A PATHWAY FOR ADOPTION OF NEW APPROACH METHODOLOGIES

Translational relevance¹ is the anchor for any nonclinical model system, animal or otherwise, that is intended to help understand, prevent, or treat a human condition. The principles that define translational relevance include replication of human physiology and pathophysiology (organ responses or disease state/pathogenesis), genetics, epigenetics, and/or other aspects of the biology of human cells. New approach methodologies are expected to meet translational expectations if they are to be broadly adopted to decrease reliance on NHPs in biomedical research. Attaining sufficient legitimacy based on its performance (track record) is key to successful adoption of a new approach methodology by scientists and organizations (Eckert et al., 2020; Marx et al., 2020; van der Zalm et al., 2022). Once a new approach methodology has demonstrated that it is equivalent or superior to other models for predicting or explaining human conditions or outcomes, it may be adopted as a model of choice (FDA, 2022b). Ultimately, the performance of a technology or approach determines whether it can be used to answer a specific research question. Although performance uncertainty is inherent in all new technologies and approaches, resource investments that can reduce that uncertainty are required to accelerate their adoption. This requirement is framed in the sections that follow as mapping out a pathway to adoption for a new technology or approach.

Key Terminology

Important terminology used to describe the components of a pathway to adoption for promising new approach methodologies include

• context of use,
• qualification,
• validation, and
• benchmarking.

These terms are defined here to support and provide guidance for those seeking to determine the status and credibility of an emerging technology or innovative approach for an intended use. More detailed discussion of these terms can be found elsewhere (Fabre et al., 2020; FDA, 2017; FDA and NIH, 2016; Patterson et al., 2021; Steger-Hartmann and Raschke, 2020; Tadenev and Burgess, 2019).

Context of Use

Context of use (COU) defines the manner and purpose of use for a technology or approach (how and when it will be used) (Baran et al., 2022; FDA, 2017, 2020). This term can generally be applied for any intended use of a methodology. COU elements include

• what is measured and in what form, and
• the purpose of the technology or approach in the testing of hypotheses or decision making/action.

The term “fit for purpose” is occasionally used synonymously with COU. However, a fit for purpose statement is meant to communicate that the intended use of a technology or approach is supported by validation/qualification information (FDA and NIH, 2016).

Qualification

Qualification is the process of confirming that a methodology is capable of yielding reproducible results that are suitable for the intended purpose based on the defined COU (Kennett, 2010). A technology or approach that is qualified for a specific COU can be relied upon to have a specific interpretation and application (FDA, 2017, 2020; FDA and NIH, 2016). The pathway for qualification of a new approach methodology is depicted in Figure 4-1, which shows that specifying the COU is a first step and defines the needs for the subsequent steps (Parish et al., 2020).

FIGURE 4-1

Pathway for qualification of the performance of a new approach methodology for a specific context of use.

Validation

Validation is the process by which the reliability and relevance of a technology or approach is established for a defined purpose using specific criteria (OECD, 2005). The process includes assessments of the methodology’s sensitivity, specificity, precision, robustness, reproducibility, and stability. Formal validation can involve intensive and costly processes that may not be necessary for all uses of a new approach methodology (FDA, 2017; ICCVAM, 2018); expectations may vary depending on the particular technology or approach.

Qualification versus Validation

Qualification and validation of a methodology denote the means used to demonstrate its suitability for its intended purpose (FDA, 2017; FDA and NIH, 2016). One way to describe the difference between the two terms is that they differ as to the extent and robustness of the parameters evaluated and the number of replicates performed for each. The extent to which a technology or approach needs to be qualified or validated depends on the decisions to be made with the data it generates, including the interpretation of the data and subsequent actions to be taken for a given outcome. For example, are the data intended for regulatory use, or to advance understanding and guide subsequent investigations? COU and assay qualification are intended to help decrease barriers to adoption by highlighting data that confirm a level of validation appropriate for the intended use of the methodology (FDA, 2017).

Benchmarking

Benchmarking entails rigorous comparison of the performance of different technologies or approaches to determine the strengths of each or to provide recommendations regarding their suitability for the purpose at hand (Weber et al., 2019). Benchmarking has several benefits, including increasing awareness of emerging technologies and approaches (NIH, 2023) and understanding how the performance of a new approach methodology compares with that of in vivo approaches. Benchmarking is also appropriate for comparing new approach methodologies relative to the same or different intended uses for a model, allowing for identification of the most appropriate technologies and approaches for specific COUs (Mangul et al., 2019; Wu et al., 2023).

NEW APPROACH METHODOLOGIES WITH THE POTENTIAL TO COMPLEMENT OR REDUCE RELIANCE ON NHP MODELS IN BIOMEDICAL RESEARCH

This section describes new approach methodologies with the potential to complement or reduce reliance on NHP models in biomedical research. These examples are intended to be illustrative and do not represent a comprehensive cataloging of all possible new approach methodologies. Although some of these technologies and approaches have shown promise in specific applications, those applications cannot necessarily be generalized to other COUs. Rather, each COU will pose specific requirements that must be addressed when any technology or approach is intended to complement or decrease reliance on NHP models. No data set can support a summary of the validation state of new approach methodologies as a whole.

