The Applications of Zebrafish Disease Models in Drug Discovery

With the advent of next-generation sequencing (NGS) technologies, variants underpinning rare genetic diseases are being discovered at an accelerated rate. However, many rare conditions lack effective treatments due to poor understanding of their pathophysiology. Therefore, there is an increasing need to develop novel experimental models of rare genetic diseases in order to validate potential pathogenic variants, study disease-causing mechanisms, and identify therapeutic targets.

Based on the large number of novel variants identified by NGS, animal models of rare diseases need to be genetically and physiologically similar to humans and well amenable to large-scale experimental manipulations. The zebrafish has become a popular model system for studying these variants, combining conserved vertebrate traits with the capacity for large-scale phenotyping and therapeutic screening.

Advances in rare disease research require the development of experimental models in which candidate variants can be validated and disease mechanisms explored. Information about the variant's function can often be gleaned using in vitro or cellular methods, or by analyzing patient tissue samples. However, these methods are insufficient to demonstrate pathogenicity at the whole organism level. Thus, for many rare diseases, in vivo models are required to confirm causality, especially for neurological diseases, where pathologically relevant patient tissues are often not available, or for disease phenotypes that require interactions between multiple cell types or organ systems.

It is important to note that some mutations may have species-specific effects, so the presence or absence of phenotypes in model organisms must be carefully interpreted. For example, some mouse models of cystic fibrosis fail to accurately recapitulate key respiratory features of the human phenotype, despite the majority of patients harboring pathogenic deletion mutations.

In recent years, zebrafish have become an attractive model organism for translational research. The zebrafish uniquely combines many of the genetic and physiological advantages of mammalian models with the high-throughput capabilities and experimental operability of invertebrate models. Their growing popularity is reflected in the continued increase in the use of zebrafish in biomedical research publications.

The zebrafish genome shares considerable homology with the human genome, with approximately 70% of human genes orthologs identified. Zebrafish are also genetically easy to tame, and tools for generating transgenic zebrafish models are continually being developed and optimized.

Anatomically and physiologically, zebrafish are more distant from humans than mice. Therefore, modeling genetic diseases that affect structures that do not exist in fish, such as the lungs, can be more challenging. Nonetheless, the simple vertebrate architecture of the zebrafish makes it simple to study disease in many organ systems and structures that zebrafish and humans share. For example, zebrafish have been successfully used to model genetic diseases affecting the human cardiovascular system, nervous system, visual system, renal system, and muscular system, among others.

More recently, powerful approaches involving the use of engineered nucleases, including zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly spaced repeat (CRISPR)/CRISPR-associated protein (Cas9) systems, have been used to generate stable human disease model that enables targeted mutations to be generated in specific zebrafish orthologs of interest. Both ZFNs and TALENs require the generation of custom protein components for each target locus, an expensive and laborious process that makes these systems less compatible for large-scale applications. In contrast, the CRISPR/Cas9 system relies on the recognition of target sites by custom guide RNA (gRNA) molecules and requires only one oligonucleotide to be designed for each target site.

Zebrafish can be used for large-scale screening of phenotypes associated with many conserved vertebrate organ systems and structures, and they possess the ability to perform high-throughput testing of therapeutic compounds, which is often impractical in other vertebrate model systems. This unique combination of properties gives zebrafish a potential unmatched by other classical model systems in advancing the understanding of rare genetic diseases.