Establishing a Transgenic Chicken Line Expressing Human Teriparatide Therapeutic Protein for Treating Osteoporosis

Abstract

Osteoporosis is an emerging public health burden in India, affecting around 61 million people, while post-menopausal women accounting for 80% of the affected population. Teriparatide, a recombinant human parathyroid hormone (PTH 1–34), is commonly used for the treatment of severe osteoporosis. It has an anabolic effect on bone by increasing bone mineral density and stability to bone. At present, teriparatide is mainly produced in Escherichia coli, with limited production potential, leading to excessive production cost. In Indian market, the cost of single vial varies ₹ 3,000 to ₹ 30,000, posing unaffordability to patients and becoming as an unmet clinical need. Thus, we hypothesize that chicken could be served as a bioreactor to produce bio-functional and potent-glycosylated teriparatide in egg white albumin thus by reducing manufacturing cost and will be cheaper and affordable to clinically need patients. To achieve this goal, day 10 incubated chicken embryos from Kadaknath indigenous chicken were used to produce a reliable chicken embryonic fibroblast cell (CEFs) line. The sgRNAs were designed at the translational start site of ovoalumin (OVA) locus for introducing the double strand breaks (DSBs) and generated the cloned CRISPR/Cas9 vector. Additionally, the teriparatide targeting vector containing the hPTH 1-34 cDNA sequence was designed and constructed to integrate into the DSB site. Sticky end cloning was performed to ligate respective sgRNAs into the CRISPR/Cas9 vectors and further confirmed its ligation by the colony PCR and Sanger sequencing. Similarly, constructed targeting vector was validated by the double restriction digestion with SacI-HF. Subsequently, CEFs were co-transfected with the ovalbumin CRISPR/Cas9 and human teriparatide targeting vector and antibiotic selection was carried out to forcefully integration of targeting construct. Interestingly, the integration of the donor cassette at the OVA loci was confirmed by the transgenic genotyping PCR band size of 626 bp and further confirmed its site-specific integration by the presence of 5’ and 3’ junction PCRs band size 354 bp and 213 bp in post transfected CEFs. Next, the teriparatide gene copy number and expression pattern will be carried out using southern, western blotting, and ELISA. Overall, this study provides a preliminary framework using CEFs as a genome-editing platform for the development of transgenic chicken bioreactor. In the future, this strategy may be extended to primordial germline stem cells (PGC) and eventually to the generation of transgenic chickens capable of producing teriparatide in egg white, providing a potential cost-effective approach for large-scale therapeutic protein production. Keywords: Osteoporosis; Teriparatide; CRISPR/Cas9 mediated homology-directed repair; Primordial germ cells (PGCs); Transgenic chicken bioreactor.