As discussed in Chapter 1, the committee did not include animal models within its definition of new approach methodologies, but with the advancement of genome editing technologies and cell/tissue transplant approaches, it should be acknowledged that engineered animal models also have the potential to complement and reduce reliance on NHP models in biomedical research.

In Vitro Cell Culture Models

The sections below provide examples of in vitro assays as well as two-dimensional (2D) and three-dimensional (3D) human cell culture models with great potential to complement NHP research and, in some cases, reduce reliance on NHPs.

In Vitro Immunoassays

In vitro immunoassays that use cultured human cells, such as assays that predict the risk of cytokine release syndrome,² provide a clear example of how in vitro models can address a question related to human safety (Finco et al., 2014). Cytokine release syndrome is an acute systemic inflammatory syndrome that may be triggered by biologic therapeutics, such as monoclonal antibodies (Eastwood et al., 2010), including those used to treat COVID-19. There are several validated methods for assessing the potential risk that immune-modulating drugs or other treatments will trigger exaggerated levels of cytokine release in vivo (Vessillier et al., 2020). Use of in vitro cytokine release assays can aid in the prediction of human physiological responses (Joubert et al., 2016), thus reducing reliance on NHPs for this purpose.

2D and 3D Stem Cell–Based Cell Culture Models

Cell culture models may use immortalized cell lines or primary cells isolated from animals, and both sources have been used to create in vitro models for biomedical research. Given that the use of NHPs in research is driven mainly by their translational relevance to humans, this section focuses primarily on stem cell–based 2D and 3D cellular models with completely human genomes. These 2D and 3D models offer opportunities for translational relevance and may be used in specific contexts to complement or reduce reliance on NHP models, particularly if they are qualified for use and can be relied upon for reproducible results of high quality. While the committee distinguishes between 2D and 3D cell culture models for the descriptions found in this section, appropriate spatial organization and replication of organ functions are important features for both.

The discovery of human induced pluripotent stem cells (iPSCs) in 2006 by Dr. Shinya Yamanaka (2012) transformed cell culture systems and their use to answer critical human health questions. These cells are generated by introducing the Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc) into skin fibroblasts or peripheral blood mononuclear cells, resulting in cells that can be differentiated into various cell types (Lam and Wu, 2021). Under specific differentiation conditions, most tissue/organ cell types can now be reliably generated from human iPSCs. Whereas iPSCs have the potential to generate all cell types in a human body, adult human tissue stem cells (ATSCs) have a more limited differentiation capacity (Chang et al., 2019). These tractable and renewable cell systems contain the complete human genome and can be used to study human gene functions, biochemistry, physiology, and molecular mechanisms. Both iPSCs and ATSCs can be derived from individuals of various genetic and ethnic backgrounds, offering unique advantages in understanding genetic and molecular mechanisms governing human cell biology and physiology. Moreover, iPSCs and ATSCs can be generated from patients, providing unprecedented opportunities for revealing disease-related phenotypes; understanding pathology; identifying disease-specific biomarkers; and screening for potential treatments, including personalized treatment strategies (Chang et al., 2019; Moradi et al., 2019; Son et al., 2017).

2D cell cultures are the most simplified cellular model systems. They are composed of a single or a few cell types and lack the molecular and cellular complexity and interactions of human organs under physiological conditions (Jensen and Tang, 2020). In contrast, in vitro systems that contain multiple cell types representing cells from various organs are used in attempts to recapitulate human organs and are being connected to represent multiple organs. However, since an organ is a functional entity with a need for specific structural context, formally defining an in vitro system as an organ requires that functionality and structure be recapitulated to some extent. Multiple cell types have been placed together in elegantly engineered platforms to form “organ-like” structures, and a few have recapitulated functionalities and structural context to some extent (Hofer and Lutolf, 2021; Huh et al., 2011). For example, with the application of tissue-patterning factors and self-organization capabilities, human tissue–specific 3D organoids can be generated from iPSCs (as depicted in Figure 4-2), as well as some ATSCs (Artegiani and Clevers, 2018). Organoids may be phenotypically similar to human tissues/organs in cell-type composition; architecture; and, to a certain degree, function. In general, ATSC-derived organoids specialize in mimicking the physiology of functionally more mature organs, whereas iPSC-derived organoids are useful for modeling organogenesis in a developmental context (Schutgens and Clevers, 2020).

FIGURE 4-2

Generation and differentiation of induced pluripotent stem cells. SOURCE: Image reproduced with permission from Ashutosh Agarwal and created with BioRender.com.

