Methods for Using Small Non-Coding RNAs to Improve Recombinant Protein Expression in Mammalian Cells
The ability to produce recombinant proteins by utilizing different "cell factories" revolutionized the biotherapeutic and pharmaceutical industry. Chinese hamster ovary (CHO) cells are the dominant industrial producer, especially for antibodies. Human embryonic kidney cells (HEK), while not being as widely used as CHO cells, are used where CHO cells are unable to meet the needs for expression, such as growth factors. Therefore, improving recombinant protein expression from mammalian cells is a
... riority, and continuing effort is being devoted to this topic. Non-coding RNAs are RNA segments that are not translated into a protein and often have a regulatory role. Since their discovery, major progress has been made towards understanding their functions. Non-coding RNA has been investigated extensively in relation to disease, especially cancer, and recently they have also been used as a method for engineering cells to improve their protein expression capability. In this review, we provide information about methods used to identify non-coding RNAs with the potential of improving recombinant protein expression in mammalian cell lines. Small non-coding RNAs are primarily short RNA segments that are not translated into a protein. Since their discovery, a great deal of progress has been made towards understanding their function    . The microRNA is an example of a small regulatory non-coding RNA that is approximately 23 nucleotides long in mature form  . The sequence includes a seed region that can promiscuously bind to multiple mRNA molecules and most often represses them by initiating degradation or translation inhibition. Small interfering RNA (siRNA) and short hairpin RNA (shRNA) are also types of non-coding RNA molecules, among many others  . Non-coding RNA has been investigated extensively in relation to disease, especially cancer, and recently have also been used as a method for engineering cells to improve protein expression [19, 20] . Several reviews have been published on the utilization of microRNA for optimizing protein expression from CHO cells. These reviews focused on using microRNAs to engineer process improvements, such as cell growth improvement and apoptosis reduction      . In this review, we provide a discussion of the methods that are used to identify non-coding RNAs with the potential of improving recombinant protein expression in mammalian cell lines. MicroRNA Screening Tools MicroRNAs are currently the most frequently used non-coding RNA for improving CHO and HEK cell protein production capabilities. MicroRNAs can target multiple genes in the same pathway, making them good targets of a specific cell process, such as reducing apoptosis, leading to improved protein production  . Initial work was done in 2007 by Gammell et al.  profiling microRNAs of CHO-K1 suspension cells during batch culture, at two different temperatures by using cross-species microRNA microarrays. Following this work, other investigator began researching the possibilities using microRNAs to improve protein expression and multiple microRNAs were evaluated for the expression of a range of recombinant proteins, as described in the following sections. A variety of screening methods were utilized for identifying specific microRNAs that can potentially improve the expression of proteins. These methods include using previously identified microRNAs, microarrays, microRNA screens, and next generation sequencing (NGS) (See Table 1 ).