus scrofa domestica) fed such a diet is the most common porcine model for atherosclerosis (Granada et al., 2009). This pig develops increased plasma LDL cholesterol and exhibits simple human-like lesions at 30 weeks (Chatzizisis et al., 2008). More complex lesions resembling human coronary plaques (with calcification and hemorrhaging) can take up to 2 years to develop, during which time the pig reaches a mean body mass of more than 200 kg (Prescott et al., 1991). The unwieldiness and cost of this large pig have hindered its use for research. In 2010 a downsized version (containing a natural mutation in the LDL receptor genes) was adopted for atherosclerosis research. This animal, called the familial hypercholesterolaemia Bretoncelles Meishan pig or “minipig,” when fed a high-cholesterol diet with cholic acid, rapidly develops lesions, either spontaneously or following balloon injury (Thim et al., 2010). The lesions resemble those seen in humans, including some that are vulnerable to rupture (Shim et al., 2016).

Diabetes significantly increases the risk for atherosclerotic disease, and the development of animal models to explore this risk has been a priority. In 2001 DM was superimposed (via injection of a pancreatic cytotoxin) on a domestic pig model fed a hypercholesterolemic (HC) diet. This reduced insulin-producing pancreatic β cells by more than 80 percent (Gerrity et al., 2001). The combination of DM and HC resulted in multiple severe, complex lesions with human-like morphology at 9 months (Gerrity et al., 2001; Mohler et al., 2008; Wilensky et al., 2008; Zhang et al., 2003).

Genetically engineered porcine models represent the newest tools in the domain of experimental atherosclerosis. For example, a porcine model of FH was created by inactivation of the low-density Ldlr gene in Yucatan miniature pigs. Ldlr-/- homozygotes developed severe hypercholesterolemia lesions in coronary arteries and aorta, and the disease severity was increased with a high-fat/high-cholesterol diet (Davis et al., 2014). Another Yucatan miniature pig FH model was created through the introduction of the human proprotein convertase subtilisin/kexin type 9 (PCSK9) gain-of-function (D374Y) mutation (Al-Mashhadi et al., 2013). Neither of these engineered models showed evidence of plaque rupture, but given the rapid evolution of gene-editing techniques, future porcine models may surmount this shortcoming.

Given the VA’s use of dogs to study arrhythmia, the committee was curious about the extent to which pigs have been used to model specific mechanisms associated with this disorder. As noted earlier in this report, while the pig’s coronary anatomy resembles that of humans, certain electrophysiological differences—including the pig’s transmural Purkinje system, which is not present in man or dog—can make the pig a questionable choice for research on acute ischemia. Nonetheless, pigs have been used successfully to model acute ischemia as well as heart failure, heart attack, and resuscitation (Piktel and Wilson, 2019). Pigs have also been used for modeling gene therapy strategies to treat atrial fibrillation (AF) and to reduce the susceptibility to ventricular tachycardia after heart attack (Greener et al., 2012; Liu and Donahue, 2014).

Two recently published reviews compare the various large animal models, including goats and sheep, currently used in arrhythmia translational research (Clauss et al., 2019; Piktel and Wilson, 2019). In a detailed analysis, Clauss et al. (2019) recommend pigs as the primary choice for studying myocardial ischemia and atrial tachycardia and suggest further research to characterize pigs as models for ventricular tachycardia. The authors conclude by suggesting a “practical trio” of three species for most arrhythmia research: mice for initial investigations and genetics, rabbits for electrophysiology and validation of initial findings, and pigs for translation and preclinical testing.

Spinal Cord Injury

Considerable investment in research on SCI has advanced the fundamental understanding of the cellular mechanisms guiding injury and recovery as well as the response to therapeutic interventions on both cellular and whole-animal levels. Nonetheless, for research aimed at the recovery of sensory and motor function, there has been limited translation of successful results from animals to humans (Floyd, 2019; Moore, 2019). Various laboratory animal models have been employed, and one human trial is recruiting participants as of the writing of this report (NLM, 2019).

Rodents

Both rats and mice have been vital for understanding the fundamental cellular mechanisms and systemic responses involved in recovery from investigator-induced trauma (Cheriyan et al., 2014). These studies enable the standardization of injury as well as a detailed and invasive measurement of recovery. Acute or partial SCI that impairs breathing can be investigated in rodents, but there are limits. Long-term studies are possible for lower (thoracic and lumbar) SCI, but studies of cervical SCI are limited by the difficulty (and ethics) of maintaining these animals on long-term

ventilator support (Alilain, 2019). Additionally, unlike injuries induced in the laboratory, the precise causes and manifestations of SCI in humans are of course diverse. Likewise, human genetics is more diverse than that of inbred rodents. Due to these and other constraints, the translation of SCI treatments directly from rodents to humans, in the absence of a large-animal intermediate, has been rare (Floyd, 2019; Guest, 2019; Jeffery, 2019). Nonetheless, the use of theophylline to stimulate respiration can be attributed to rodent studies (Alilain, 2019), Schwann cell transplantation has moved from rodent studies to human trials (NLM, 2019), and there is hope for other pharmacologic or genetic approaches.