While recreating a whole organ or human body (which contains 78 organs) in vitro is appreciated as a futuristic goal, in the past decade human organoids with endoderm, mesoderm, and ectoderm origins have been generated that represent complex organs, including brain, liver, lung, airway, kidney, intestine, and sensory organs, among others (Artegiani and Clevers, 2018; Ho et al., 2018; Schutgens and Clevers, 2020). Compared with 2D cell culture models, human organoids are in general more complex and, in many cases, more physiologically relevant because they preserve or reconstitute the cell-type diversity of the organs in vivo. In some cases, they also maintain similarity to human organs at the molecular, cellular, and functional levels (Artegiani and Clevers, 2018; Schutgens and Clevers, 2020). Certain cells, such as milk-producing mammary gland cells, function as in vivo counterparts only when cultured under 3D conditions (Barcellos-Hoff et al., 1989). Patterned, self-assembled 3D organoids contain major cell types from the same lineage; cell types originating from different lineages, such as mesenchymal cells, vasculature cells, or immune cells, are usually not present (Ho et al., 2018; Qian et al., 2019).

Advances in 3D culture technology, microfluidics,³ and bioengineering have led to the generation of more complex microphysiological systems (MPS). MPS are in vitro platforms composed of 3D constructs (including spheroids, organoids, bioprinted tissue constructs, and tissue/organs-on-chips) of human or animal origin that mimic the biochemical, electrical, and/or mechanical properties of organ or tissue function. The design of MPS platforms allows researchers to control the composition of the tissue and its architecture using cellular and extracellular cues—molecular, structural, and mechanical—found within the human organ system (Hargrove-Grimes et al., 2021). Vasculature or immune cells, for example, can be cocultured with various types of organoids (Sun et al., 2022; Trujillo and Muotri, 2018; Yu, 2021). More complex models can be created by fusing different types of organoids generated separately to create assembloids that reconstitute the interaction of multiple tissues (Kanton and Paşca, 2022; Paşca, 2019; Paşca et al., 2022). For example, brain, spinal cord, and muscle organoids have been assembled together to model the motor control from brain to muscle (Andersen et al., 2020). Through the integration of organoids and microfluidic chips, MPS enable the engineering of a more physiologically relevant in vitro model and permit closer control over cell–cell and cell–matrix interactions, biomechanical cues, nutrient delivery, and waste removal (Huh et al., 2011; Ingber, 2022). Box 4-1 describes biomedical applications for tissue-specific organoids and MPS.

BOX 4-1

BIOMEDICAL APPLICATIONS OF TISSUE-SPECIFIC ORGANOIDS AND MPS.

The real power of MPS technology lies in the capacity of singular organ chips to reveal not only drug effects or biological response on primary human organs but also secondary effects stemming from organ–organ interactions. Therefore, multiorgan MPS are being designed to be as physiologically realistic as possible by connecting individual organ chips in a modular fashion (Ronaldson et al., 2022; Sung et al., 2019); factoring in functional organ scaling in the human body; and replicating those scaling parameters, such as organ volumes, volumetric perfusion rates, and fluid-to-cell ratios (Malik et al., 2021). While multiorgan MPS with as many as 10 organ-specific components have been generated as proof of concept (Novak et al., 2020), MPS models being used in biomedical research are generally less complex. For example, since liver is the primary site for human metabolism, several “liver–other tissue” (e.g., liver–gut, liver–heart, liver–skin, liver–kidney, liver–muscle, liver–bone marrow–tumor tissue) MPS platforms have been developed to study potential side effects of both the parent compound and its metabolites on multiple downstream organs (Chang et al., 2017; Edington et al., 2018; Maschmeyer et al., 2015; McAleer et al., 2019; Sung et al., 2010; Tsamandouras et al., 2017; Vernetti et al., 2017; Wagner et al., 2013). Outside of the drug development space, multiorgan MPS are also being used to better understand human physiology and disease mechanisms. For example, a multiorgan MPS with liver, gut, and cerebral components has been used to gain insight into pathogenic mechanisms for Parkinson’s disease (Trapecar et al., 2021). Another notable example of a complex multiorgan MPS is the EVATAR system, which includes separate organ modules for the ovary, fallopian tube, uterus, cervix, and liver within a microfluidic system to simulate the in vivo female reproductive tract. The system includes endocrine loops with a sustained circulating flow between all tissues and has demonstrated that the human female menstrual cycle can be modeled in vitro (Xiao et al., 2017).

Several scientifically rich hurdles remain. Finding an in vitro blood substitute that could perfuse all organ chips and regulation of the blood surrogate volume to mimic physiological levels are critical to obtain precise concentrations of drug/hormone/chemical metabolites in the blood surrogate volume (Malik et al., 2021; Herland et al., 2020). It is also important to build organ-specific endothelial barriers so that nutrients, oxygen, drugs, and other solutes must circulate through a common vascular conduit (Herland et al., 2020; Ronaldson et al., 2022). Ideally, each chip also requires on-chip analytical technologies so that effective functional readouts (in addition to viability and morphological analyses) can be carried out to assess the cellular responses to a specific challenge (Jalili-Firoozinezhad et al., 2019; Kujala et al., 2016; Odijk et al., 2015; van der Helm et al., 2019). If these technical challenges can be addressed, multiorgan MPS technology is poised to offer useful, cost-effective guidance in the early determination of drug toxicity and efficacy, response to hormones and endogenous metabolites, and modeling of human disease. Ultimately, the goal of multiorgan MPS efforts is not to build a perfect replicate of the human body but to provide a predictive model that is superior to animal models, including NHPs.

