Antisense delivery to Atlantic salmon eggs for large scale sterilization

Cultivation of sterile-only fish in aquaculture offers multiple benefits of environmental, economical, and social value. A reliable method for efficient sterilization without affecting fish welfare and performance traits would have significant impact on fish production practices. Previously, we have developed a method for production of sterile Atlantic salmon by ablating the germ cells population at embryonic stages. For the prove-of-principal experiments, antisense oligos (ASOs) were delivered with microinjections to fertilized salmon eggs, which resulted in the production of morphologically normal sterile fish. However, microinjections are laborious and imprecise method unsuitable for large-scale field production. The aim of the current project was to explore alternative protocols for efficient large-scale delivery, mainly based on bath incubation of salmon eggs and sperm. To this end, we tested ASOs with several backbone modifications and determined the most potent configuration. We designed fluorescently labelled ASOs for in-vivo tracing of the delivery process and established protocols for safe incubation of sperm, unfertilized and freshly fertilized eggs. Further, based on recent drug delivery studies, we designed nano-carriers and verified their ASOs-loading capacities. Over the course of the project, we performed several incubation trials with bare ASOs, tagged ASOs and ASOs encapsulated in different nano-carriers. The delivery success in each case was examined by direct fluorescence measurements and functional effect evaluation, reflected by the germ cells count. In most cases, the fluorescently tagged ASOs were largely trapped at the egg shells (chorion) and only a small portion of the fluorescence was transmitted to the egg proper. Successful delivery to mature sperm, on the other hand, appeared possible under standard cell transfection conditions. Unfortunately, despite the multiple variations in concentration, charge, nanocarriers type and incubation conditions, we did not observe full elimination of the primordial germ cells in any of our experiments and, consequently, we could not perform the initially planned large-scale trials with sterilized fish. In summary, although we could not establish a fully functional protocol for bath-sterilization of salmon eggs, we obtained valuable information on the ASOs structure and function, the nano-carriers formulations and loading, and conditions and media necessary for the incubations. Overall, the results from the project could serve as a foundation for further attempts to achieve large-scale sterilization using ASOs.

1. Determined an optimal configuration of Gapmer anti-sense oligos for achieving gene knock down and germ cell ablation in salmon eggs. 2. Developed and applied protocols for loading different types of nano-carriers with Gapmers. 3. Developed and utilized protocols for incubation of salmon eggs and sperm with nano-carriers filled with Gapmers. 4. Achieved delivery of small amounts of fluorescently labelled Gapmers to the salmon embryo, using bath-incubation protocols; no functional effect.

One of the biggest threats for wild salmon populations is the genetic effect escaped farmed salmon have on native populations affecting their functional traits and leading to reduced fitness. Farming only sterile salmon would decrease the negative effect of escapees, resulting in the recovery of native populations. Additional benefits of farming sterile fish will be the elimination of the sex maturation triggered negative impacts on growth, flesh quality, hypoosmoregulatory ability and immune function. Finally, control over the propagation of the genetic material is essential for protecting the intellectual property and value creation of Norwegian salmon breeders. The use of sterile fish in salmon farming would thus contribute to a more sustainable and profitable industry. Triploid salmon are currently the only commercially produced type of sterile salmon. They perform poorly under suboptimal environmental conditions and are only farmed by few commercial producers. This project aims to develop an alternative method for large scale sterilization by delivering antisense-oligonucleotides (ASO) mediating mRNA degradation of a gene controlling the survival of primordial germ cells, progenitor cells of sperm and egg cells. While this method has been proven to result in sterile fish, an efficient throughput delivery method for large scale sterilization is lacking. In this project we aim to develop an efficient delivery method by exploring different nanocarriers to deliver ASOs to eggs.