Pigs

The pig spinal cord is similar to that of humans in size and anatomy, making the pig an attractive model for testing surgical and other interventions (Guest, 2019). As with rodents and all other laboratory animals, long-term maintenance that requires ventilator support is not feasible. This, in concert with the continual growth of the pig, renders pigs of little use for studying long-term recovery from respiratory compromise. Nonetheless, as described in Chapter 3, the Yucatan minipig is proving to be a useful large-animal bridge from rodent to human studies and an alternative to laboratory dogs. One example is the transplantation of Schwann cells to facilitate spinal cord regeneration, which was shown to be successful first in rodents and then in pigs (Santamaria et al., 2018), prior to the initiation of human clinical trials.

Cancer

Rodents

The mouse is one of the most commonly used small animal cancer models. As just one example of a mouse that was genetically engineered to closely resemble human disease progression, the KPC pancreatic cancer mouse model was introduced in 2005 (Hingorani et al., 2005). Compared with other mouse models, KPC mice possess more clinical and histopathological features akin to humans, making it the gold standard murine model for preclinical pancreatic cancer research (Lee et al., 2016). There are dozens of genetically engineered mouse models of melanoma; the majority contain oncogenic mutations in the pathways most likely to be altered in human disease (RAS/RAF/MEK/ERK and phosphoinositide-3 kinase), while others carry mutations in genes regulating cell cycle progression (reviewed in Perez-Guijarro et al., 2017). Mouse models have made significant contributions to the understanding of the molecular basis of melanoma formation. They are also being used in the discovery of biomarkers for both diagnosis and response and in the preclinical testing of drug efficacy as well as to investigate the mechanisms of drug resistance. Importantly, genetically altered mice currently represent the only preclinical platform for the development of melanoma immunomodulatory therapies (Day et al., 2015).

Rats are also commonly used as a preclinical cancer model; their larger size makes them better suited for studies that require imaging or surgery. Rats are frequently the model of choice for studies of colon and bone cancers (Rubio, 2017; Zhang et al., 2019). Rodent models of cancer have limitations, however. Because humans live up to 50 times longer than mice and are 3,000 times larger, with proportionately more cells, humans undergo 10⁵ more cell divisions in a lifetime than mice (Rangarajan and Weinberg, 2003), potentially resulting in fundamentally different risk profiles (Tomasetti and Vogelstein, 2015). Many rat models do not mimic certain human cancer pathologies (Szpirer, 2010).

Rabbits

Rabbits are also used as cancer models; however, their use is often limited to a single type of cancer and has important limitations. For example, the rabbit VX2 model developed by Rous and Beard (1935) (Kidd and Rous, 1940) is used to study hepatocellular carcinoma (Ko et al., 2001; Parvinian et al., 2014). Tumors are induced in the liver by injecting virally infected VX2 carcinoma cells. The tumors exhibit a high rate of spontaneous necrosis, confounding studies of treatment response (Parvinian et al., 2014), although modified lines with greater viability have been developed (Pascale et al., 2012). Rabbit models have also been used in studies of metastatic colorectal cancer (Prieto et al., 2017), oral cancers (Chen and Lin, 2010), skin cancers (Breitburd et al., 1997), and breast cancer (Zhang et al., 2017).

Pigs

In general, cancer cell biology in pigs and dogs is more analogous to that in humans than are small animal models, while the larger animals’ size and anatomy make them well-suited for interventional studies, including device testing and surgical practice (Flisikowska et al., 2013; Gardner et al., 2016).

Pigs offer a large-animal alternative to laboratory dog models, with outbred populations displaying genetic diversity akin to human populations (Schachtschneider et al., 2017a), thereby sidestepping the inbreeding critique leveled at domestic dogs. Pigs’ many similarities to humans (in terms of cancer pathophysiology as well as size, anatomy, genetics, and epigenetics) as well as their low cost compared to primates (Groenen et al., 2012; Schook et al., 2005; Swindle et al., 2012) make them attractive subjects. Pigs age at three to five times the rate of humans but can live up to 10 years; this enables the relatively rapid detection of disease while allowing researchers to monitor tumor progression over long time periods (Flisikowska et al., 2013; Watson et al., 2016). Anatomical similarities with humans facilitate the use of imaging technologies (positron emission tomography, computed tomography, magnetic resonance imaging) developed for humans (Sieren et al., 2014). Pig anatomy allows drugs to be administered via the same routes used in humans, and pig blood can be drawn in sufficient size and frequency to enable pharmacokinetic analysis. Pig cytochrome P450 enzymes metabolize four of the six most common probe substrates with activities similar to those of human cytochrome P450, thereby supporting the use of pigs for a range of drug metabolism studies as well (Schelstraete et al., 2019). Comorbidities common in human disease (e.g., nonalcoholic steatohepatitis, alcoholic cirrhosis) can also be induced in pigs (Gaba et al., 2018; Lee et al., 2009).

