Animal models are established, important tools for preclinical safety and efficacy testing. Companies are advancing more “humanized” models to better reflect human responses, while at the same time focusing on responsible and ethical use of these models. This latter task aligns with the animal welfare principles known as the “3Rs”: reduction, replacement, and refinement. An alternative set of principles, the “4Rs,” adds another “R”: responsibility. Whichever set of “Rs” is applied, the idea is to solve ethical problems in functional animal experimentation.
Meanwhile, the FDA Modernization Act 2.0 challenges the entrenchment of in vivo animal models in preclinical work and supports development of alternative models. And yet another piece of legislation, the Biosecure Act, if passed, will significantly impact the sources U.S. researchers use to obtain their animal models.
As animal models become increasingly disease specific, alternative models—organ-chip and microphysiological systems—emerge alongside them, finding “best fit” applications. For example, advanced alternative models are being introduced that resemble functional organs and tissue.
Embracing the future
Committed to the 4Rs, Charles River Laboratories explores operational and commercial innovations to support ethical and responsible animal model use. For instance, the 2022 launch of PathogenBinder, a novel soiled-bedding sampling method for detecting rodent pathogens, eliminated the need for a sentinel animal. Last year, the company also began introducing next-generation NCG models, each of which offers a specific utility to enhance the flexibility of study design. The portfolio includes the NCG-M mouse strain, which expresses human cytokines that alter the human cell populations, and the NCG-B2m KO, which resists graft-versus-host disease. More strains will become available later this year.
At Charles River, a corporate initiative called the Alternative Methods Advancement Project (AMAP) is dedicated to developing alternative models to reduce the use of animal models in testing. The company has invested over $200 million in AMAP over the past four years.
“We pursue scientific and technological innovations to explore new ways to further reduce animal use,” says Julie Frearson, PhD, Charles River’s CSO “The guidance of AMAP will help us align our investments, partnerships, product and service initiatives, and advocacy efforts. Our goal is to invest $300 million over the next five years to further enhance this critical mission and drive industry-wide adoption of alternatives.”
In addition, a new multidisciplinary program was launched to develop an in vitro alternative to inhalation toxicology studies in collaboration with MatTek Corporation, Battelle, and Greek Creek Toxicokinetics Consulting. The collaboration aims to develop a New Approach Methodology (NAM) inhalation toxicology test, an animal-free alternative to a traditionally in vivo method.
Frearson notes that regulatory and market trends are driving the development of both animal models and alternative models. On the regulatory side, the FDA Modernization Act 2.0 will likely encourage the development of novel cell-based assays and computer models. Charles River, through strategic partnerships with companies such as Cypre and Aitia, is positioning itself to contribute.
Acting responsibly
“The Biosecure Act, if passed, will impact from where and from whom U.S. academic and biopharma researchers can source animal models in the near future,” notes Monika Buczek, PhD, director, Humanized Immune Model Core, Taconic Biosciences. She advises researchers to start looking at U.S.-based suppliers like Taconic now in anticipation of the Act’s passage.
In 2023, Taconic launched the FcResolv NOG model with murine Fc gamma receptors (FcgRs) knocked out on the triple immunodeficient NOG background. This animal model facilitates antibody-based therapeutic research, as the presence of murine FcgRs can cause false positives or false negatives in both antibody clearance as well as in efficacy data. Taking the model a step further, Taconic scientists created the FcResolv NOG-EXL, which has been modified to include human genes for interleukin-3 and tranulocyte-macrophage colony-stimulating factor. This model allows for a more complex human immune system, including the myeloid compartment, to be onboarded. “We are producing the humanized immune system version of this model,” Buczek notes. “Donors are matched to the NOG-EXL parent strain for side-by-side comparisons.”
In addition to the engineered murine models, the company is also addressing recent cases where the genetics of a CD34+ hematopoietic stem cell (HSC) donor was to blame for unwanted variability in therapeutic responses. “We now leverage an extensive HSC donor catalog to allow researchers to select donors based on HLA Class I and Class II matches or mismatches,” Buczek says. “Researchers can also reserve a specific donor for future studies.” (She adds that none of Taconic’s models utilize fetal tissue in any form.) Another recently launched service provides customers with access to the Wild Mouse Microbiome (WildR), a gut microbiota profile that represents natural mice instead of microorganism-deficient laboratory mice.
