June 1, 2015 (Vol. 35, No. 11)
GEN Recently Asked a Number of Experts about the Applicability of Different Technologies and Their Limitations
Transfection is the definitive method for deliberately introducing DNA or RNA into cells, most often for gene therapy purposes. There are several technical approaches available for performing a transfection experiment either in vitro or in vivo.
GEN recently asked a number of transfection experts about the applicability of these different technologies as well as their limitations. Their responses appear on the following pages.
GEN: Which transfection techniques (viral vector, gene gun, cationic lipid) seem to work best, and for what tissues? For what genes or diseases?
Mr. Banks: With regard to transfection of small double-stranded RNA for gene knockdown or using plasmids and/or synthetic RNAs for gene knockout into cell lines, the most effective transient transfection method tends to be cationic lipid. This method allows for the widest application across normal and disease cell lines, with low impact on cell viability. For longer term RNA interference (RNAi) or applications in cells that are difficult to transfect, the use of lentiviral vectors for transduction is the most efficient method currently being utilized for cultured cells.
Dr. de Mollerat du Jeu: For the study of Immune diseases, the best technique available is electroporation. Viruses work very well, but they require a lot of upfront work to produce the virus as well as special biosafety labs to handle them. For cancer research, transfection reagents are the method of choice. They are inexpensive, easy to use, and work well in a large spectrum of cancer cells. In neurobiology, viruses (AAV and LV) and transfection reagents seems to dominate the field. In stem cell research, transfection reagents and electroporation are preferred, while in plants, gene gun and viruses (agrobacterium) approaches are mostly used.
Dr. Juckem: Primary cells are often extracted from tissues and frequently used in an in vitro setting, which is more amenable to overexpression and knockdown studies. Chemical transfection methods, broadly categorized as cationic lipids and/or polymers, work best with cells that continue to divide in vitro. It is believed that breakdown of the nuclear envelope during cell division assists plasmid DNA entry into the nucleus. Recombinant virus technologies are important tools for introducing exogenous DNA into nondividing cells. Other physical methods, such as electroporation, excel with cells of hematopoietic origin.
Dr. Nelson: All three techniques have distinct roles in today’s cell biology labs. Cationic lipid transfection is widely used because it is simple, convenient, and works for most cultured mammalian cell lines. X-tremeGENE reagents from Roche are non-liposomal, which promotes high-efficiency and low-toxicity transfection. Viral vector transfections (AAV or lentivirus) offer high transfection efficiency, even with cells refractory to cationic lipid transfections and can be used to drive expression in vivo. Finally, gene gun transfections have low efficiency but excel in environments such as primary tissue, allowing the study of cells with altered gene expression in a more physiologically relevant preparation.
Dr. Peshwa: MaxCyte has developed a scalable flow electroporation technology that is a rapid, automated, regulatory-compliant, cGMP, closed system for cell transfection. MaxCyte’s approach enables control over the on-target, off-tumor toxicity. It is reproducible and enables customized potency [biological activity] of cells. Multiple stem/progenitor and immune cell products, in human clinical trials, validate the ability to obtain high cell viability and transfection efficiencies. Additionally, flow electroporation is routinely used by multiple pharma/biotech companies to transfect physiologically relevant cells for small molecule drug discovery and cell lines for rapid, multigram-scale production of proteins, antibodies, vaccines, virus-like particles, and viral vectors.
Dr. Toell: In vitro transfection of primary animal or human cells can be a valuable step toward therapeutic approaches. It may help reduce animal testing and allow for extrapolation of animal model findings to humans. A growing number of potential therapeutic approaches involve ex vivo transfection of human pluripotent or adult stem cells, or primary cells (e.g., human T cells). Achieving high transfection efficiencies in such cells can be challenging with lipid reagents and might be best achieved by using viruses. An optimized electroporation technique, such as Lonza’s Nucleofector™ technology, could serve as a nonviral alternative because it is as easy to use as lipids but achieves transfection efficiencies closer to virus.
GEN: What are the limitations of the three abovementioned approaches to gene therapy?
Mr. Banks: Delivery of genetic material for gene modulation, either up- or down-regulation, in gene therapy is challenging. Methods of cell disruption, such as gene gun, can prove to be damaging to cells and/or tissues. As stated above, the main hurdle with lipid-induced transfection is the transient nature and the need to repeat the process for a more long-term effect. As with most use of viral particles, the issue can be viral-induced toxicity; however, replication-incompetent viruses can be produced to help minimize this limitation.
Dr. de Mollerat du Jeu: The AAV virus-based approach is the dominating technique in gene therapy, because of their safety profile and efficacy. Electroporation is the method that is used in cell therapy and also vaccine research. Lipid-based approaches are also making an appearance in the field of gene therapy with several clinical trials in progress.
Dr. Juckem: Gene therapy often begins with in vitro models where simpler directed studies can be performed. The efficiency of transfection by chemical methods is dictated by the cell type, whereas many recombinant virus technologies incorporate the G protein from vesicular stomatitis virus, which allows for broad tropism and high transduction levels. Generation of the recombinant virus is labor intensive and depending on the virus system may have size limitations for the gene of interest. Electroporation can frequently yield high transgene efficiency but is hindered by high cellular toxicity and low throughput.
Dr. Nelson: Gene gun transfection is too inefficient and inconsistent for most gene therapy applications, and cationic lipids aren’t cell-type selective (though intriguing for transfections ex vivo). Both adeno-associated virus (AAV) and lentivirus are very efficient, and by selecting viral serotypes and transgene promoters, they can be tuned to affect cell types of interest. Of the two, AAV is limited for packaging exogenous sequence (~5 kb limit vs. ~10 kb for lentivirus), but it diffuses better through the extracellular matrix. One should also consider temporal needs as AAV is transient, but lentivirus integrates into the genome.
Dr. Pshwa: An ideal transfection technology must meet the following performance specifications: it is safe and non-toxic (cell viability, integrity, phenotype, physiology, immunology, and function are not impacted by the transfection method); it results in reproducible, consistent, high-efficiency transfection with robust ability to control targeted cellular bioactivity/function (viz. potency); it is scalable and results in comparable product quality from discovery research (~105 cells) to clinical/commercial (~1011 cells) scale; and it is easy to deploy and cost-effectively operate in cGMP and regulatory environments. MaxCyte’s technology meets all of these.
Dr. Toell: At least for in vitro applications, lipid-based transfection may provide only limited efficiencies for primary or stem cells. While it is very efficient, working with viruses is laborious. In addition, some viruses have limitations in regard to insert size, can cause immunogenicity, or influence cell functionality due to stable integration or induction of interferon responses. Gene gun usually comes with high cell mortality rates and is not suited for suspension cells.