Non-clinical models for Cell & Gene Therapies

Non-clinical means conducted in vitro, in silico, or in chemico, or a non-human in vivo test related to the investigation of the safety and efficacy of a medicinal product. Non-clinical test may include simple and complex human cell-based assays, microphysiological systems including organ-on-chip, computer modelling, other non-human or human biology-based test methods, and animal-based tests. Cells, organ-on-chip, computer algorithm or lab animals are commonly used as non-clinical or preclinical models mimicking different aspects of human physiology and diseases. The terms pre-clinical and non-clinical are generally used interchangeably even if non-clinical is the preferred option in regulatory documents such as clinical trial applications. Non-clinical or preclinical models are essential for evaluating the safety, efficacy, and biodistribution of Cell and Gene Therapy (CGT) products before human clinical trials. These models inform dosing, route of administration, toxicity profiles, biological mechanisms and potential side effects depending on the product's attributes and the target disease. Preclinical models are used for discovery activities, proof of principle experiments and Clinical Trials Applications (CTA, corresponding to Investigational New Drug (IND) applications in the USA) enabling studies before initiating proof of concept clinical studies also called ‘first in human’. The non-clinical (or pre-clinical) development phase of Advanced Therapy Medicinal Products (ATMPs) aims at assessing at therapeutic candidate in terms of safety and efficacy thanks to in silico, molecular, cellular, organoid, or animal based non-clinical models. Data are presented according to the Common Technical Document (CTD) format as defined by the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). See figure 1.

Figure 1: Common Technical Document (CTD) format

EUPATI - Non-clinical development: Basic principles

The CTD presents the advantage to assemble all the Quality, Safety, and Efficacy information in this common format. This is a set of specifications for an application dossier for the registration of medicine, designed for use across Europe, Japan, the United States, and beyond. Safety studies must be performed under Good Laboratory Practice (GLP) to ensure the reliability, integrity, and reproducibility of non-clinical studies. GLP isn’t just about maintaining testing quality. It’s also about ensuring the ethical treatment of animals used in these studies. 

Non-clinical studies use in vitro and in vivo models of human physiology and human diseases. They aim at evaluating the activity, the mode of action, the efficacy, the biodistribution and the potential toxicity of medicinal product candidates. See figure 2.

Data from process development, manufacturing and control and combined with results from non-clinical studies assessing efficacy, safety and biodistribution to file clinical trial applications and initiate first-in-human studies under Good Clinical Practice (GCP) conditions. 

Figure 2: Main steps in ATMP development

A Regulatory Risk-Based Approach to ATMP/CGT Development: Integrating Scientific Challenges With Current Regulatory Expectations, Laura I. Salazar-Fontana, Front. Med., Sec. Regulatory Science, 13 May 2022

Animal models of human diseases can be used for dose range selection related to efficacy and toxicology before application in human clinical trials. Stem cell therapies (e.g. HSC, MSC, iPSC) and cellular immunotherapies (e.g. CAR-T, NK, DC) are commonly tested in immunodeficient mice (nude mice, SCID mice, NOD SCID mice, NSG mice, NOG mice, BRGSF mice, NCG mice, etc…) or humanized mouse models (e.g. expression of human cytokines, human HLA alleles, humanized TCR). 

Acronyms used:

• HSC: Hematopoietic Stem Cell
• MSC: Mesenchymal Stem Cell
• iPSC: Induced Pluripotent Stem Cell
• CAR-T: Chimeric Antigenic Receptor-T
• NK: Natural Killer
• DC: Dendritic Cell
• SCID: Severe Combined Immunodeficiency
• NOD: Non-Obese Diabetic
• NSG: NOD Scid Gamma
• BRGSF: stands for a mouse model known as the BRGSF mouse, a highly immunodeficient model used in research
• NCG: NOD Scid Gamma

Based on the effect to be evaluated, the characteristics of the product to be tested and the criteria chosen for the assessment, different types of models can be used.