Genetically engineered pig cancer models exhibit disease courses similar to those observed in humans. The Oncopig cancer model (OCM) is a notable recent example (Schook et al., 2015). The OCM can be made to develop site-specific tumors via the induced expression of KRAS^G12D and TP53^R167H transgenes, an oncogene and a tumor suppressor, respectively, both of which are commonly observed in human cancers (Schook et al., 2015). Schachtschneider and colleagues (2017a) outlined a number of unmet clinical needs that OCM research could potentially fill. For example, in the quest to improve early detection of cancers, the OCM offers an ideal platform to investigate the prognostic value of candidate cancer biomarkers in liquid biopsies. Thanks to similarities between OCM and human metabolic pathways, the OCM is also a strong candidate for exploring drug dynamics, kinetics, and toxicity.

The OCM has been used to model a variety of both hematologic and solid tumor cancers, including soft tissue sarcoma, or STS (Diaz et al., 2016; Schachtschneider et al., 2017a,b; Schook et al., 2015). An OCM STS model was used to test the efficacy of a real-time, image-guided technique to precisely place catheters in tumors for thermal ablation (Schachtschneider et al., 2017a). A

porcine osteosarcoma model is being studied to obtain mechanistic insights into human bone cancer (Saalfrank et al., 2016), and models for pancreatic and other cancers, currently under development, may provide tools that lead to earlier diagnosis as well as new surgical interventions (Bailey and Carlson, 2019; Diaz et al., 2016; Kalla et al., 2020; Leuchs et al., 2012; Schachtschneider et al., 2017a). Pig size and anatomy also make the pig an attractive model for liver cancer. OCM hepatocarcinoma cell lines recapitulate human hepatocellular carcinoma (HCC) and may prove useful for developing immunotherapy. The antitumor immune response in the OCM includes both innate and adaptive recognition of induced tumors as well as tumor-induced suppression of T-cell effector functions, all of which are relevant to the human condition (Overgaard et al., 2018).

Given the VA’s interest in melanoma, the committee reviewed pig models for this most deadly form of skin cancer. Hereditary metastatic melanoma is modeled by three strains of miniature pig, most recently the melanoma-bearing Libechof minipig (MeLiM) (Bourneuf, 2017). While the MeLiM tumors display histological, immunohistochemical, and hematological similarity to human melanomas, they also show significant differences, including their non-UV-dependent origin and a higher rate of spontaneous regression (although a higher rate of melanoma progression is observed in MeLim than in the two other strains) (Horak et al., 2019). The availability of the OCM, with its potential to generate a variety of cancer types (including melanoma) through targeted mutation, opens up new possibilities for using pigs to model melanoma at all stages of the disease, from tumorigenesis to pathogenesis to therapeutics (Cuoto et al., 2019).

Non-Mammalian Models

In addition to the mammalian models mentioned above, it should be noted that non-mammalian species, including fruit flies and a variety of fish, play significant roles in the elucidation of genetic pathways associated with tumor development (Cagan et al., 2019). Recombinant zebrafish (Danio rerio) are used to study the genetic basis of melanoma initiation and progression and are also used in the validation of targeted treatments (Cuoto et al., 2019; Fernandez Del Ama et al., 2016). Nonetheless, zebrafish have limited utility for phenotypic modeling of human cancers (Schachtschneider et al., 2017a).

Diabetes

While laboratory dogs are no longer used in large numbers to study diabetes (and published research is more likely to use companion dogs with naturally acquired disease), they continue to be employed in targeted studies that rely on their physiology and similarity to particular human disease states.

Rodents

Rodents have been the primary species for experimental diabetes research for many years (King and Bowe, 2016; Rees and Alcolado, 2005). The non-obese diabetic (NOD) mouse and the biobreeding diabetes-prone rat have been favored (Yang and Santamaria, 2006) for type 1 diabetes, while the Lep^ob/ob mouse (deficient in leptin), and the Lep^db/db mouse and Zucker Diabetic Fatty rat (both deficient in leptin receptor) are commonly used in the study of type 2 diabetes (King, 2012). Many factors that make rodents attractive models for cardiovascular disease research, such as their low cost and rapid reproduction, also apply in the context of diabetes. Mouse models have provided valuable insights into diabetic disease mechanisms and treatment by, for example, identifying signaling pathways and genetic factors that can lead to type 1 diabetes (Driver et al., 2012; Wallis et al., 2009) and testing type 2 diabetes therapies (Gault et al., 2011; Park et al., 2011).