Cultured Tissue Slice Models

Cultured human tissue explants,⁴ such as precision-cut-tissue slices (PCTS), offer some advantages over the 3D stem cell–based MPS models described above in that the natural architecture of the human tissue/organ is retained, all cell types found in the tissue are present and in their original tissue–matrix configuration, and tissue- or organ-specific functions (e.g., metabolic and some aspects of immunologic activity) are preserved to a certain extent (Liu et al., 2019; Majorova et al., 2021). While tissue slices have been used in biomedical research for decades, technological advances over the years that have enabled precision cutting of slices and extended cell viability during culture have increased the utility of this model system for answering specific research questions (Alsafadi et al., 2020). PCTS models can be generated from healthy or diseased human tissues (e.g., liver, kidney, brain, lung), enabling their use in understanding fundamental human anatomy and physiology, as well as disease modeling. Their ability to closely recapitulate in vivo conditions and enable the study of interactions at the molecular, cellular, and extracellular levels has made PCTS useful human-relevant models for research related to drug discovery, toxicology, and host–pathogen interactions, among other purposes (Alsafadi et al., 2020; Majorova et al., 2021). However, finite culture viability and limited accessibility of qualified human tissue constrain the scalability of this approach.

Examples Demonstrating Potential to Complement or Reduce Reliance on NHP Models

This section provides examples of the application of 2D and 3D human cellular models in research areas in which NHPs are commonly used. These examples include research aimed at generating fundamental knowledge regarding normal human biology and translational research focused on understanding disease mechanisms, identifying potential therapeutic targets, and evaluating drug and vaccine candidates. Note that these examples are intended to be illustrative and do not represent a comprehensive cataloging of ways in which these models have complemented NHP research or reduced reliance on NHPs. Note also that, while the examples focus primarily on human cell culture models, animal cell–based models have a role in informing the development of and building confidence in human cell–based MPS, as well as in assisting in the interpretation of existing animal model data and refining the use of future animal models (NASEM, 2021).

As detailed in Chapter 2, NHPs are widely used for research on infectious diseases such as COVID-19, Zika, and malaria to understand pathogen tropism (i.e., the types of cells the pathogen infects), immune responses, and pathological outcomes, as well as for vaccine and drug testing (Doritchamou et al., 2017; Galinski, 2022; Haese et al., 2021; Liang et al., 2021; Miner and Diamond, 2017; Osuna and Whitney, 2017; Rutkai et al., 2022). With rapid advances in human stem cell research, many 2D and 3D human cell culture systems have been applied to address similar questions.

The COVID-19 pandemic in particular spurred collaborative efforts to develop and apply human cellular models to address the urgent need to understand SARS-CoV-2 pathogenesis and to develop effective therapeutic and prophylactic products (Kleinstreuer and Holmes, 2021). In the past 3 years, many studies have reported on the use of such models to elucidate cellular tropism, to study immune responses to SARS-CoV-2 and the pathology in different tissue and organs, and to facilitate drug development for COVID-19 (Busquet et al., 2020; Clevers, 2020; Monteil et al., 2020). Examples include cardiac cells recapitulating patient cardiac cytopathic features (Perez-Bermejo et al., 2021); examining immune responses in lung lineage cells (Huang et al., 2020; Youk et al., 2020); demonstrating infection and viral replication within the intestinal epithelium using intestinal organoids (Lamers et al., 2020); and studying neurotropism identified in brain organoids (Jacob et al., 2020; Samudyata et al., 2022; Wang et al., 2021). Si and colleagues (2021) demonstrated the utility of an MPS model (human airway chip) for screening antiviral therapeutics for SARS-CoV-2 and the potential to accelerate the identification of drugs previously approved for other indications that have repurposing potential.

In research on Zika virus, human brain organoids were used to suggest the causality of infection and microcephaly (Qian et al., 2016). Human iPSC–derived 2D neural cultures and 3D brain organoids were used to identify the neurotropism and pathogenesis of Zika virus infection (Qian et al., 2016; Tang et al., 2016) and to screen protective treatments (Xu et al., 2016). The Centers for Disease Control and Prevention used human stem cell–based in vitro findings, along with evidence from animal models and clinical and epidemiological evidence in humans, to conclude that Zika virus causes microcephaly (Rasmussen et al., 2016). Importantly, neural progenitor cells were found to be one of the main cellular targets in vitro (Tang et al., 2016), a finding that has been replicated in many animal models, including NHP models.

