For the generation of stable cell lines expressing multiple genes such as the light and heavy chains of IgGs, it is often desirable and necessary to express multiple genes from a single vector. The major advantage of multicistronic vectors is that they ensure multiple genes expressed in the same cells transfected. In this regard, multicistronic vectors are superior than co-transfection of monocistronic vectors. An added benefit of multicistronic vectors is the convenience and lower cost of DNA manufacturing. In the cases of cGMP DNA production for gene therapy or DNA vaccines, production of one multicistronic plasmid DNA is significantly cheaper and faster than production of multiple monocistronic plasmids.
There are several technical challenges for multicistronic vectors. The major challenge is transcriptional interference among the gene expression cassettes from the same multicistronic vectors. Gene expression cassettes often interfere with one another when placed closely together. How to arrange multiple gene expression cassettes in a manner that ensure independent and efficient expression is so far more art than science. The second issue is the size of the vector. To accommodate multiple promoters, enhancers, and poly A sequences, the vectors can become very large, perhaps 10 kb or greater. Larger size may lead to difficulty in plasmid DNA scale up and mammalian cell transfection. Thirdly, as multiple pieces of DNA fragments needed to be cloned in the vector, convenience restriction enzyme sites often become very limited.
LakePharma's multicistronic gene expression vector technology can effectively overcome these challenges. Having generated and tested numerous expression cassettes in various orientations and configurations, LakePharma has a clear grasp of how expression cassettes affect one another, and the rank and hierarchy of different positions. More importantly, LakePharma has established multicistronic vectors that keep transcritional interference to the minimal. As of press, LakePharma has successfully constructed vectors producing up to four different genes of interest, and has observed efficient expression for each of the four genes. Even with four expression cassettes, these vectors but still managed relatively small in size (~6 kb). Restriction enzyme sites are conveniently positioned so incorporation of genes of interest is not a difficulty at all.