• Complexicity of models

Different types of non-clinical or preclinical models can be distinguished based on their level of complexity, recapitulating the functions of a cell, of a tissue, of an organ or of the human body (estimated to contain about 30 to 40 thousand billion cells). See figure 3.

Figure 3: Complexity of current living models available for non-clinical studies

Organoids as host models for infection biology – a review of methods, Aguilar et al. Experimental & Molecular Medicine (2021) 53:1471–1482

Individual cells are practical but lack multicellular structure. Organoids fill the gap between monotypic cells and complex organisms. The term “organoid” has been used in the past to describe the histological characteristics of tumors or other tissues and generally meant “resembling an organ”. Organ-on-chip devices can recapitulate vascular flow, tissue–tissue interactions, and organ-relevant mechanical movements. Animal models recapitulate the complexity of organisms such as mammals without perfectly mimicking the human physiology. 

• In vitro models such as human cells in liquid culture, on feeder cells (2D), in organoids (3D), or on chips 

Primary cells and cells lines are used in liquid culture to perform preclinical studies such as biochemistry, cell biology and immunology assays. Cultures on feeder cells benefit from growth factors and interactions with the layer of feeders supporting the growth and the differentiation of the cells of interest for preclinical studies. 

Organoids are miniature 3D models mimicking organs or tissues, enabling scientists to investigate the causes of diseases and test new treatments. e.g. brain organoids to evaluate cellular therapies (Brain Organoids to Evaluate Cellular Therapies, García-Delgado AB, Campos-Cuerva R, Rosell-Valle C, Martin-López M, Casado C, Ferrari D, Márquez-Rivas J, Sánchez-Pernaute R, Fernández-Muñoz B. Brain Organoids to Evaluate Cellular Therapies. Animals (Basel). 2022 Nov 15;12(22):3150) or organoid models for assessing cancer immunotherapy (Breakthroughs and challenges of organoid models for assessing cancer immunotherapy: a cutting-edge tool for advancing personalised treatments, Wang, Q., Yuan, F., Zuo, X. et al. Breakthroughs and challenges of organoid models for assessing cancer immunotherapy: a cutting-edge tool for advancing personalised treatments. Cell Death Discov. 11, 222 (2025)). See figure 4.

Figure 4: Two-dimensional (2D) cell culture, three-dimensional (3D) brain organoid models. Int. J. Mol. Sci. 2023, 24, 12528. Application of Human Brain Organoids—Opportunities and Challenges in Modeling Human Brain Development and Neurodevelopmental Diseases.

Comparison of characteristics among two-dimensional (2D) cell culture, three-dimensional (3D) brain organoid models, and animal models. Brain organoids become alternatives to animal models or classical 2D culture systems in experiments and their significance as valuable models for studying brain developmental disorders.

Cells on chips aim to miniaturize and complete assays on lab-on-a-chip systems allowing control of culture conditions and measurements of biomarkers e.g. pancreas on chip.

Organoids as preclinical models of human disease: progress and applications, Chen B, Du C, Wang M, Guo J, Liu X. Organoids as preclinical models of human disease: progress and applications. Med Rev (2021). 2024 Mar 14;4(2):129-153.

Pancreatic islet organoids-on-a-chip: how far have we gone?, Yin J, Meng H, Lin J, Ji W, Xu T, Liu H. Pancreatic islet organoids-on-a-chip: how far have we gone? J Nanobiotechnology. 2022 Jun 28;20(1):308.

• Ex vivo models such as perfused organs or organ-on-chips

Organs perfused ex vivo offer an excellent experimental platform for studying organ physiology and testing new treatments.

Ex-Vivo Human-Sized Organ Machine Perfusion: A Systematic Review on the Added Value of Medical Imaging for Organ Condition Assessment, Van Der Hoek JL, Krommendijk ME, Manohar S, Arens J and Groot Jebbink E (2024)

Organs-on-chips are systems containing engineered or natural miniature tissues grown inside microfluidic chips. To better mimic human physiology, the chips are designed to control cell microenvironments and maintain tissue-specific functions.