Taconic continuously evaluates alternative models, such as advanced 3D cell models and organoids, as opportunities to screen or enhance preliminary data to best design and inform animal studies. While the industry is focused on creating the next best preclinical model, genetic integrity of rodent models is paramount at the company in maintaining the quality and reliability of preclinical research. Buczek recommends that a robust breeding program incorporating best practices in husbandry, colony refreshment, and monitoring techniques is essential to mitigate the risks posed by strain contamination, genetic drift, and expression change.
Market-driven models
“Humans of a broad age range and diverse genetic backgrounds are administered therapeutics,” says Sierra Kent, PhD, business unit manager, The Jackson Laboratory (JAX). “The genetically diverse aged HET3 model recapitulates that, bridging the gap between mice and humans.” These mice are available up to 90 weeks of age to enable aging, age-related, and longevity studies. The model complements and allows expansion beyond the genetically homogeneous inbred strain, the aged black 6 model (C57BL/6J).
New mouse models or strains that can be humanized through cell engraftment are also being introduced. Two recent models are Hu-NSG-S15 and Hu-NSG-FLT3, both enabling the engraftment of human HSCs and the development of diverse human immune cell populations. The Hu-NSG-S15 model combines the popular NSG-SGM3 model with NSG-IL15 to add mature natural killer cells to the broad human immune system that can develop following HSC engraftment.
Hu-NSG-FLT3 builds on the humanized NSG model with the addition of human FLT3 ligand and knockout of the mouse Flt3 receptor, creating an innate immunity humanized mouse model with the development of a diverse myeloid population and functional dendritic cells. The models enable nonhuman in vivo studies of therapeutics in the presence of a functional human immune system.
As immunology and oncology therapeutics continue to dominate the current global pharmaceutical, JAX is launching three new products: another advanced humanized mouse model (NSG-FLT3-IL15), the Atlas mouse platform in partnership with AbTherx, and a new FcRn model that expresses human albumin. Adding human interleukin-15 to the latest NSG-FLT3 model enables the development of natural killer cells in the FLT3 model following HSC engraftment. The Atlas model is used to produce antibodies with human variable regions. The FcRn human albumin model will enable more accurate pharmacokinetic testing of albumin-based therapeutics.
In addition, JAX offers a continuously expanding catalog of human induced pluripotent stem cells for Alzheimer’s and related dementias (ADRDs), amyotrophic lateral sclerosis, and other neurodegenerative disorders. This collection is built on one high-quality, highly characterized cell lineage, KOLF2.1J. The consistent lineage and use of CRISPR-Cas technology allows creation of homozygous, heterozygous, and revertant (mutation-corrected) lines for each disease-related genetic target.
Reducing use of nonhuman primates
Emulate is developing new toolkits for cell-based therapies and biologics. “We have a breakthrough new application for CAR T cells targeting solid tumors,” says Daniel Levner, PhD, the company’s co-founder and CTO. “Very human-specific technology CAR T cells are hard to test, even in humanized mice. Nonhuman primates or humans are typically needed to get relevant data.” The application allows researchers to put their target cancer tissue and CAR T cells into Emulate Organ-Chips to study the entire immune cascade. A protocol is provided as well as guidance on customizing the workflow based on a researcher’s target tissue of interest.
Another modeling tool from Emulate is the new Chip-A1 Accessible Chip, which enables the modeling of more complex 3D tissue. Chip-A1 can be opened from the top to access the tissue, facilitating experimentation with aerosols and topical treatments. More broadly, it empowers the creation of interesting research models that have stromal compartments and layered structures.
Organoid technology complements organ-on-a-chip technology. “Organoids are true to their source,” Levner notes. “So, we took these cells and put them on our platform to synergize the best of both worlds.” RNA-seq studies have demonstrated that organoid-derived cells in the microenvironment of the Organ-Chips exhibit behavior even more consistent with human gene expression than is seen with organoids alone.
Moderna has been using Emulate Organ-Chips to prescreen and capture more clinically relevant data on lipid nanoparticles, which are used for mRNA delivery, before moving into nonhuman primate studies. They successfully used the Liver-Chip to gauge the risk of liver fibrosis by assessing collagen remodeling and pro-fibrotic gene expression. This allowed the team to screen a large number of lipid nanoparticles and move forward with only the most promising candidates.
“Many cell-based therapies have to utilize nonhuman primate models, which are slow and very expensive, and which researchers would like to minimize,” Levner remarks. “Our Organ-Chips slot very nicely into these prescreening workflows and show economic and time savings.”
Levner believes that the optimistic regulatory discussions resulting from the FDA Modernization Act 2.0, along with the application of prescreening prior to nonhuman primate studies, are setting the stage for a watershed moment for alternative models.
