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  • Review Article
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Towards safe, non-viral therapeutic gene expression in humans

Nature Reviews Genetics volume 6, pages 299–310 (2005)Cite this article

Key Points

  • The development of a leukaemia-like disorder in three patients in a recent retroviral-based clinical trial underlines the need for safe and efficient approaches for achieving gene restoration.

  • Non-viral vectors, with their safety and manufacturing advantages, have been held back from clinical use because of their poor in vivo efficiency and their inability to confer long-term expression of therapeutic genes. Recent advances in our understanding of viral integrases and episomal replication might be applied to non-viral vectors, offering, for the first time, the possibility of achieving sustained non-viral gene expression.

  • Site-specific recombinases from bacteriophages and other sources have been exploited — in conjunction with non-viral vectors — to catalyze the integration of therapeutic genes into safe, defined genome locations. This approach allows genetic disorders to be corrected but avoids the risk of insertional mutagenesis.

  • So that they can be used in mammalian cells, the activity of site-specific recombinases from prokaryotic origins may be enhanced by the addition of nuclear import sequences, and by using directed evolution to alter the specificity of the recognized site of integration.

  • Rep integrase isolated from the adeno-associated virus is able to mediated efficient site-specific integration into the human genome at a safe site on chromosome 19. Recent studies have focused on overcoming problems of Rep cytotoxicity.

  • By using scaffold/nuclear matrix attachment region sequences, plasmids have been created that can replicate and segregate during mitosis in an extrachromosomal (episomal) form without either requiring viral proteins or interfering with the host DNA.

  • Most engineered human artificial chromosomes are either unstable or are difficult to fully characterize, but neocentromere-based minichromosomes have recently been created that are stable and structurally defined, showing great potential for gene therapy.

  • The various new advances make it possible for the first time to achieve long-term therapeutic gene expression in humans through non-viral approaches.

Abstract

The potential dangers of using viruses to deliver and integrate DNA into host cells in gene therapy have been poignantly highlighted in recent clinical trials. Safer, non-viral gene delivery approaches have been largely ignored in the past because of their inefficient delivery and the resulting transient transgene expression. However, recent advances indicate that efficient, long-term gene expression can be achieved by non-viral means. In particular, integration of DNA can be targeted to specific genomic sites without deleterious consequences and it is possible to maintain transgenes as small episomal plasmids or artificial chromosomes. The application of these approaches to human gene therapy is gradually becoming a reality.

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Figure 1: Site-directed integration of foreign DNA.
Figure 2: Site-specific recombination mechanisms.
Figure 3: Site-specific integration mediated by the adeno-associated virus Rep protein.
Figure 4: Structure and visualization of an episomally replicating plasmid (pEPI) and a human artificial chromosome (HAC).

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Acknowledgements

The authors acknowledge the support of the National Health and Medical Research Council to D.A.J., and of the Deutsche Forschungsgemeinschaft and the European Union to H.-J.L. The authors are also indebted to Michele Calos for critical reading of the manuscript, and to Isa M. Stehle, Lee Wong and Richard Saffery for the FISH illustrations shown in Figure 4.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Nuclear Signalling Laboratory, Monash University, 3800, Victoria, Australia

    Dominic J. Glover & David A. Jans

  2. Institute of Cell Biology, Witten/Herdecke University, Witten, 58448, Germany

    Hans J. Lipps

  3. Australian Research Council Centre of Excellence for Biotechnology and Development, Australia

    David A. Jans

Authors
  1. Dominic J. Glover
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  2. Hans J. Lipps
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Correspondence to David A. Jans.

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DATABASES

OMIM

cystic fibrosis

Duchenne muscular dystrophy

Lesch-Nyhan syndrome

Nijmegen breakage syndrome

RDEB

SCIDX1

FURTHER INFORMATION

Agence Française de Sécurité Sanitaire des Produits de Santé press release

David Jans' laboratory

Hans J. Lipps' laboratory

Journal of Gene Medicine Clinical Trials web site (Wiley Database)

Glossary

RETROVIRUS

A family of RNA viruses that replicate by reverse transcription and then integrate into the host genome.

ADENOVIRUS

A DNA-virus that replicates in the nucleus in an extra-chromosomal form.

X-LINKED SEVERE COMBINED IMMUNE DEFICIENCY

(SCIDX1). Disorder characterised by the absence of an immune system, caused by mutations that result in a failure to make a protein essential for T- and B-cell function.

LONG TERMINAL REPEAT

(LTR). A long DNA sequence, repeated at each end of retroviral DNA. An LTR contains regulatory sequences that are required to initiate transcription of the viral DNA into an RNA form that is inserted into new viral particles.

PROVIRUS

A stage in the life cycle of some viruses in which viral DNA has been inserted into the chromosome of the host cell.

INTERLEUKIN 2 RECEPTOR γ

The IL2RG protein is located on the surface of blood-forming cells and is a receptor for the ligand IL2, crucial for directing the growth and activation of cells of the immune system.

LIPOSOME

A spherical lipid-bilayer vesicle that can enclose DNA for transport into cells.

DNA COMPACTION

The reduction in the volume occupied by a DNA molecule caused by the addition of multivalent cations such as polyamine to the DNA.

EPISOME

A stable DNA molecule that persists in the nucleus without integrating into the cellular genome.

LYSOGENIC

A phase of the virus life cycle during which the virus integrates into the host chromosome of the infected cell, often remaining dormant for some period of time.

PARTIAL HEPATECTOMY

Removal of two-thirds of the liver, which stimulates liver cell division resulting in the removal of almost all unintergrated DNA from the liver.

RECESSIVE DYSTROPHIC EPIDERMOLYSIS BULLOSA

A blistering skin condition in which the filaments that anchor the epidermis to the underlying dermis are either absent or do not function. This is due to defects in the gene for type VII collagen, a fibrous protein that is the main component of the anchoring filaments.

FLUORESCENCE-ACTIVATED CELL SORTING

A cell-separation technique that is based on differences in physical and chemical properties of cells, such as the degree of fluorescence conferred by cell surface labelling.

INVERTED TERMINAL REPEATS

Repeat sequences in reverse orientation that serve as the viral origins of replication. ITRs are essential for virus packaging and rescue of the integrated viral genome.

CYTOSTATIC

Inhibiting or suppressing cellular growth and multiplication.

NUCLEAR MATRIX

The dense fibrillar network within the nucleus to which loops of chromatin attach.

ALPHA-SATELLITE DNA

A class of ∼170 bp repeating sequences of nucleotide pairs, found at centromeres.

MICROCELL-MEDIATED CHROMOSOME TRANSFER

A technique to deliver chromosomes into cells, whereby the chromosomes of donor cells are partitioned into discrete subnuclear packets. These microcells can be fused with recipient cells, resulting in transfer of chromosomes.

CYTOSINE METHYLATION

The addition of methyl groups (-CH3) to cytosine residues; This has a role in transcriptional silencing in higher organisms.

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Glover, D., Lipps, H. & Jans, D. Towards safe, non-viral therapeutic gene expression in humans. Nat Rev Genet 6, 299–310 (2005). https://doi.org/10.1038/nrg1577

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