The Zebrafish Embryo as a Model Organism for Testing mRNA-Based Therapeutics

Abstract

mRNA-based therapeutics have revolutionized the world of molecular therapy and have proven their potential in the vaccination campaigns for SARS-CoV2 and clinical trials for hereditary disorders. Preclinical studies have mainly focused on in vitro and rodent studies. However, research in rodents is costly and labour intensive, and requires ethical approval for all interventions. Zebrafish embryonic disease models are not always classified as laboratory animals and have been shown to be extremely valuable for high-throughput drug testing. Zebrafish larvae are characterized by their small size, optical transparency and high number of embryos, and are therefore also suited for the study of mRNA-based therapeutics. First, the one-cell stage injection of naked mRNA can be used to assess the effectivity of gene addition in vivo. Second, the intravascular injection in older larvae can be used to assess tissue targeting efficiency of (packaged) mRNA. In this review, we describe how zebrafish can be used as a steppingstone prior to testing mRNA in rodent models. We define the procedures that can be employed for both the one-cell stage and later-stage injections, as well as the appropriate procedures for post-injection follow-up.

Evaluation of the efficacy of cystinosin supplementation through CTNS mRNA delivery in experimental models for cystinosis

“…While our experiments and previous studies using the injection of human mRNA in fish did not show any toxicity 22 , only innate immune responses can be taken into account in the zebrafish model. Whether adaptive reactions can also occur, will be heavily influenced by the delivery vehicle, which will be the subject of future research and will require mammalian models 23 . The stability of mRNA can be further improved through careful selection of UTRs and as poly-A tail shortening destabilizes the RNA with every translation round, a sufficiently long (150A) poly-A tail was chosen 24 , 25 .…”

“…As we injected mRNA at the one-cell stage, this study was not suitable to evaluate the effect of the therapy on the fertility or hatching rate, nor to study the potential dosing regimens as the mRNA is quickly diluted in the dividing embryo. In addition, studying zebrafish larvae does not allow for prediction of the full immunogenic response, as only innate immunity responses can be studied in the zebrafish larvae 23 . In the future, a full safety study will have to be performed in a rodent model, after identification of the proper delivery vehicle, which will also greatly influence safety.…”

Evaluating the In Vivo Virulence of Environmental Pseudomonas aeruginosa Using Microinjection Model of Zebrafish (Danio rerio)

“…Compared to other vertebrate models, a major advantage of zebrafish larvae testing is that, according to European legislation, before the independent feeding stage of the larvae, zebrafish larval models are not regulated as experiments with laboratory animals [63,64]. Therefore, zebrafish models using <120 hpf embryos have low ethical considerations [65]. Moreover, similar to C. elegans and D. melanogaster, zebrafish larvae microinjection tests have a short timeframe, low maintenance cost and high throughput [52] which are major advantages in virulence testing [48].…”