Biology subjects

Wells, D. N.

Publications and source records attributed to Wells, D. N..

4 recordsLinked to original sources

bioRxiv2026

Morula complementation restores fetal kidneys in xenocompatible SALL1 null sheep

To meet the global shortage of organs, extensive genome modifications have been performed to "humanize" livestock tissues for xenotransplantation. However, residual immune rejection remains a problem, prompting alternative approaches that explore the use of animals as hosts for growing human organs. This requires genome editing to disable organogenesis in the host and embryo complementation with suitable donor cells to fill the empty organ niche in chimaeric animals. Pigs have been the predominant livestock species investigated for this approach. Here, we used domestic sheep as hosts for donor-derived kidney formation. Spalt-Like Transcription Factor 1 (SALL1) was targeted in male fibroblasts lacking xenoantigens CMAH and GGTA1, using either one gRNA within zinc finger cluster (ZFC) 2 or two gRNAs to remove all ZF domains. Following somatic cell cloning and embryo transfer of triple knockout strains, fetuses were collected on gestational day 48 to analyze the SALL1 KO phenotypes. Single gRNA editing produced a hypomorph with different degrees of metanephric hypoplasia, while the dual-gRNA deletion resulted in a null allele which completely abolished nephrogenesis. Female donor cells carrying high vs low copy numbers of an mCherry transgene, as well as CMAH and GGTA1 edits, were used for morula complementation. Fetal kidney development was anatomically and histologically restored in sex-chimaeric hosts, providing proof-of- concept for using sheep as a new model species for in vivo organ generation.

cell biology↗

bioRxiv2025

Generation of cloned sheep lacking galactose-α1,3-galactose and Nglycolylneuraminic acid antigens

Livestock have long been regarded as a potential source of donor organs to alleviate the global organ shortage for transplantation. Sheep have a similar physiology and anatomy to humans, providing the standard model for demonstrating biocompatibility and performance of biological heart valves to obtain regulatory approval for their use in transplantation. Like most mammals, sheep cells contain two well-characterized carbohydrate epitopes, galactose-1,3-galactose (-Gal) and N-glycolylneuraminic acid (Neu5Gc), which are absent in humans. Formation of these xenoantigens is catalysed by two enzymes, namely (1,3) galactosyltransferase (GGTA1) and CMP-Neu5Gc hydroxylase (CMAH), respectively. Towards generating new sheep models, we used Cas9-mediated genome editing in both male and female ovine fetal fibroblasts to knockout (KO) both alleles of CMAH and GGTA1. Selected double KO (DKO) fibroblast strains were used for somatic cell transfer cloning to produce blastocysts that were transferred into gestational surrogate ewes. Following transfer of 128 male and 40 female cloned blastocysts, 6 male and 8 female lambs were born, however, none of the males survived. Molecular analyses of cells from the five surviving ewes confirmed their compound heterozygous state, resulting in a functional DKO phenotype and subsequent immune rejection of embryonic tissues during natural breeding with wild-type rams. This new DKO sheep model better mimics the human immune status, offering greater physiological relevance for preclinical testing of biological heart valves, alleviation of red meat allergy syndrome due to the presence of dietary xenoantigens, and an alternative source of donor organs that may be culturally more widely accepted than pigs.

cell biology↗

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↗