Biology subjects

Laible, G.

Publications and source records attributed to Laible, G..

4 recordsLinked to original sources

bioRxiv2021

The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis

Animal health and welfare are at the forefront of public concern and the agricultural sector is responding by prioritising the selection of welfare-relevant traits in their breeding schemes. In some cases, welfare-enhancing traits such as horn-status (i.e., polled) or diluted coat colour, which could enhance heat tolerance, may not segregate in breeds of primary interest, highlighting gene-editing tools such as the CRISPR-Cas9 technology as an approach to rapidly introduce variation into these populations. A major limitation preventing the acceptance of CRISPR-Cas9 mediated gene-editing, however, is the potential for off-target mutagenesis, which has raised concerns about the safety and ultimate applicability of this technology. Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour. No off-target events were detected from high depth whole genome sequencing performed in precursor cell-lines and resultant calves cloned from those edited and non-edited cell lines. Long molecule sequencing at the edited site and plasmid-specific PCRs did not reveal structural variations and/or plasmid integration events in edited samples. Furthermore, an in-depth analysis of de novo mutations across samples revealed that the mutation frequency and spectra were unaffected by editing status. Cells in culture, however, had a distinct mutation signature where de novo mutations were predominantly C>A mutations, and in cloned calves they were predominantly T>G mutations, deviating from the expected excess of C>T mutations. We conclude that the gene-edited cells and calves in this study did not present a higher mutation load than unedited controls. Cell culture and somatic cell nuclear transfer cloning processes contributed the major source of contrast in mutational profile between samples.

genomics↗

bioRxiv2020

Holstein Friesian dairy cattle edited for diluted coat color as adaptation to climate change

High-producing Holstein Friesian dairy cattle have a characteristic black and white coat pattern where black frequently comprises a large proportion of the coat. Compared to a light coat color, black absorbs more solar radiation translating into radiative heat gain which is a contributing factor to heat stress in cattle, negatively impacting on their production levels, fertility and welfare. To better adapt dairy cattle to the rapidly changing climatic conditions with predictions for more frequent and prolonged hot temperature patterns, we aimed to lighten their coat color by genome editing. Using gRNA/Cas9-mediated editing, we introduced a three base pair (bp) deletion in the pre-melanosomal protein 17 gene (PMEL) proposed as the causative variant responsible for the semi-dominant color dilution phenotype seen in Galloway and Highland cattle. Calves generated from cells homozygous for the edited mutation revealed a strong color dilution effect. Instead of the characteristic black and white coat color patterning of control calves generated from unedited parental cells, the edited calves displayed a novel pattern of grey and white markings and absence of any black areas. This, for the first time, verified the causative nature of the PMEL mutation for diluting the black coat color in cattle. With these edited animals, it is now possible to dissect the effects of the introgressed edit and other interfering allelic variants that might exist in individual cattle and accurately determine the impact of only the three bp change on important health, welfare and production traits. In addition, our study proved targeted editing as a promising approach for the rapid adaptation of livestock to changing climatic conditions.

genetics↗

bioRxiv2020

Transgenic goats producing an improved version of cetuximab in milk

Therapeutic monoclonal antibodies (mAbs) represent one of the most important classes of pharmaceutical proteins to treat human diseases. Most are produced in cultured mammalian cells which is expensive, limiting their availability. Goats, striking a good balance between a relatively short generation time and copious milk yield, present an alternative platform for the cost-effective, flexible, large-scale production of therapeutic mAbs. Here, we focused on cetuximab, a mAb against epidermal growth factor receptor, that is commercially produced under the brand name Erbitux and approved for anti-cancer treatments. We generated several transgenic goat lines that produce cetuximab in their milk. Two lines were selected for detailed characterization. Both showed stable genotypes and cetuximab production levels of up to 10g/L. The mAb could be readily purified and showed improved characteristics compared to Erbitux. The goat-produced cetuximab (gCetuximab) lacked a highly immunogenic epitope that is part of Erbitux. Moreover, it showed enhanced binding to CD16 and increased antibody-dependent cell-dependent cytotoxicity compared to Erbitux. This indicates that these goats produce an improved cetuximab version with the potential for enhanced effectiveness and better safety profile compared to treatments with Erbitux. In addition, our study validates transgenic goats as an excellent platform for large-scale production of therapeutic mAbs.

molecular biology↗

bioRxiv2020

Cetuximab produced from a goat mammary gland expression system is equally efficacious as innovator cetuximab in model systems of cancer.

Humanised monoclonal antibodies have proven a very effective mode of therapy for a wide range of conditions. With many of the monoclonal antibody drugs now coming off patent there is an increasing interest in developing biosimilar, or even biobetter, forms of these drugs. With the commercial competition associated with such generic products there is increasing demand for improved production and purification system for biosimilars. Cetuximab, also known as Erbitux, is widely used in cancer therapy, especially in advanced colorecal cancer, metastatic non-small cell lung cancer and head and neck cancer and it will come off patent in the near future. We have previously reported on a genetically engineered goat system to produce cetuximab (gCetuximab) in milk. Herein we now report on the further charactization of the gCetuximab produced utilizing additional and more sophisticated biological assays. There is similar bioactivity of the gCetuximab compared with the commercial product produced in mammalian cell culture. In particular both cetuximab antibodies selectively target EGFR and induce its internalization to down regulate EGFR signaling. Both forms have very similar half life in animals and in a HT29 colorectal cancer xenograft model have similar efficacy. We also show that the toxin MMAE can be conjugated to gCetuximab, that this targets it to cells and that this results in direct cell killing in HT29 cells. This demonstrates that the gCetuximab will also be a viable vehicle for antibody drug conjugate based therapies. Taken together, this shows that the goat milk monoclonal antibody production system is an effective way of producing a biosimilar form of cetuximab.

bioengineering↗