A guide to the organ-on-a-chip, Leung, C.M., de Haan, P., Ronaldson-Bouchard, K. et al. Nat Rev Methods Primers 2, 33 (2022).

• In vivo models such as animal models mimicking human disease conditions

Animal models mimicking human disease conditions are very useful at the preclinical stage before embarking on a clinical trial. In gene therapy, for instance, they are useful in the assessment of variables related to the use of viral vectors such as safety, efficacy, dosage and localization of transgene expression.

Contemporary Animal Models For Human Gene Therapy Applications, Gopinath C, Nathar TJ, Ghosh A, Hickstein DD, Nelson EJR. Curr Gene Ther. 2015;15(6):531-40.

Small animals such as mice are often used to develop advanced therapies related to hematology or immunology, e.g. development cellular immunotherapies like CAR-T cells. Transgenic mice are generated to create models of human diseases. Immunodeficient and humanized mouse models are generated to test human cells in xenogeneic studies. Cellular therapies based on human hematopoietic stem cells (therapies for Adenosine Deaminase Deficient Severe Combined Immunodeficiency (ADA-SCID), transfusion-dependent thalassemia (TDT), Metachromatic Leukodystrophy (MLD), Sickle Cell Disease (SCD) … ) or immune cells (therapies for Multiple Myeloma (MM), B-cell Acute Lymphoblastic Leukemia (B-ALL), Acute Myeloid Leukemia (AML) …) are often tested in immunodeficient mice (nude mice, SCID mice, NOD SCID mice, NSG mice, NOG mice, BRGSF mice, NCG mice, etc…) or in humanized mouse models (usually generated to better recapitulate the development of human innate and adaptive immunity)  before being assessed in clinical trials.

Larger animals such as dogs or non-human primate are used for preclinical studies on muscles and central nervous system, e.g. test of Adeno-Associated Virus (AAV) vectors to treat myopathies or neurodegenerative diseases.

• In silico models

In silico models are developed by computer programmers and data scientists and built with real world data sets such as clinical data, genomics, proteomics, transcriptomics … Artificial intelligence algorithms are often used to navigate big data sets and interact with end users like wet lab scientists and clinicians.

In silico regenerative medicine: how computational tools allow regulatory and financial challenges to be addressed in a volatile market, Geris L, Guyot Y, Schrooten J, Papantoniou I. Interface Focus. 2016 Apr 6;6(2):20150105.

The most advanced in silico models aim at becoming digital twins of patients and be used to accelerate the selection of drug candidates and the development of medicinal products. See figure 5.

Figure 5: the concept of digital twins 

Figures adapted from Targeting MAPK Signaling in Cancer: Mechanisms of Drug Resistance and Sensitivity, Lee S, Rauch J, Kolch W. Targeting MAPK Signaling in Cancer: Mechanisms of Drug Resistance and Sensitivity. International Journal of Molecular Sciences. 2020; 21(3):1102.

Integrating molecular, clinical and imaging data through computational models will allow to generate digital twins of tissues, organs and patients. New treatments could be first tried on digital twin and the best option selected for clinical trials (Targeting MAPK Signaling in Cancer: Mechanisms of Drug Resistance and Sensitivity). Creating a digital twin of a patient is highly challenging. If 100 million parts have to be considered for a space shuttle, 1000 000 million cells should be considered for a human body. 

In the European Union (EU), the European Medicines Agency (EMA) published the ICH S6 (R1) on preclinical safety evaluation of biotechnology-derived pharmaceuticals.

The document describes non-clinical activities and is divided in two parts. In the first part, after an introduction and a section on the specification of the test material, the guidelines discuss preclinical safety testing, including biological activity/pharmacodynamics, animal species/model selection, number/gender of animals, administration/dose selection, immunogenicity. Then the section on specific considerations addresses safety pharmacology, exposure assessment, pharmacokinetic and toxicokinetic assays, metabolism, single dose toxicity studies, repeated dose toxicity studies, immunogenicity studies, reproductive performance and developmental toxicity studies, genotoxicity studies, carcinogenicity studies and local tolerance studies. 

