The introduction of biomolecules into cells is a key technology for research in biological sciences. Transfection, which consists of introducing nucleic acids into eukaryotic cells, has enabled advances in many fields, including cell and molecular biology, gene function and regulation studies, and drug target identification and validation. This methodology, based on a simple, yet sophisticated concept, is also the basis for nucleic acid therapeutics, such as gene therapy and DNA vaccination.

Figure 1

Transfection is a standard tool that has been widely used in research for many years. While the introduction of DNA into cells is routine in most laboratories, it is also possible to deliver other biomolecules, such as messenger RNA (mRNA), small interfering RNA (siRNA), micro RNA, small hairpin RNA (shRNA) and antisense oligonucleotides, with comparable efficiencies— in particular using synthetic transfection reagents. The delivery mechanism with a common carrier, such as the cationic polymer polyethylenimine (PEI), consists of several steps that can be broadly divided into uptake, intracytoplasmic delivery and nuclear import (Figure 1). Recent developments have also enabled the delivery of proteins and antibodies to live cells,¹ a novel approach for the study of gene products and signaling pathways. Although widespread, transfection methods are constantly evolving to provide adapted solutions depending on the applications and the delivered biomolecules. The reagents should also offer high-transfection efficiencies and low toxicity, and have minimal impact on basic cellular functions and metabolism upon transfection. They should also overcome several hurdles, including cell entry (Figure 2), release from the endosomes and nuclear transport, when required.

Figure 2

With the fast pace of scientific progress, transfection remains a fast moving technology. The growing use of different cell types, including primary cells, and the use of novel nucleic acids and other biomolecules with therapeutic potential, place significant demands on existing techniques. In addition, increasing requests by researchers encourage novel developments.

Gene therapy

Figure 3

Gene therapy — defined as a method for transferring functional genetic material to cure diseases or to improve the clinical status of a patient — requires therapeutic DNA to be delivered to the nucleus of the cells of a particular organ using a safe and efficient delivery system, adding a level of complexity compared to in vitro transfection. To date, viral-based vectors have been used extensively because of their innate ability to deliver DNA to cells as they naturally infect host cells to replicate. These vectors consist of genetically modified viruses such as replication-incompetent viruses that contain the gene of interest. The most commonly used ones are retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses and pox viruses. The efficiency at which they transduce cells and the potential long-term expression of the transgene explain, in part, why viral vectors represent approximately 68% of ongoing clinical trials to date (Figure 3).²

Despite progress leading to the generation of safer viral vectors, safety considerations, such as pathogenicity, oncogenicity and immune responses in the host, remain a concern. For example, despite clear successes, serious adverse effects have been observed in several SCID-X1 gene therapy trials because of overexpression of a proto-oncogene, which has led to the development of leukemia caused by retroviral vector insertions.³ Other serious adverse effects have been observed with adenoviruses and adeno-associated viruses.³ This has, in part, focused development on physical and nonviral gene delivery systems, which represent 27% of gene therapy clinical trials (Figure 3).^4,5