The increasing global demand for biotherapeutics, including recombinant proteins, monoclonal antibodies, cytokines, growth factors, and next-generation biologics, has created an urgent need for efficient, scalable, and cost-effective biomanufacturing platforms. Mammalian cell systems remain the preferred choice for therapeutic protein production because of their ability to perform complex post-translational modifications, ensure proper protein folding, and secrete biologically active molecules. Among these systems, Chinese hamster ovary (CHO) cells have dominated the biopharmaceutical industry for several decades. However, conventional production hosts still face significant limitations, including genomic instability, heterogeneous glycosylation, metabolic inefficiency, productivity decline during long-term culture, and expensive manufacturing processes.

To address these challenges, our work focuses on developing a biologically specialized mammalian expression platform. We focused on generating 1) an efficient ubiquitous promoter for robust expression of proteins, 2) an efficient signal peptide for better posttranslational modification and increased secretion of proteins, and 3) a better cellular system to increase yield.

The existing widely used viral ubiquitous promoters face various limitations in driving robust transgene expression in vitro and in vivo. To overcome this, the human elongation factor 1 alpha 1 (hEF1α) promoter is used as an alternative, but it has also been shown to be silenced epigenetically in vivo in mice. In this study, we explored the possibility of using the elongation factor 1 alpha 1 promoter from the Indian River Buffalo (bbEF1α1) as an alternative. In silico analysis revealed that the bbEF1α1 promoter has numerous transcription factor-binding sites and fewer CpG islands, suggesting reduced susceptibility to silencing and robust transgene expression. Our experiments showed that bbEF1α1 drove significantly stronger EGFP expression than CMV and hEF1α promoters in across different cell lines. Additionally, transgenic mice generated with bbEF1α1 promoter showed robust EGFP expression in most of the major organs highlighting its potential for reliable gene expression.

The yield of therapeutic proteins in mammalian cell-based production systems is often limited by inefficient secretion and reduced bioactivity caused by incompatible secretory signal peptides (SPs). Therefore, a broadly applicable signal peptide that can be fused to different proteins of interest to enhance their secretion and overall production is needed. To address this challenge, we engineered a robust, broadly applicable Unique Secretory signal PepTide (UniSePT) by combining SP sequences from β-casein (CSN2) and β-lactoglobulin (BLG) proteins of Indian river buffalo. UniSePT demonstrated higher secretion efficiency than commonly used SPs, such as human serum albumin and preproinsulin SP. Importantly, this improved secretion does not compromise the bioactivity of the therapeutic proteins produced. Additionally, UniSePT showed minimal cleavage variability, supporting its compatibility with various therapeutic proteins. The newly developed UniSePT enhances the titer and bioactivity of therapeutic proteins in mammalian expression systems. Its broad applicability and improved secretion efficiency make it a promising tool for industrial-scale production, potentially reducing the cost of therapeutic protein production.

Mammary epithelial cells are naturally adapted for high-level protein synthesis and secretion during lactation, making them highly attractive candidates for therapeutic protein manufacturing. Their intrinsic secretory machinery, epithelial polarity, and capability for complex post-translational processing provide a strong biological foundation for advanced biomanufacturing applications.

Using precision CRISPR/Cas9 genome engineering, we developed an immortalized goat mammary luminal epithelial cell platform (ImGMLEC) through targeted integration of goat telomerase reverse transcriptase (gTERT) into the endogenous KRT19 locus using a microhomology-mediated end-joining (MMEJ)-assisted knock-in strategy. Unlike traditional immortalization approaches that rely on random genomic integration or oncogenic transformation, our lineage-specific genome engineering strategy preserves epithelial identity while enabling stable long-term proliferation.

The KRT19 locus was specifically selected because it is highly enriched in mammary luminal secretory epithelial cells and associated with active protein synthesis and secretion. By targeting immortalization to this lineage-specific locus, we established a homogeneous and functionally specialized epithelial cell population optimized for recombinant protein production.

The engineered ImGMLEC platform demonstrated:

• Stable long-term proliferation across extended passages

• Preservation of luminal epithelial characteristics

• Maintenance of functional cellular responses

• Enhanced recombinant therapeutic protein secretion

• Improved production efficiency compared with conventional mammalian hosts

• Sustained genomic and phenotypic stability during prolonged culture

To evaluate its industrial potential, the platform was tested for recombinant human interferon-gamma (hIFNγ) production and showed significantly higher secretion levels compared with widely used mammalian cell systems such as CHO, HEK293T, and HepG2 cells under comparable transgene conditions. Importantly, the secreted proteins retained biological functionality, confirming proper folding and post-translational processing within the engineered cells.

Beyond recombinant protein production, this platform provides a versatile foundation for future synthetic biology, metabolic engineering, and precision bioprocess optimization strategies. The system can potentially be adapted for:

• Monoclonal antibody production

• Cytokine and growth factor manufacturing

• Biosimilar development

• Vaccine antigen expression

• Cell-based bioreactor engineering

• Precision glycoengineering

• Secretome and exosome-based therapeutics

Our long-term vision is to establish biologically specialized mammalian cell factories capable of supporting next-generation biopharmaceutical manufacturing with improved productivity, scalability, and cost efficiency. This work represents a significant step toward transforming lineage-specific primary cells into robust industrial biomanufacturing platforms through precision genome engineering.