In the second part, the guidelines give an overview of key elements to be considered: the species selection, the study design for dose selection, the immunogenicity, the reproductive and developmental toxicity and the carcinogenicity. 

ICH guideline S6 (R1) – preclinical safety evaluation of biotechnology-derived pharmaceuticals

Moreover, the EMA is also offering Scientific Advice and Opinions to support the qualification of novel methodologies for medicines development. This is particularly relevant in the context of Research & Development, both for non-clinical and clinical studies. 

More information on the EMA website here: 

Qualification of novel methodologies for medicine development

Finally, it should be highlighted that beyond regulatory guidelines, two sets of legislations of the EU may be applicable.

On the one hand, the legislation on genetically modified organisms and/or on genetically modified micro-organisms may be applicable at the non-clinical stage of GCT development depending on the National implementations of the EU directives on this topic. More information here.

• On the other hand, the legislation on GLP as well as the guidance from the European Commission and from the Organisation for Economic Cooperation and Development (OECD) apply at the non clinical stage of development of GCT.

More information on the EMA website here, on the European Commission website here, and on the OECD website here.

In the USA, the Food & Drug Administration (FDA) has published guidelines on non-clinical activities that are divided in two steps: 1) discovery, development 2) preclinical research. 

Discovery: new insights into disease mechanisms allow researchers to design a product to stop or reverse the effects of the disease. Many tests are conducted to find possible beneficial effects including in animal models. Cellular and molecular analyses are usually performed. 

Step 1: Discovery and Development | FDA 

Development: once potential therapeutic modalities are identified, experiments are conducted to gather information on potential benefits, mechanisms of action, dosage, route of administration on animal models, side effects and adverse events.  

Preclinical research toward clinical trials: to assess toxicity, FDA requires researchers to use GLP. Toxicology studies are usually performed on animal models and the GLP regulations set the minimum basic requirements for study conduct, personnel, facilities, equipment, written protocols operating procedures, study reports and a system of quality assurance. These studies must provide information on dosing and toxicity levels. Data from preclinical studies performed in vitro and in vivo are aggregated and organized in the investigational new drug application that must be cleared to initiate a clinical trial. 

In April 2025, FDA announces plan to reducing animal testing in preclinical safety studies. This roadmap outlines a strategic, stepwise approach for FDA to reduce animal testing in preclinical safety studies with scientifically validated new approach methodologies (NAMs), such as organ-on-a-chip systems, computational modeling, and advanced in vitro assays.

FDA - Roadmap to reducing animal testing in preclinical safety studies

The EMA's scientific guidelines on non-clinical development help medicine developers prepare marketing authorisation applications for human medicines.

Non-clinical development guidelines

Among those guidelines, the “regulatory acceptance of 3R (Replacement, Reduction, Refinement)” aims to encourage stakeholders and authorities to initiate, support and accept development and use of 3Rs testing approaches with the aim to replace, reduce and refine in vivo animal studies for human and veterinary medicinal products.

Regulatory acceptance of 3R (replacement, reduction, refinement) testing approaches - Scientific guideline

Published: 28/10/2025

Authors:

Olivier Nègre, Member of The General Committee Management Group at EuroGCT, Board Member of the French Society of Cell and Gene Therapy, Paris, France; Co-President of the Gene and Cell Therapy Institute, Expert in Cell and Gene Therapy at Biotherapy Partners, Paris, France

Minodora Brimpari, EuroGCT ELSI Group 1 member, Project Leader in Cell Therapy at Helmholtz Zentrum München

Reviewed by:

Aurélie Mahalatchimy, EuroGCT WP4 Convenor, UMR 7318 DICE CERIC, Aix-Marseille University, CNRS, Aix-en-Provence, France