A combinatorial approach for robust transgene delivery and targeted expression in mammary gland for generating biotherapeutics in milk, bypassing germline gene integration

Abstract

Protein expression in the milk of transgenic farmed animals offers a cost-effective system for producing therapeutics. However, transgenesis in farmed animals is not only cumbersome but also involves risk of potential hazard by germline gene integration, due to interruptions caused by the transgene in the native genome. Avoiding germline gene integration, we have delivered buffalo β-casein promoter-driven transgene construct entrapped in virosomes directly in the milk gland through intraductal perfusion delivery. Virosomes were generated from purified Sendai viral membrane, containing hemagglutinin-neuraminidase (HN) and fusion factor (F) proteins on surface (HNF-Virosomes) which initiate membrane fusion, devoid of any viral nucleic acids. Intraductal delivery of HNF-Virosomes predominantly transfected luminal epithelial cells lining the milk duct and buffalo β-casein promoter of the construct ensured mammary luminal epithelial cell specific expression of the transgene. Mammary epithelial cells expressed EGFP at lactation when egfp was used as a transgene. Similarly, human interferon-γ (hIFN-γ) was expressed in the mammary gland as well as in the milk when hIFN-γ was used as a transgene. This combinatorial approach of using Sendai viral membrane-derived virosomes for entrapment and delivery of the transgene and using buffalo β-casein promoter for mammary gland specific gene expression provided a better option for generating therapeutic proteins in milk, bypassing germline gene integration avoiding risks associated with animal bioreactor generated through germline gene integration.

Goat mammary epithelial cells provide a better expression system for production of recombinant human bone morphogenetic protein 2 compared to Chinese hamster ovarian cells

“…Immunocytochemistry was performed on cultured GMECs as per the protocol described by Ganguli et al 24 Briefly, cells were seeded on glass coverslips after the coverslips were sterilised in absolute ethanol, air dried and placed in sterile 12‐well plates. Cells were grown in 37°C in a humidified CO 2 incubator until they were 70‐80% confluent.…”

An easy method for developing fusion enabled SARS-CoV2 virus fusion mimic (SCFM), bypassing the need of Bio Safety Level (BSL) facility

“…The lipid bilayer structured mammalian cell membrane [ 9 ] can be used for mimicking the viral fusogenic property if studded with additional viral proteins that help in interaction followed by fusion with host cells. Previously, we developed a nonreplicating virosome consisting of Sendai virus membrane that can fuse with mammary epithelial cells based on the specific interaction of receptor present on the viral membrane with its ligand on mammary epithelial cells for the delivery of transgene [ 10 ]. Here, we report the generation of fusion mimics of SARS-CoV2 virus based on cultured mammalian cells, which is fusion enabled with its specific target cells like SARS-CoV2 virus, through specific receptor–ligand interaction.…”