Siddiqui Asim Azhar, Peter Sabrina, Ngoh Eve Zi Xian, Wang Cheng-I, Ng Shirelle, Dangerfield John A, Gunzburg Walter H, Dröge Peter, Makhija Harshyaa
School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
Front Bioeng Biotechnol. 2023 Jun 22;11:1198465. doi: 10.3389/fbioe.2023.1198465. eCollection 2023.
Reliable cell-based platforms to test and/or produce biologics in a sustainable manner are important for the biotech industry. Utilizing enhanced λ integrase, a sequence-specific DNA recombinase, we developed a novel transgenesis platform involving a fully characterized single genomic locus as an artificial landing pad for transgene insertion in human Expi293F cells. Importantly, transgene instability and variation in expression were not observed in the absence of selection pressure, thus enabling reliable long-term biotherapeutics testing or production. The artificial landing pad for λ integrase can be targeted with multi-transgene constructs and offers future modularity involving additional genome manipulation tools to generate sequential or nearly seamless insertions. We demonstrated broad utility with expression constructs for anti PD-1 monoclonal antibodies and showed that the orientation of heavy and light chain transcription units profoundly affected antibody expression levels. In addition, we demonstrated encapsulation of our PD-1 platform cells into bio-compatible mini-bioreactors and the continued secretion of antibodies, thus providing a basis for future cell-based applications for more effective and affordable therapies.
以可持续方式测试和/或生产生物制品的可靠细胞平台对生物技术产业至关重要。利用增强型λ整合酶（一种序列特异性DNA重组酶），我们开发了一种新型转基因平台，该平台涉及一个经过充分表征的单一基因组位点，作为人类Expi293F细胞中转基因插入的人工着陆平台。重要的是，在没有选择压力的情况下未观察到转基因不稳定性和表达变化，从而能够进行可靠的长期生物治疗测试或生产。λ整合酶的人工着陆平台可以用多转基因构建体进行靶向，并提供未来的模块化，涉及额外的基因组操作工具，以产生连续或几乎无缝的插入。我们用抗PD-1单克隆抗体的表达构建体证明了其广泛的实用性，并表明重链和轻链转录单元的方向对抗体表达水平有深远影响。此外，我们展示了将我们的PD-1平台细胞封装到生物相容性微型生物反应器中以及抗体的持续分泌，从而为未来基于细胞的更有效和更经济疗法的应用提供了基础。