Human brain organoids are also being applied to study human immunodeficiency virus (HIV) infection of the central nervous system, including neuropathology and the establishment of viral reservoirs (dos Reis et al., 2020; Premeaux et al., 2021). Understanding of latent reservoirs is important to the development of effective curative strategies, which have remained elusive despite successes in the development of HIV therapeutics, as discussed in Chapter 2 (see Box 2-3). Findings from such in vitro human model systems can be used to better inform the design of experiments in NHP models for questions that cannot be addressed in vitro.

The complementarity of in vitro and NHP models has also been a critical component of the study of neurological disorders. The brain is the most evolutionarily advanced organ and is difficult to model in vitro because of its complex circuitry and ongoing development in response to experience. Compared with other conventional animal models, NHPs exhibit greater similarity to humans in brain structure and complexity, and as discussed in Chapter 2, research using NHP models therefore provides valuable insights into brain function and neurological disorders such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and stroke, many of which remain untreatable. To date, many iPSC lines have been established from patients with neuropsychiatric (Levy and Pasca, 2023; Zhang et al., 2023) and neurodegenerative disorders. Brain cells or organoids have been generated from these lines to examine the molecular and cellular changes potentially linked to disease (Mertens et al., 2018) (see also Box 4-2) and to enable “initial rapid, high-throughput screens (genetic or pharmacological) to define and characterize putative targets” (Outeiro et al., 2021, p. 835).

BOX 4-2

POTENTIAL OF HUMAN CELLULAR MODELS TO YIELD INSIGHTS INTO THE PATHOGENESIS AND TREATMENT OF ALZHEIMER’S DISEASE.

While brain organoids are enabling improved understanding of neurodevelopmental processes and diseases (Andrews and Kriegstein, 2022), these models currently have limitedapplicability to the study of experience-related refinement of neural circuitry (e.g., changes in response to visual cues or other postnatal stimuli) and neurological conditions that are emergent from the activity of neural networks (Andrews and Kriegstein, 2022; Jalink and Caiazzo, 2021; Trujillo and Muotri, 2018). Brain organoids cannot yet recapitulate the full complexity of the spatial organization of a human brain, have limited cellular diversity and connectivity between different brain regions (Arlotta and Gage, 2023), and demonstrate several shortcomings with respect to studying the development of neurodegenerative disorders that develop over long periods of time (Jalink and Caiazzo, 2021; Kim et al., 2021; Qian et al., 2019; Shou et al., 2020; Sun et al., 2021; Wang, 2018). Animal models, including NHPs, are needed to test mechanistic hypotheses for the development of typical symptoms and to test therapeutic strategies at the organism level (Outeiro et al., 2021). From these studies, it is clear that certain pathologic features are conserved in both in vitro and NHP models, which thus complement each other.

Given their similarities to humans, NHPs are important models in the area of reproductive biology and have contributed to improved understanding of endometriosis, as discussed in Chapter 2. Recently, complex, multicellular endometrial organoids have been developed that provide an in vitro model for the study of endometrial physiology (e.g., molecular and cellular regulation of dynamic cyclic tissue remodeling) and pathology (e.g., endometriosis), highlighting the opportunities for organoid models to contribute to both basic and translational research (Song and Fazleabas, 2021).

NHP research plays an important role in the development of safe and effective treatments that are translatable to humans. Given the relatively low cost and high throughput of in vitro culture systems, initial drug screening using disease-relevant human cellular models may significantly reduce the number of NHPs required for drug development research by advancing only promising candidates to NHP studies (Prior et al., 2017; SCHEER, 2017). The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative, for example, which began following a 2013 workshop hosted by the Food and Drug Administration (FDA) (CiPA Initiative, 2019), resulted in validation of the use of human iPSC–derived cardiomyocytes (hiPSC-CM) to determine the risk of drugs inducing torsade des pointes tachyarrhythmias in a clinical setting (Blinova et al., 2018). The use of iPSC-derived cardiac myocytes in the CiPA initiative is one example, specifically in toxicology, of a successful approach to clearly demonstrating the utility of a 2D hiPSC-CM system for detecting drug-induced proarrhythmic effects and reducing the use of animals, including NHPs, in such research (Pang et al., 2019). In another example, Kerns and colleagues (2021) showed that a MPS could recapitulate antibody-mediated lung toxicities observed in cynomolgus macaques, demonstrating the value of the model for evaluating translation of animal findings to human cellular models, and raising the potential for a reduction in reliance on NHP-based safety assessments.

Other Applications of In Vitro Cell Culture Models

As emphasized earlier in this chapter, the usefulness of new approach methodologies extends beyond opportunities to reduce reliance on NHPs; 2D and 3D cell culture models have many valuable applications, particularly when good animal models for human disease are lacking. For example, iPSCs derived from patients suffering from familial cancers can be used to identify the mechanisms underlying the onset of pediatric cancer and to detect some familial mutations, making it possible for these cells to serve as a valuable prognostic tool (Marin Navarro et al., 2018). Disease causality for several genetic polymorphisms in long QT syndrome and aortic valve disease were demonstrated by combining the use of cardiomyocytes differentiated from iPSCs with multiomics technologies (Evans et al., 2022). Similarly, causality was established between a specific gene mutation and disease-associated phenotype using cortical neurons differentiated from iPSCs from patients with major mental disorders and genome editing (Wen et al., 2014).