Pigs

Pigs share many anatomic, metabolic, and pathophysiologic traits with humans that render them useful for studying the complex factors involved in the initiation and progression of diabetes. Adult domestic pigs are used on occasion (e.g., for testing medical devices scaled for adult humans and when large blood volumes are needed). However, minipigs are generally preferred for their smaller size. The domestic pig’s beta-cell-mass-to-body-mass ratio—considered a good parameter for interspecific comparisons—shows a trajectory over the lifetime of the animal that closely resembles the ratio in rats (Bock et al., 2003; Montanya et al., 2000), and minipigs have been used to study the processes leading to beta-cell dysfunction (Larsen, 2009; Larsen et al., 2005). Obesity is reliably induced in pigs through diet, and genetic engineering has produced strains of pigs tailored to particular applications in diabetes and dyslipidemia research (Kleinert et al., 2018).

A variety of approaches have been used to create tailored pig strains (Cho et al., 2018; Kleinert et al., 2018; Phelps et al., 2003; Renner et al., 2010; Wang et al., 2015; Yum et al., 2016). Inbreeding can produce genetically standardized models; outbreeding can produce populations of pigs with genetic variation akin to that observed in human populations. Type 2 diabetes has been modeled in pigs by the introduction of a glucose-dependent insulinotropic polypeptide (GIP) receptor mutation that suppresses insulin secretion in response to food uptake and results in reduced glucose tolerance, reduced insulin secretion, and decreased pancreatic beta-cell mass (Renner et al., 2010). This model was used to test a promising drug, liraglutide, which compensates for the GIP deficiency by increasing downstream signaling (Renner et al., 2016; Streckel et al., 2015). Drug studies in pigs produced more consistent results than studies performed in rodents, and the pharmacokinetics in pigs more closely resembled that of humans (Renner et al., 2016; Tamura et al., 2015).

Islet transplantation is considered a potentially useful therapy for type 1 diabetes; however, shortages of human donor organs preclude widespread adoption of the protocol. Xenotransplanation has been put forth as a possible solution and is a subject of active research with recognized risks (Denner et al., 2016; FDA, 2016; Spizzo et al., 2016). There is currently much interest in developing genetically engineered pigs from which islets could be harvested, which would be done shortly after birth to reduce the risk of zoonotic infection (Cooper and Ayares, 2011). Neonatal pig islets offer additional advantages, including reduced immunogenicity, increased in vitro stability, and the tendency to proliferate better after isolation (Vanderschelden et al., 2019).

Summary

While animal studies have made notable contributions to understanding CVD pathways and uncovering therapeutic targets (Daugherty et al., 2017; Getz and Reardon, 2012; Nishida et al., 2010), all models have limitations, making the choice of animal dependent on the precise question being asked. For example, while plaque calcification (a significant component of coronary artery disease) is common in humans, it is minimal in mice (Otsuka et al., 2014). Dogs are generally resistant to atherosclerosis, although it can be induced in the laboratory with a special diet (Moghadasian et al., 2001). In contrast, dogs arguably display the strongest physiological similarity to humans for a particular, relatively small suite of cardiovascular research needs related to arrhythmia (Nishida et al., 2010).

Despite the variety of approaches currently available for studying SCI, there is no laboratory animal model that mimics the diversity of injuries and the diversity of the human population. Long-term recovery from SCI that compromises respiration cannot be studied in animals due to the practical and ethical concerns regarding the maintenance of an immobilized, sedated, or paralyzed laboratory animal for long periods. This type of SCI is nonetheless common in humans, and its impact is devastating. Indeed, the suffering that human survivors of cervical SCI must endure is too

distressing to be considered humane for laboratory animals. Advancing these studies will require an accelerated effort to conduct human trials, even pilot studies, for interventions that cannot be tested on large animals in the laboratory.

Laboratory dogs have largely been supplanted by other animals, primarily mice, for the study of cancer. The same holds true for diabetes, although dogs remain the most well established model for the quantification of liver glucose uptake, which cannot be measured directly in humans or rodents (Kleinert et al., 2018). Recent developments enabling targeted gene editing in pigs show promise for improved translatability to the human disease state.

Conclusion 4-3: With respect to other animal models, rats and mice are the predominant species used for biomedical research in the fields of cardiovascular disease, spinal cord injury, cancer, and diabetes. For studies that cannot be performed in rodents (due to constraints of size, anatomy, or physiology), the pig has become the large animal translational model of choice. While pigs are not tractable for all areas, their potential uses are likely to expand in the near future as genetically modified strains become more widely available.

NON-ANIMAL MODELS: NEW APPROACH METHODOLOGIES

The transition from the exclusive use of animal models to an increasing reliance on NAMs requires first establishing confidence that NAMs can adequately address the scientific questions being posed. In this context, while NAMs are not yet able to recapitulate the complexity of whole animal systems, some are now capable of interrogating specific aspects of biology that are confined to a limited physiological space (at the molecular, cellular, or organ level) and that take place in well-defined contexts of use.

