The distribution of glycans on a monoclonal antibody impacts its function and half-life. Earlier this month, Masue Marbiah, PhD, a postdoctoral researcher at Imperial College London who develops biosensors intended for improving the efficiency of producing biotherapeutics, and her colleagues reported: “To facilitate glycoengineering of IgG-producing Chinese hamster ovary cells, we present a rapid protocol that involves the use of RNA interference for the knockdown of genes of interest coupled with capillary gel electrophoresis and laser-induced fluorescence detection (CGE-LIF) for fast, high-throughput glycan analysis.”
The key to this work was the implementation of a design-of-experiments approach, which was used “to find the optimal conditions for siRNA-mediated gene silencing,” Marbiah explains.
“Interestingly, the reduction of core fucosylation reflected incremental changes in siRNA concentration and duration of the experiment,” she adds. “This observation highlights the monogenic nature of core fucosylation and also provides a window into the kinetics of siRNA processing and Fut8 protein function.”
Using RNA interference comes with crucial benefits. “Gene modification tools are typically comprised of two parts: a template and an effector protein,” Marbiah says. “RNAi uses endogenous effector proteins—RNA-induced silencing complex, RISC, thereby circumventing the need to introduce an effector such as Cas9 in CRISPR systems.” Consequently, there is no need to optimize the levels of the effector protein.
In addition, scientists can purchase siRNA “as custom or predesigned sequences in a variety of scales and delivery formats,” Marbiah notes. “This means someone with little experience in siRNA design can easily perform RNAi screening experiments.”
No perfect approach
But no method is perfect. “Like other templated gene-modification tools, RNA interference has the potential for off-target effects,” Marbiah says. “Design algorithms use bioinformatics to minimize unintentional gene targeting but this should be confirmed experimentally by the end user.”
Plus, siRNA degrades over time. Still, Marbiah points out: “Experiments like ours with a limited duration are unaffected by this inadequacy, but degradation is considered one of the major hindrances to the suitability of siRNA as a clinical therapeutic.”
Shortcomings aside, the siRNA-based method developed by Marbiah and her colleagues could be used to make more effective therapeutic antibodies. These biotherapeutics could also be designed for longer-term potency.