Building confidence in non-protected zebrafish embryo-larvae as a viable alternative to mammalian DART assessment

Why did we fund this project?

This award aims to develop an embryonic zebrafish model for embryo-fetal developmental toxicity tests replacing the use of rabbits and rodents in human drug safety assessments.

Developmental and reproductive toxicity (DART) studies are part of a suite of tests required by regulatory bodies to assess the safety of new chemicals and drugs. Typically, pregnant rats and rabbits are treated with compounds before undergoing caesarean section to examine the placenta, corpora lutea and developing foetuses. While there is increasing interest in using larval zebrafish to replace the use of mammals in DART testing, their use is currently limited as zebrafish are not sensitive to Immunomodulatory imide Drugs (IMiDs), a group of chemicals with potential teratogenic effects such as thalidomide. The student, with Dr Jonathan Ball, will establish a transgenic zebrafish model containing an IMiD sensitising modification. They will then determine the effects of IMiDs on the developing larvae life to build confidence in the use of larval zebrafish for DART studies. At this early life stage, prior to independent feeding, larval zebrafish are not considered capable of suffering based on current scientific thinking.

Human drug development involves clinical assessments for efficacy and safety operating through three phases (I-III). During Phase II, a developmental and reproductive toxicity (DART) package is conducted on two mammalian species (normally, rat and rabbit) in order to assess risk for women of childbearing potential in clinical trials. Eighty five percent of drugs which complete Phase II are not approved for market use by the FDA and for the period 2015-2022 this equates to 2100 drugs, requiring the use of 500,000 adult rabbits or rats for the DART assessment alone. These tests also require caesarean section to examine the placenta, corpora lutea, and developing fetuses, and they are extremely time consuming. Thus, there is urgent need for trusted alternatives.

In the latest revision (2020) of the guideline which governs reproductive toxicology detection for medicinal products, trusted New Approach Methodologies can be used to justify a reduced DART with a major (63%) reduction in adult animal usage. They  potentially also allow for the postponement of DART assessment into Phase III with a further considerable reduction in animals tested (only 29% of compounds completing Phase II progress to Phase III). Application of the reduced DART could remove the need for testing on over 43,000 adult rats/rabbits for DART annually (approx. 50 FDA drugs are approved each year).

Zebrafish is a highly promising alternative for determination of DART, however, like all non-primate species except rabbit, zebrafish lack the sensitivity to identify teratogenicity associated with a key group of chemicals, namely, ImmunoModulatory imide Drugs (IMiDs), epitomised by thalidomide. This shortfall has contributed to the lack of regulatory and industry confidence in zebrafish as an alternative to mammals for DART assessment.

Recent work, including our own, has strongly implicated that down-regulation of the Sall4 gene (via ubiquitisation through cereblon protein, Crbn) drives toxicities/malformations associated with IMiD exposure. Cross species homology has identified a difference of only one amino acid (isoleucine vs valine in humans) in the CRBN protein binding site for IMiD drugs as being the crucial difference between sensitive and non-sensitive species. Generation of an IMiD sensitive transgenic mouse corroborates this, where a single point substitution of isoleucine for valine results in rabbit and human-like responsiveness to IMiD exposure in the mouse.

In this studentship, we will apply cutting edge gene editing technology (Prime Editing (PE); developed by our industry partner AstraZeneca, and now established in our laboratory, to produce a CrbnI393V knock-in zebrafish and assess the ability of this model to detect developmental exposure effects of IMiDs in non-protected embryo-larval life stages. The model will be compared with the existing wild type zebrafish test for responses to a wider cross-section of known developmental toxins to demonstrate consistency in predictive performance, and importantly from a 3Rs perspective, to demonstrate its application using a non-protected (4dpf) life stage. Ultimately, through this studentship application of a new gene editing technology will create a new zebrafish model with the potential for contributing very significantly to the 3Rs principles and building greater confidence in the value of the zebrafish as an alternative for DART assessment. Furthermore, the existing industry and academic networks of the team will ensure widespread communication and dissemination of the new model and associated information to facilitate the model uptake. Importantly, the student will receive an exceptional training experience in the very latest molecular biology, in-life studies and advanced imaging techniques and 3Rs applications in a working partnership with industry, with the associated benefits this will bring for training and career development for the student.