Advances in the cultivation and differentiation of human embryonic and induced pluripotent stem cells are providing ready access to one of the most important tools known to biomedical research, enabling tissue-, disease-, and even patient-specific modeling using a range of in vitro systems. Concurrently, the fields of three-dimensional (bio) printing and biomedical engineering are providing advanced scaffolds (i.e., micro physiological systems) to support cellular systems with physiological complexity never before observed outside the human body. The vast amount of biological information being generated by these and other innovative technologies has in turn fueled the development of new computational tools using the power of machine learning/artificial intelligence and in silico modeling. In addition to in vitro and computational approaches, significant advances are also being made in the ability to preserve and use organs and tissues from human donors. Although none of these technologies can currently “replace” the dog, they do offer the potential to provide more human-relevant mechanistic insights and may therefore warrant consideration as valuable resources for VA research.

Here the committee provides a few examples of existing or emerging technologies that could potentially be used to address biomedical research needs identified by the VA in the areas of CVD, SCI, and cancer. These examples are not intended to be comprehensive, but rather to highlight some of the remarkable advances being made in fields as complex as in vitro biology, computational modeling, and ex vivo use of human organs, with relevance to VA clinical research interests. Several of the NAMs currently under development may be capable of addressing certain clinical research needs of the VA, but their evaluation and adoption will require a coordinated effort from both NAM developers and VA researchers.

Cardiovascular Disease

There is a growing arsenal of non-animal tools and approaches with potential applicability to the study of cardiovascular function, disease, and treatment. A subset of these tools are described here, with a focus on areas of VA research relevant to this report: AF, premature ventricular contractions, cardiac contractility, and cardiomyopathy.

Stem Cells

Human pluripotent stem cell–derived cardiomyocytes have revolutionized the field of cardiovascular research. Methods for differentiating human induced pluripotent stem cells (hiPSCs) into beating cardiomyocytes (hiPSC-CMs) have been standardized and commercialized, providing the research community with wide access to these tools. However, most hiPSC-CMs used in research remain predominantly in an immature state with regards to their physiological structure and function, demonstrating fetal gene expression as well as morphological, metabolic, and contractile characteristics that differ from those of adult cardiomyocytes (Machiraju and Greenway, 2019). As a result, disease modeling using hiPSC-CMs may be of limited value if the cells are not adequately characterized with respect to their electrophysiology, contractility, kinetics, etc. Fortunately, rapid progress toward more physiologically relevant systems continues to be made, with, for example, recent success in the maturation of hiPSC-CMs using a diverse array of approaches (Machiraju and Greenway, 2019).

Researchers have used human embryonic stem cells (hESCs) to generate confluent sheets of atrial-like cardiomyocytes (hESCs-CMs) with spontaneous pacemakers, allowing for the induction of abnormal rhythms (i.e., simulated AF) using chemicals or hormones with pacemaking restored to normal rhythm after the stimulus is removed (Laksman, 2019; Laksman et al., 2017). Voltage-sensitive dyes make it possible to map out the electrical signals as they propagate through the tissue in a spiral pattern. Using this system, researchers have demonstrated that antiarrhythmic drugs modulate the rotor activation patterns in a manner consistent with their known efficacy in treating and preventing AF.

Organoids and Microphysiological Systems

The ability to direct the differentiation of hiPSCs into specific tissues has been foundational to the development of organoids, which are three-dimensional (3D) structures of self-organizing, tissue-specific cells that can recapitulate the physiological functions of human tissues far better than two-dimensional (2D) cultures of the same cells (Mills et al., 2017; Park et al., 2019b). The construction of organoids that use iPSC-CMs from human patients with known genotypes and phenotypes of clinical interest has enabled the development of more physiologically relevant in vitro tools for modeling a spectrum of conditions, from disease pathophysiology to drug screening (Mills et al., 2017; Nugraha et al., 2018). A further extension of this approach has been the development of engineered 3D microphysiological systems (tissue or organ chips) that support the integration of multiple cell types into an organ-like configuration, with the goal of obtaining more organ-like functions. Researchers have developed numerous 3D working models of human heart tissue (Feric et al., 2019; Guyette et al., 2015; Kitsara et al., 2019; Noor et al., 2019), including an intact scale model of a human ventricle (MacQueen et al., 2018), which offer the potential to study heart disease as well as drug safety and efficacy in patient-derived cells.

Computational Models

Physiologic computer models of the human heart are proving to be an effective and efficient alternative to animal-based experimentation in understanding and predicting cardiac adverse events during drug development (Passini et al., 2019) and have been used to develop virtual in silico drug trials incorporating variability in response seen across an entire population of patients (Britton et al., 2013; Passini et al., 2017). Physiologic computer models are also being developed to enable patient-specific diagnosis and treatment of cardiovascular disease. As noted in a recent review (Gray and Pathmanathan, 2018, p. 82):

Stem Cells and In Silico Modeling for Regulatory Use

The Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative was established to update the paradigm for assessing the proarrhythmic risk of pharmaceuticals, with broad applicability to other sectors (Blinova et al., 2018; Colatsky et al., 2016; Park et al., 2019a). This project seeks to develop a standardized in silico version of a human ventricular cell in which to evaluate the risk of cardiac toxicity. The evaluation of proarrhythmic risk is based on an electrophysiologic understanding of proarrhythmia with two primary components: (1) in vitro drug effects on multiple cardiac channels plus in silico reconstruction of cardiac action potential, and (2) confirmation using human stem cell–derived cardiomyocytes.