A notable area of application for in vitro cell culture models is personalized medicine. In the past decade, innovative 3D culture approaches have resulted in successful derivation and cultivation of tumor organoids, including colon, breast, liver, lung, pancreas, ovary, bladder, esophagus, and brain cancer organoids. These tumor organoids have shown some similarity in drug responses to the responses of patients, making them a valuable tool for studying personalized disease progression and therapies (Veninga and Voest, 2021). In a recent example, Schuster and colleagues (2020) developed a microfluidic platform to study pancreatic tumor organoids. Using this system, hundreds of patient-derived cultures were treated with combinations of drug treatments, and an automated, high-throughput analysis of the microfluidic 3D organoids enabled the identification of potentially effective treatments for individual patients, demonstrating the potential of these culture systems to support personalized precision medicine. Importantly, the results showed significant differences in drug response among different patients, a feature that cannot be studied using NHPs. In another example, human rectal organoids from cystic fibrosis patients were used to predict drug response, enabling the identification of responders to CFTR-modulating drug therapy even for those patients with very rare genetic mutations in the CFTR gene (Dekkers et al., 2016).

Advantages and Limitations of In Vitro Models

Significant advances have been made in deriving and culturing human cells, tissues, and organs from stem cells to study basic biology and certain disease-related cellular pathologies. For example, the molecular mechanisms underlying cortical layer formation were delineated using brain organoids involving Wnt signaling, which is disrupted by a psychiatric disorder–associated genetic mutation in brain organoids derived from patient iPSCs (Qian et al., 2020). In another example, human iPSC–derived neural cells were used to identify the fundamental molecular and cellular mechanisms underlying human neoteny (Iwata et al., 2023; Linker et al., 2022).

The ability to derive ATSCs and iPSCs from patients, combined with targeted differentiation into disease-relevant cell types, enables use of these cellular models to reveal disease-related phenotypes, understand pathology, and identify potential biomarkers. The more complex cell models, such as organoids and MPS, allow better understanding of disease pathology and mechanisms using relevant human cell and tissue types, as well as physiologic niches important for disease development and progression, especially in hereditary diseases. ATSCs and iPSCs also are amenable to CRISPR/Cas-based genetic editing, thus making it possible to understand the functional contribution of specific genes involved in normal function or under diseased conditions, and to establish a causal relationship between genotype and phenotype (Evans et al., 2022; Wen et al., 2014).

However, not all human cell types can be isolated or generated from ATSCs or iPSCs, and the integrity of the cells (e.g., low rate of genotype discordance between parent cell and iPSC cell DNA, genomic structural integrity, and transcriptomic similarity between the derived cell type and the cells that reside in tissues in vivo) must be confirmed before the cells can be used reliably (Assou et al., 2018; Kammers et al., 2017; Kanchan et al., 2020). As with all models, predictivity and validation principles must be established. For translational purposes, qualification and a well-defined COU for these model systems are essential (Hargrove-Grimes et al., 2021, 2022; Ingber, 2022; Low et al., 2021) and can significantly expand the types of critical human health–related questions they can be used to explore.

While organoids and MPS have successfully mimicked many aspects of the complex physiology of human organs, 3D culture systems in their current state cannot replicate all of the complex interactions that occur in vivo (Low et al., 2021; Marx et al., 2020), nor can they be used to study processes that require systemic regulation. Therefore, for human health and safety questions that require this level of complexity, these in vitro systems cannot replace NHPs. The use of in vitro systems to study surgical interventions is limited as well (Ruspi et al., 2019). Furthermore, certain outcomes, such as behavior, can be studied only in the full context that is present in vivo. However, results from studies using in vitro models can inform improvements in the design of experiments using NHPs to reduce reliance on these animal models, such as with drug screening, as discussed earlier in this chapter.

REGULATORY GUIDANCE ON TECHNOLOGIES AND APPROACHES TO COMPLEMENT OR REDUCE RELIANCE ON NHP MODELS

The National Institutes of Health (NIH) has supported a few programmatic efforts with the expressed intent of enabling investigational new drug applications (INDs) (see, for example, the Bridging Interventional Development Gaps program supported by the National Center for Advancing Translational Sciences [NIH, 2022]). These efforts represent a very small percentage of the total resource allocation of the NIH institutes and centers, and do not necessarily involve research conducted with NHPs. With this in mind, this section provides some relevant information about the regulatory landscape for new approach methodologies. Notably, the regulatory views included in this section are not relevant to the use of NHPs in basic disease research and are focused on informing human safety.

