Advances and perspectives in intracellular biomolecule delivery

July 1, 2008
Jeanne-Francoise Williamson

Pharmaceutical Technology Europe

Pharmaceutical Technology Europe, Pharmaceutical Technology Europe-07-01-2008, Volume 20, Issue 7

The introduction of biomolecules into cells is a key technology for research in biological sciences.

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.

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 1

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.

Figure 2

Gene therapy

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

Figure 3

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

Where possible, researchers apply physical methods to deliver naked DNA or RNA molecules without a carrier, such as electroporation, gene gun and direct injection. These methods are limited to certain organs and have led to other nonviral approaches when increased DNA protection is needed, and when target cells are unable to internalize naked DNA efficiently. There are two major classes of nonviral vectors: cationic lipids and cationic polymers.6 Both molecules condense the DNA and form cationic complexes that recognize anionic proteoglycan and, thus, allow binding to the cell surface receptors and subsequent endocytosis (Figure 1).

The differences between the two mainly lie in downstream processes, including intracellular trafficking through the endosomal pathway. Cationic lipids are able to carry nucleic acids across the various intracellular compartments, but require 'helpers', such as fusogenic lipids or peptides, for the release of the DNA from the endosomes into the cytoplasm. Cationic lipid-based formulations contain several compounds, but most are hydrophobic and induce self-aggregation. In contrast, polymers, such as PEI, present the intrinsic advantage of enhancing endosomal release and nuclear import.7 Moreover, the stability of the DNA/polymer complexes is relatively well controlled, improving in vivo diffusion within the target organs, and delivery efficiency and reproducibility. Such synthetic cationic vectors are less efficient than their viral counterparts and are usually used when transient gene expression is required. However, they are suitable for cancer applications and the treatment of viral infections.

The choice of a delivery system for any experiment requires safety issues and delivery requirements to be counterbalanced for a specific therapy. Novel developments lie in multiple approaches; for example, combining cationic molecules with fusogenic peptides or nanoparticles based on scaffolding technology with cationic lipids or polymers.

DNA vaccination

In addition to gene therapy, a novel approach that has received considerable attention in recent years is the use of DNA for vaccination, where DNA represents a prophylactic drug rather than a therapeutic treatment. Classical vaccination procedures consist of introducing antigens at small doses in the patient to elicit an immune response. With DNA vaccination, the DNA molecule encoding the antigen is delivered to the patient and the protein is produced in situ .8 One major advantage of this method is that it removes the need for production of high quantities of vaccines, and offers interesting openings where other vaccination strategies have failed. However, immunization with plasmid DNA and a nonviral carrier raises novel safety issues, such as indirect protein dose control and overall tolerability, that need to be addressed carefully.

RNA therapeutics

Another rapidly expanding field in line with gene therapy is RNA interference (RNAi) therapeutics. This potential treatment has boomed following the sequential discovery of RNAi in plants,9 worms and mammalian cells.10,11 These studies have uncovered novel gene regulatory mechanisms that have revolutionized the understanding of gene expression and provided powerful tools for biological research and drug discovery.12,13

One approach consists of using nucleic acid molecules, such as siRNA or shRNA, to inhibit gene expression. Such small RNA-based therapeutics may allow the treatment of several human diseases, which are resistant to current drugs, by addressing other targets.14,15

Transfer from research discovery to drug development has been rapid in this field, and several Phase I and II clinical trials have already been completed using local routes of administration to treat ocular neurodegenerative diseases, viral infections and cancer.16,17

The clinical trial using siRNA therapeutics for treatment of age-related macular degeneration has now entered Phase III and has demonstrated proof of concept. Despite these successes, hurdles remain to be overcome to translate the wide therapeutic potential of RNAi to clinical reality. The principal challenge to broaden the application of RNAi in therapeutics consists of overcoming targeting and delivery issues. The most common delivery methods for siRNA in the clinical and preclinical studies are naked siRNA, but several investigational new drug applications that use viruses and synthetic vectors (lipid-or polymer-based) are pending, leaving room for vector development.

Finding success

Several biotech and pharmaceutical companies have exploited the technological progress in molecular and cell biology, as well as in therapeutics, but the determination of leading compounds remains a crucial choice. While some companies have based their expansion on using lipofectans, which are limited by their administration route, others have expanded from a solid in vitro basis using PEI.18 The versatility of PEI greatly facilitates the transfer from cells to the clinic.

Increasing scientific successes with nonviral gene delivery methods, in conjunction with safety concerns regarding currently used viral methods, have advanced preclinical projects and clinical trials with carriers such as licensed PEI. This has led to GMP manufacturing demands earlier in the developmental pipeline that, in addition to evolving regulatory guidance in the industry regarding purity, quality control and analytical requirements for clinical-grade reagents, have imposed stringent rules for suppliers. To set a standard for GMP-grade reagents based on cationic polymer technology for therapeutic or prophylactic clinical trials using nucleic acid based therapy, suppliers need to take these requirements into account early during the development of innovative carriers.