The U.S. Food and Drug Administration’s (FDA’s) Division of Applied Regulatory Science has a goal of moving new methods, including alternatives to animals, into the drug review process (FDA, 2019). This includes the development of in vitro cellular microsystems for the early stages of drug testing, with cardiac contractility as one example of a functional endpoint that can predict drug effects (Ribeiro, 2019; Ribeiro et al., 2015). These systems use micropatterning of hiPSC-CMs on engineered material that enables the contractility of the muscle fibers to be used as a physiologically relevant endpoint of cellular function downstream of an action potential.

Human Heart Tissue

Although laboratory animals provide a convenient and consistent medium for research into mammalian biology, the historical difficulty of translating these findings into (human) clinical practice is well documented and universally acknowledged. When considering approaches to improve translational impact and efficiency, the use of intact human tissues (in situ or ex vivo) offers perhaps some of the best opportunities. The use of human donor hearts not suitable for transplantation and those from patients with end-stage heart failure receiving donor hearts offers an extremely valuable translational platform for advancing research on human heart function and disease (Efimov, 2019; Gloschat et al., 2016). The use of human hearts for biomedical research,

as with all human tissues, will become even more impactful as processes for their procurement, distribution, and storage continue to improve.

The physiology and cellular integrity of human hearts maintained ex vivo deteriorates rapidly once the heart is removed from the host. However, thin slices of freshly harvested heart tissue offer a promising model of intermediate structural/mechanical complexity with the innate 3D tissue architecture and extracellular matrix preserved (Kang et al., 2016). This allows intact myocardium to be used for a wide range of research purposes, from the visualization of 3D collagen distribution and micro/macrovascular networks to probing the effects of novel conductive biomaterials on cardiac physiology and testing pharmacological safety and efficacy (Watson et al., 2019a,b). Thin slices preserve normal electrophysiology, enabling the testing of pharmacological interventions, gene therapy, cellular therapies, and medical devices, among others (Kang et al., 2016). Although they are much shorter-lived than slices, larger (wedge) preparations of intact heart tissue have also proved useful. Wedges of human ventricles were used to optimize thermal ablation techniques for creating lesions to block reentrant wavefronts, thus mitigating the tachycardia that can lead to ventricular fibrillation and sudden cardiac death (Sulkin et al., 2018).

Using image-based analysis and multicellular mathematical modeling of electrical activation, Stephenson et al. (2018) showed the 3D disposition of the cardiac conduction system for the first time in an intact human heart with congenital atrioventricular septal defects. The advance raises new possibilities for understanding arrhythmogenesis and ablation strategies by studying the congenitally malformed heart. A high-throughput left ventricular myocardial slice model has been developed that is physiologically stable for at least 3 days, enabling the investigation of ion signaling and myofiber contraction at scale (Thomas et al., 2016). More recently, a medium-throughput methodology retained physiological functionality in both pig and human heart slices for up to 6 days (Ou et al., 2019). The heart slice model has been further refined using electromechanical stimulation to prevent the onset of myocardial dedifferentiation that begins to occur when the tissue slices are placed in culture (Watson et al., 2019a), bringing researchers another important step closer to recapitulating the complexity of the in situ human heart.

Spinal Cord Injury

Bioprinting

The complexity of the central nervous system presents a significant challenge to the construction of non-animal systems for studying SCI. However, advances in 3D bioprinting are beginning to overcome these constraints, enabling the study of neuroregeneration in a context that is faithful to the anatomy and biochemistry of the human condition (Joung et al., 2020). Bioengineered scaffolds are being developed for implantation into the site of injury and for stimulation of central nervous system regeneration (Koffler et al., 2019); they also offer broad research applications (Joung et al., 2020). Spinal cord spheroids, bioprinted into 3D hydrogel, can be mass produced for use in the preclinical safety and efficacy screening of pharmacological interventions (Bowser and Moore, 2020).

Microphysiological Systems

Even more complex than regeneration, the locomotion circuit requires multiple cell types organized in a precise arrangement and capable of acting in a coordinated fashion. No non-animal model has yet succeeded in replicating this motor circuit. However, the combination of compartmentalized

microfluidic culture with 3D culture techniques and the use of hiPSCs may ultimately produce a model capable of mimicking human neuromuscular disease in vitro (Badiola-Mateos et al., 2018).

Organotypic Modeling with Ex Vivo Tissue

Recent studies have obtained physiologically relevant data using ex vivo spinal cords from rodents. Adult mouse spinal cords were used to identify populations of axons recruited by spinal cord stimulation, a method employed to reduce chronic pain in the clinic (Idlett et al., 2019). In a study by Pandamooz et al. (2019), slices of adult rat spinal cord were maintained in culture for 1 week prior to being subjected to mechanical damage simulating an SCI. Treatment of damaged slices with valproic acid, a drug that shows potential for promoting SCI recovery in the clinic, reduced expression of the inflammatory cytokine TNF-a and increased expression of the neurotrophic factor BDNF (Pandamooz et al., 2019), supporting the potential utility of slice models for studies of pathophysiology and drug screening.