In general practice, NHPs are used to answer questions about human safety when it is scientifically demonstrated and clear that NHPs are the most relevant species for human translation, and no other approach is appropriate for the purpose of the study (SCHEER, 2017). Scientific demonstration “may include metabolic, pharmacokinetic, and pharmacologic similarities to humans as well as sensitivity to particular types of toxicity, among other factors” (FDA, 2022c, p.3). The committee found evidence that the FDA and other regulatory agencies are supportive of new approach methodologies and have communicated strategies that are being considered to put this acceptance into practice (FDA, 2021a, 2022a). To this end, the FDA formed an Alternative Methods Working Group⁵ focused on the advancement of both new and existing models to support regulatory decision making in toxicology. The working group also facilitates interactions with global regulatory bodies that are considering the application of alternative methodologies in toxicology (FDA, 2022a). This effort and others are focused on ensuring that the data generated will be comparable and of the highest quality, and that studies using these models and the data generated from them will be robust, reproducible, relevant, and fit for purpose. In response to the 21st Century Cures Act of 2016,⁶ the FDA also launched its Innovative Science and Technology Approaches for New Drugs (ISTAND) pilot program, which is designed to support the development and, in some cases, qualification of novel approaches to drug development—including MPS and AI/ML-based algorithms—for use in regulatory decision making (FDA, 2021c). All of these efforts have culminated in the FDA Modernization Act 2.0,⁷ enacted in December 2022, which amended the Federal Food, Drug, and Cosmetic Act to explicitly permit the use of new approach methodologies, including in vitro and in silico methods, as alternatives to in vivo nonclinical testing. Taken together with the efforts already implemented at the FDA, this new law encourages the application of new approach methodologies and documents policy efforts to facilitate their use as they are qualified and/or validated.

New FDA guidance with direct relevance to the reliance on NHPs for conducting nonclinical toxicity assessments is included in Nonclinical Considerations for Mitigating Nonhuman Primate Supply Constraints Arising from the COVID-19 Pandemic (FDA, 2022c). The FDA provided this guidance to address challenges related to the limited NHP supply resulting from the pandemic. The guidance states: “FDA supports the principles of the 3Rs, to reduce, refine, and replace animal use in testing when feasible. We encourage sponsors to consult with us if they wish to use a nonanimal testing method they believe is suitable, adequate, validated, and feasible. We will consider if such an alternative method is adequate to meet the regulatory need.” Although this guidance was prompted by the COVID-19 pandemic, the core issue was the reduction in the already limited NHP supply available for nonclinical toxicity assessments, together with the simultaneous increase in demand for NHPs for evaluating candidate COVID-19 treatments and vaccines. In response, the FDA acknowledged the restricted availability of NHPs for other pharmaceutical development programs and potential delays in the development of new medications for diseases that currently lack any effective treatment options. Specifically, this guidance discourages “the use of NHPs for the general toxicology assessment of small molecule drugs unless the sponsor can provide a scientifically compelling reason why NHPs must be used.” The guidance also states:

• “If the biological product is active in other nonrodent species, the sponsor should conduct any warranted general toxicity studies in a nonrodent species other than the NHP, whenever scientifically justified.”
• “On a case-by-case basis, if the biological product is active in a rodent and acts on a well-characterized target (e.g., vascular endothelial growth factor, or its receptor), it may be scientifically appropriate for sponsors to conduct warranted general toxicity studies only in the rodent.”
• “Sponsors should not use sexually mature NHPs in toxicity studies designed specifically to assess fertility by histopathological examination when fertility parameters can be assessed in rodents.”
• “Consistent with current FDA guidance on the assessment of developmental and reproductive toxicity (DART), FDA considers NHPs to be a nonroutine test species for the DART assessment of small molecule drugs. FDA strongly discourages sponsors from using NHPs for assessing DART endpoints for their small molecule drug development programs.”
• “Consistent with current FDA guidance, sponsors should only use NHPs for the DART assessment of biological products if they are the only relevant species. For the duration of the COVID-19-related disruption in the supply of NHPs, sponsors should also consider the following when planning approaches to address DART for biological products that are pharmacologically inactive in non-primates: […] while the supply of NHPs is disrupted, we strongly encourage the use of appropriate alternative models for assessing DART endpoints (e.g., species-specific surrogates in rodents, genetically modified rodents) when scientifically justified.” (FDA, 2022c).

In general, this guidance provides many approaches that can be considered to reduce reliance on NHPs even after the end of the COVID-19 public health crisis.

Another relevant FDA source is Advancing Regulatory Science at FDA: Focus Areas of Regulatory Science (FDA, 2021b). The section of this document on “Novel Technologies to Improve Predictivity of Non-clinical Studies and Replace, Reduce, and Refine Reliance on Animal Testing” (p. 31) describes the FDA’s intention “to replace, reduce, and refine (the 3 Rs) dependence on animal studies by advancing development of, and evaluating new, fit-for-purpose nonclinical tools, standards, and approaches that may someday improve predictability.” In the document, it is recognized that in silico modeling, such as using “available information in computational science approaches to predict safety issues, can be used to supplement and may potentially replace some risk analyses that are currently based on animal data.” Of note, the document also expresses that these advances will depend on the development of performance criteria for the realistic assessment of the potential use of these new tools for safety and efficacy testing, and the conduct of “large multilaboratory studies to assess the reliability, sensitivity, specificity, and reproducibility of in vitro alternatives to in vivo assays (e.g., to assess the potency of certain vaccines).”