With an increasing number of preclinical studies using in vivo jetPEI and clinical trials moving into late phases, the therapeutic and vaccination market for licensed PEI is expanding. Such high-grade products only represent a component of therapeutic agents, namely the drug substance, providing a nonviral carrier for the delivery of therapeutic nucleic acids, such as DNA and siRNA. With DNA-based therapy being up and running with Phase I and II clinical trials using in vivo jetPEI,19 it is time for small biotechnology companies to focus on innovative programmes to develop siRNA delivery in vivo in collaboration with well-established partners.20

However, therapeutic approaches using nucleic acids must adapt to the challenges of emerging diseases that are resistant to current treatments. One method consists of exploiting combinatorial therapies, where conventional treatments, such as surgery, chemotherapy and radiotherapy, are coupled with nucleic acid DNA/siRNA-based drugs.

Another route for alternative therapeutics consists of individually tailored treatments. The availability of gene banks and gene profiling enables the development of custom-produced gene therapies in situ and ex vivo . The use of viral vectors is too expensive and time consuming for such approaches, and versatile nonviral carriers represent more amenable methods.

The demand for GMP-grade reagents is increasing steadily in biomanufacturing; for example, high-quality transfection reagents are required to manufacture therapeutic viruses used in the clinic where production protocols have become more stringent. Moreover, transfection and biomolecule delivery to mammalian cells represent a means of producing a range of human therapeutic molecules, such as recombinant proteins, monoclonal antibodies and vaccine antigens, on a large scale.

Maintaining quality

Pharmaceutical drugs produced from biotechnology represent a rapidly growing market and currently account for 10–20% of all marketed drugs. During the next few years, this is expected to rise to more than 50%, stimulating a demand for improved production and manufacturing techniques. Given the suitability of chemically defined transfection reagents, such as licensed PEI for biomanufacturing, suppliers must ensure high-quality production as the market develops.

The rapidly growing nucleic acid therapeutics market is currently most active in the US (67%) and Europe (27%), where fundamental research provides a solid basis for clinical development. However, rapidly emerging economies in the Far East and Asia (currently 3%) are likely to soon become active players. Limitations may depend on regulatory requirements in the different countries and on the ability to transfer clinical trials into marketable drugs.

Amazing advances have been made during the last two decades in intracellular biomolecule delivery in vitro . The translation to therapeutics and to the clinic, where biological complexity adds to the hurdles at the cellular level, is proving challenging and further technological developments remain to be made.

Jeanne-Françoise Williamson is the Technical Support and Scientific Communication Manager at Polyplus-transfection (France).


1. C.O. Weill et al ., Cytotechnology, 56(1), 41–48 (2008).

2. M. Cavazzana-Calvo et al ., Science, 288 (1), 669–672 (2000).

3. J.M. Wilson, Human Gene Therapy, 19(1), 1–2 (2008).

4. D.J. Glover, H.J. Lipps and D.A. Jans, Nat. Rev. Genet ., 6(4), 299–310 (2005).

5. S.D Li and L. Huang, Gene Ther., 13(18), 1313–1319 (2006).

6. T.G. Park et al ., Adv. Drug Deliv. Rev., 58(4), 467–486 (2006).

7. S. Bunner et al., Molecular Therapy, 5(1), 80–86 (2002).

8. M. Foldvari, S. Babiuk and I. Badea, Curr. Drug Deliv ., 3(1), 17–28 (2006).

9. C. Napoli, C. Lemieux and R. Jorgensen, The Plant Cell , 2(4), 279–289 (1990).

10. A. Fire et al., Nature, 391(6669, 806–811 (1998).

11. S.M. Elbashir et al., Nature, 411(6836), 494–498 (2001).

12. D. Bumcrot et al., Nat. Chem. Biol., 2(12), 711–719 (2006).

13. E. Iorns et al., Nat. Rev. Drug Discov., 6(7), 556–568 (2007).

14. J. Hall, Nat. Rev. Genet., 5(7), 552–557 (2004).

15. D.H. Kim and J.J. Rossi, Nat. Rev. Genet., 8(3), 173–184 (2007).

16. A. Aigner, J. Biotechnol., 124(1), 12–25 (2006).

17. A. de Fougerolles et al., Nat. Rev. Drug Discov., 6(6), 443–453 (2007).

18. O. Boussif et al., Proc. Natl. Acad. Sci. USA, 92(16), 7297–7301 (1995).

19. P. Ohana et al., Gene Therapy and Molecular Biology, 8(1), 181–192 (2004).

20. A.L. Bolcato-Bellemin et al., PNAS, 104(41), 16050–16055 (2007).


Thank you to Patrick Erbacher for his input on this paper, and to Anne-Lise Monjanel, Géraldine Guérin-Peyrou, Anne-Laure Bellemin and Claire Weill for proofing the manuscript.