Computational Modeling

Computational models are being developed to study discrete aspects of SCI, such as pressure ulcer formation (Ziraldo et al., 2015) and cough stimulation (Pitts et al., 2016). It should be noted that the development of these models involved validation against biological data, which was collected in humans with SCI for the first study and in cats for the second.

Cancer

Microphysiological Systems and Organ Chips

Organ chips have been used to model multiple steps in the cancer cascade, from tumor growth and angiogenesis to the epithelial–mesenchymal transition, invasion, and metastasis. Organ chips enable the growth of multiple tissue types on layers of extracellular matrix seeded on a flexible, see-through substrate under conditions of constant perfusion, with a medium or blood flowing through an endothelium-lined vasculature (Sontheimer-Phelps et al., 2019). Partitions within the device enable researchers to study cell migration and cell–cell communication, including trans-endothelial migration of immune cells. Organ chips have enabled the recapitulation of complex drug-resistance profiles seen in vivo, which do not find expression in more simplified in vitro systems (Sontheimer-Phelps et al., 2019).

Organ chips and related biomimetic systems have begun to make significant contributions to the study of melanoma. In a 2D model, metastatic melanomas demonstrated directed migration when co-cultured with epithelium or fibroblasts from different organs, providing an avenue for interrogating the factors responsible for organ tropism (Zhang et al., 2015). Melanoma cell growth and morphology are strongly dependent on the mechanical properties of the substrate (Prauzner-Bechcicki et al., 2015). One 3D melanoma model recapitulates a stratified epidermis, human extracellular matrix, and blood and lymphatic capillaries; this model has shown promise for testing new anticancer compounds in vitro (Bourland et al., 2018). In time it should be possible to model metastatic spread by combining multiple fluidically linked organ chips to create human “body-on-chip” models (McAleer et al., 2019; Sontheimer-Phelps et al., 2019).

In comparing organ chips to animal models, it is worth noting that as cancer treatment becomes increasingly specific—for example, with the use of therapeutic monoclonal antibodies—the ability to evaluate therapies in non-human species becomes more challenging (Sontheimer-Phelps et al.,

2019). Multi-organ models built on organ chips offer a possible way forward. NIH is contributing to this effort on several fronts, including NCI’s Cancer Tissue Engineering Collaborative Research Program as well as a grant instrument supporting research to test the utility of incorporating “tissue chip” models into clinical trial design (HHS, 2019; NCI, 2019).

Organ chip technology is still young, relatively expensive, and not as well characterized as other 3D culture systems. The successful incorporation of organ chip systems into research will therefore require dedicated and innovative efforts to establish confidence in these systems. As new commercial sources become available, however, their technical robustness is expected to increase and their costs to decrease (Sontheimer-Phelps et al., 2019).

Computational Modeling

In the realm of cancer, a major aim of computational modeling is to improve the success of drug trials by streamlining the process of translation (McKenna et al., 2018). Researchers are pursuing this goal through multiple avenues. For example, a 2018 study developed simulations of nanoparticle flow through blood vessels in order to help drug designers predict the optimal particle shape for drug delivery; real-world experiments validated these predictions (Shah et al., 2018).

Patient-to-patient variability in drug response presents a critical challenge for cancer treatment. In one case a model of breast cancer, personalized with details from histopathology, imaging, and molecular profiling, showed success in simulating the responses of individual tumors to a 12-week treatment regimen (Lai et al., 2019). In another, high-resolution metabolic models of colorectal cancer cells, constructed using an RNA sequence dataset, were able to detect patterns of metabolic rewiring in the individual cancers and thereby identify three potential new drugs for colorectal cancer (Pacheco et al., 2019). The optimal approach to drug discovery for cancer will likely include a combination of computational modeling and biological testing (Nagaraj et al., 2018).

Incorporation of New Approach Methodologies at the VA

The Animal Welfare Act and associated U.S. Public Health Service (PHS) policies set forth federal requirements designed to ensure that investigators in the United States have appropriately considered alternatives to procedures that can cause more than slight or momentary pain or distress in animals.² Additional guidance for VA researchers and institutional animal care and use committees (IACUCs) on the evaluation of non-animal alternatives is provided in the Veterans Health Administration handbook 1200.07, Use of Animals in Research, which requires researchers to perform one or more database searches for alternatives and indicate if any of the animal procedures can be replaced by computer models or in vitro techniques (VHA, 2011; Appendix D section 1.v.). Although the intent of this requirement is laudable, the fact that NAMs cannot (and are not intended to) replace the physiological complexity of intact animals leaves ample opportunity to exclude their consideration if so desired.