A transitional moment
CN Bio recently secured $21 million in Series B investment to expand their design and manufacture of 3D human organ and tissue microphysiological systems (MPSs). The company’s PhysioMimix Single-Organ Higher Throughput system and associated Multi-Chip Liver-48 plate was developed in response to market demand. The new plate features a standard SLAS-footprint with 48 miniaturized chips. Three plates can be run simultaneously.
“Importantly, supporting data from the PhysioMimix platform were incorporated into a successful 2023 regulatory submission,” says Emily Richardson, PhD, lead scientist, CN Bio. “Inipharm’s INI-822 for a common fibrotic disorder is now in clinical trials. Plus, collaborators at Charles River developed and published a novel genotoxicity application using the system.”
CN Bio also launched a primary gut/liver dual-organ model for improved human ADME and bioavailability estimations. The model was developed with Altis Biosystems, which recreated the intestinal barrier using primary cells isolated from the human jejunum on a biomimetic scaffold (RepliGut). When connected to the Liver-on-a-Chip model in the Multi-Chip Dual-Organ plate, an improved predictive capacity for profiling the ADME behavior of oral drugs was seen as compared to an equivalent Caco-2 Gut/Liver MPS.
“We are expanding the range of our ‘in a box’ kit,” Richardson asserts. “The kit contains everything required to recreate our models and applications, and to develop preclinical animal MPS models to flag potential interspecies differences and minimize the risk of unforeseen clinical issues.” Initial results demonstrate the translatability of animal MPS data.
Richardson has seen an upward trend in acceptance following the FDA Modernization Act 2.0 and increased 3R project funding. This trend reflects a growing awareness that ethical considerations can be addressed while practical benefits are pursued, benefits such as lower workflow costs, higher efficiency, and faster clinical translation. The company participates actively in regulatory and standards groups, including the Critical Path Institute, the European Committee for Electrotechnical Standardization, and the IQ-MPS affiliate of the International Consortium for Innovation and Quality in Pharmaceutical Development.
“A growing body of evidence proves that the approach is more predictive than traditional methods and that technologies are being used in a complementary manner,” Richardson declares. “Standardization, a crucial area, needs to be addressed. Existing models will become more comprehensive, and the breadth of models and applications will expand along with more automation solutions.”
Advancing organ design
According to Paul Vulto, PhD, CEO at Mimetas, demand is increasing for services in oncology, immunology, and metabolic diseases. The oncology drivers are novel immunotherapeutic angles, including CAR T-cell therapies and bispecific antibodies. Focus is on the activation, extravasation, migration, killing, and inhibition of immune cell activity in assays that capture the full tumor microenvironment, that is, tumor and stromal tissue, immune cells, and vasculature. Similar assays are being applied in immunological diseases such as inflammatory bowel disease or renal diseases like lupus nephritis.
“Our assays,” Vulto points out, “modulate immune cell invasion, cytokine production, stromal activation, barrier function, and complement activation.”
The company has made remarkable progress in liver modeling for metabolic diseases, leading to a life-like liver model that comprises a true liver sinusoidal structure with polarized hepatocytes, stellate cells, and perfusable liver vasculature. The model is applicable to a broad range of assays in fibrosis, steatosis, and nonalcoholic steatohepatitis (NASH), as well as aspects of gene therapies.
OrganoReady products—OrganoPlates that have the tissue models already prepared—are being launched and shipped live to the end user. The user simply removes the blister and starts pipetting. Products in this format include OrganoReady Colon Organoid, Collagen, Colon Caco‑2, BBB HBMEC, Blood Vessel HUVEC, and Angiogenesis HUVEC.
“Our most impressive advance is the OrganoReady Colon Organoid product,” Vulto asserts. “It makes use of adult stem cell technology developed by Utrecht University Professor Hans Clevers.”
The company continues to address the pharma industry’s pressing need for robust, representative disease models. “If you can model disease, you can cure it,” Vulto insists. The message is resonating, particularly with the emergence of advanced drug modalities that are pushing beyond the limits of current in vitro and in vivo models.
Although Vulto sees a consistent increase in uptake of Mimetas’s products and services, he also sees a lot of fragmentation. The sheer variety of offerings is bewildering to end users. But he predicts that a wave of consolidation is coming that will change the modeling landscape. Applications will become more robust and routine, and they will be offered by fewer, larger companies. He believes that as a result, end users will have more clarity, and innovators will be more accountable.