NEED FOR COLLABORATION BETWEEN NHP RESEARCHERS AND THOSE DEVELOPING AND USING NEW APPROACH METHODOLOGIES

The establishment of collaborations between developers of new approach methodologies and those who currently use NHPs may reduce barriers to the adoption of new approach methodologies (ICCVAM, 2018). These collaborations might entail training NHP researchers to implement new technologies and approaches in their own laboratories or creating a partnership whereby research with different model systems would be conducted in different laboratories. Effective collaborations involving multiple laboratories with expertise in different model systems are facilitated by supportive funding mechanisms. For example, the Collaborative Research in Computational Neuroscience program—a joint program of the National Science Foundation and NIH—supports collaborative research projects in theoretical and experimental neuroscience that involve multiple investigators using both animal models (including NHPs) and computational models (Flanders, 2022). Coordination within NIH—for example, between programs supporting NHP research and those developing MPS for Alzheimer’s disease research—could facilitate the pursuit of promising research goals in this space. Additionally, the inclusion of experts in new approach methodologies in the review process for NIH research proposals involving NHP models (including review of the consideration of alternatives in the vertebrate animals section of the proposal) could help identify opportunities for collaborative research that might complement the proposed NHP study or reduce the need for NHPs.

At present, interactions between research groups developing and using new approach methodologies and those using NHP models are limited. Such interactions could help raise awareness among NHP researchers regarding the evolving capabilities of in vitro and in silico systems, and could educate investigators developing new approach methodologies on the needs and priorities of NHP researchers, thereby enabling improvements in the design of in vitro systems such that they would be better suited to answering research questions for which NHPs are currently used. For example, this approach could be used to define COUs to ensure that systems would be designed to be fit for purpose. Accordingly, there is an urgent need for mechanisms that can facilitate such direct interaction and collaboration, including

• multilaboratory funding opportunities, such as those supported by the Collaborative Research in Computational Neuroscience program;
• challenge programs that encourage cross-sector collaborations, such as that supporting the application of data from experimental dog studies to the development of a virtual “second species” for toxicology studies;
• cross-training programs; and
• conferences, symposiums, and other events designed to provide opportunities for interaction between those investigators with expertise in the evolving capabilities of new approach methodologies and those using animal models, and to catalyze collaborations.

CONCLUSIONS

In its examination of new approach methodologies, the committee identified numerous ways in which in vitro and in silico models are being used to complement NHP research, but few concrete examples of a demonstrated role for those models in reducing reliance on NHPs. While references to replacement of NHPs are aspirational at this time, the committee envisions the potential for new approach methodologies to reduce reliance on NHPs in the future, especially as technology and science continue to advance, and as investments are made in the development of fit-for-purpose model systems and in their validation and qualification. In the absence of validation, enthusiasm for new technologies and approaches must be tempered to avoid overpromising their capabilities as valid replacements for necessary and proven experimental systems. Based on its evaluation of the research and development status of new approach methodologies, the committee reached the following conclusions:

Conclusion 4-1: Based on the current state of the science, there are no alternative approaches that can replace nonhuman primate (NHP) models to answer research questions that require complete multiorgan interactions and integrated biology. Thus, NHPs continue to be essential for the conduct of National Institutes of Health–supported biomedical research.

Conclusion 4-2: Select new approach methodologies (in vitro and in silico models) can replicate certain complex cellular interactions and functions. As such, these new approach methodologies may be used to answer specific research questions that contribute to understanding human biology to prevent and treat human disease. Although there currently exist no alternatives that can fully replace nonhuman primates, it is reasonable to be optimistic that this may change in the years ahead as new approach methodologies continue to advance.

Conclusion 4-3: Furthering the adoption of new approach methodologies (including in vitro and in silico model systems and approaches) with the intent of reducing reliance on nonhuman primate models will require planning and support for studies that can demonstrate adequate performance for specific contexts of use or intended purposes. Expectations for qualification or validation of new approach methodologies depend on the decisions to be made using the data derived from their use and the potential human health consequences of those decisions.

Conclusion 4-4: While nonhuman primates have been regarded as preeminent models for the evaluation of human safety and efficacy, recent guidance demonstrates that the Food and Drug Administration and other regulatory agencies are supportive of the use of new technologies and approaches for regulatory decision making once they have been adequately qualified or validated.

Conclusion 4-5: Efforts to reduce reliance on nonhuman primates (NHPs) in biomedical research will require investment in opportunities to facilitate direct interaction and collaborative research among investigators using NHP models and those developing in vitro and in silico approaches to expand the applicability of new approach methodologies to research questions for which NHPs are currently needed. At present, however, few mechanisms for fostering such interaction and collaborative research are available.

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