Indeed, a review of the animal component of research protocol (ACORP) forms associated with the 14 protocols for VA biomedical research in laboratory dogs that were active as of June 1, 2017, revealed that most database searches appeared to have been conducted using narrow definitions, as opposed to casting a broad net capable of revealing all possible alternative approaches to addressing the question. A protocol review process that requires the principal investigators and IACUCs to explore broader contexts for using NAMs would serve to encourage the advancement and uptake of new promising technologies.

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² U.S. Code 7, Chapter 54: Transportation, sale, and handling of certain animals; CFR Title 9, Subchapter A: Animal welfare. Both available at https://www.nal.usda.gov/awic/animal-welfare-act (accessed December 30, 2019).

The effective incorporation of NAMs into any given area of biomedical research will require VA investigators accustomed to animal-based research to consider reframing the scientific question, as opposed to evaluating NAMs purely based on their ability to fully replace a laboratory animal. Some questions that are currently studied in dogs and other large animals may be partially addressable with less complex systems, such as NAMs that could offer more detailed interrogation of mechanistic information germane to the research question, and even when large animal use cannot be eliminated entirely, the use of NAMS could reduce the numbers of animals required for translation. However, the appropriate evaluation and use of NAMs will necessitate collaborations among diverse research communities (in vivo, in vitro, in silico), which are not always accustomed to such interactions. Overcoming cultural and historical barriers to changing the way research is done could pose a barrier to NAM adoption if it is not sufficiently incentivized by the VA (ICCVAM, 2017).

Conclusion 4-4: While the scientific and institutional animal care and use committee review processes at the U.S. Department of Veterans Affairs adhere to all relevant policies established by the U.S. government (as described in Chapters 2 and 5), compliance with these standards on its own may not be sufficient to ensure adequate identification and consideration of new approach methodologies (NAMs). Even in the case of protocols that still require the use of laboratory animals, researchers need to be encouraged to evaluate and incorporate NAMs where feasible.

Summary of New Approach Methodologies

U.S. federal agencies are placing an increased emphasis on ensuring that researchers adequately consider alternatives to animal use (GAO, 2019). Although none of the NAMs highlighted above can serve as an immediate or complete replacement for animals, they do enable researchers to interrogate aspects of human biology that cannot be addressed in dogs or other animals and thereby offer the potential to enhance the translational impact of VA research. In cases where the current research is focused on discrete mechanisms, NAMs can address the ethical imperative to reduce the number of animals used and may eventually replace the current dog model for conducting mechanistic studies.

However, the incorporation of NAMs into any animal-based research program will not be successful without a dedicated and comprehensive effort to do so. To facilitate their adoption, institutions need to identify and address institutional and cultural practices that may impede the evaluation and adoption of NAMs. In this context, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) published A Strategic Roadmap for Establishing New Approaches to Evaluate the Safety of Chemicals and Medical Products in the United States (2017), which describes the processes necessary for developing, establishing confidence in, and effectively incorporating the use of NAMs to replace or complement existing animal-based test methods. ICCVAM member agencies such as FDA and the U.S. Environmental Protection Agency have subsequently incorporated these principles into strategic plans tailored to address the particular needs of each agency and its stakeholders. The VA now has an opportunity to establish itself as a leader in expediting the incorporation of human-relevant technologies that could enhance the translational relevance of VA-funded research (shortening the time from bench to bedside) while reducing the reliance on laboratory animals and, most importantly, could improve and protect the health of veterans.

HUMAN CLINICAL TRIALS

During the time frame that this National Academies of Sciences, Engineering, and Medicine committee was charged with reviewing VA biomedical research in laboratory dogs, one protocol that had originally proposed to use laboratory dogs, which was designed to study the ability of targeted nanoporphyrin to potentiate immunotherapy for bladder cancer, was changed to a human clinical trial. The committee did not request details from the VA regarding the precise reasoning or justification for the decision to move from laboratory dogs to human trials in this instance. The committee notes the importance of always considering human clinical trials as an alternative to laboratory dogs and providing explicit justification for why human clinical trials are inappropriate to ethically meet the study objectives.

The VA ACORP forms reviewed by the committee include a number of opportunities and areas for an explanation of animal model selection but do not require a justification for excluding human clinical trials from consideration. Perhaps this justification for excluding humans appears on other research review and approval forms the committee did not see. In any case, the committee believes that human clinical trials need to be fully considered and that reasons for excluding humans need to be explained in supporting research documentation.

Recommendation 5: Establish long-term external collaborations to optimize the use of companion dogs and humans in biomedical research.

The U.S. Department of Veterans Affairs should prioritize the development and continuation of external multi-disciplinary collaborations to develop, validate, and apply alternatives to the laboratory dog in biomedical research. This effort should result in the following:

• Increased collaborations with external scientists and use of public–private partnerships to promote cross-sector communication and cooperation.
• The fostering of collaborations with researchers conducting clinical trials in companion dogs to identify or develop trials to benefit veterans and dogs.
• The encouragement of the use of human organs and tissues from human organ banks whenever possible.

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