Stable CNS Gene Delivery with Sleeping Beauty Armed with a High-capacity HSV Virion (2006)

The central nervous system (CNS) comprises the brain and spinal cord. It is the most complex of all somatic organs and among the most critical for life. Because of the anatomical and functional complexity of the CNS, as well as its very limited innate capacity for regeneration and repair, gene therapy and/or stem cell-based replacement therapy cannot be easily undertaken to treat neurological disorders. However, in this issue of Molecular Therapy, Bowers and colleagues report a new hybrid gene delivery system that possesses tremendous potential for CNS therapy and neuroscience research [1Bowers W.J. et al.Neuronal Precursor-Restricted Transduction via in Utero CNS Gene Delivery of a Novel Bipartite HSV Amplicon/Transposase Hybrid Vector.Mol. Ther. 2006; 13 (this issue.)Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar]. Herpes simplex virus (HSV) is a neurotropic virus that possesses several features suitable for application in the CNS. The virus encodes a large, 150-kb double-stranded DNA genome. Enveloped HSV virions can be actively transported anterogradely and retrogradely in neuronal axons. Finally, the virus has the ability to establish latent infection in sensory neurons [2Frampton A.R. et al.HSV trafficking and development of gene therapy vectors with applications in the nervous system.Gene Ther. 2005; 12: 891-901Crossref PubMed Scopus (78) Google Scholar]. The HSV amplicon vector (see [3Oehmig A. Fraefel C. Breakefield X.O. Update on herpesvirus amplicon vectors.Mol. Ther. 2004; 10: 630-643Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar] for a recent review) is a plasmid-based expression system equipped with the full infection machinery of its parental virus. Although the recent development of the so-called “helper virus-free” packaging systems has eliminated some of the concerns over safety and toxicity [4Fraefel C. et al.Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.J. Virol. 1996; 70: 7190-7197PubMed Google Scholar, 5Saeki Y. et al.Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome.Mol. Ther. 2001; 3: 591-601Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 6Zaupa C. Revol-Guyot V. Epstein A.L. Improved packaging system for generation of high-level noncytotoxic HSV-1 amplicon vectors using Cre-loxP site-specific recombination to delete the packaging signals of defective helper genomes.Hum. Gene Ther. 2003; 14: 1049-1063Crossref PubMed Scopus (56) Google Scholar], the application of the HSV amplicon vector for human gene therapy has been hampered by the transient nature of amplicon-mediated transgene expression, as well as by the absence of a scalable method to produce a high-titer vector. Bowers and colleagues reasoned that the episomal nature of the HSV amplicon vector DNA limited the duration of its transgene expression. They therefore incorporated the well characterized Sleeping Beauty (SB) DNA transposon system [7Izsvak Z. Ivics Z. Sleeping Beauty Transposition: Biology and Applications for Molecular Therapy.Mol. Ther. 2004; 9: 147-156Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar] into the vector, with the aim of inducing stable, long-term transgene expression. The hybrid SB-HSV amplicon system reported in this study comprises two separate amplicon vectors, one expressing the SB transposase and the other carrying an expression cassette flanked by an inverted repeat/direct repeat (IR/DR) from the SB transposon. In experiments using baby hamster kidney (BHK) cells in vitro, the workers demonstrated an approximately 25-fold increase in the frequency of stable transduction using this integration-competent amplicon system compared to conventional amplicons. They also demonstrated that the vector system efficiently integrated the IR/DR-flanked expression cassette randomly into the genomes of nondividing, primary mouse neurons in culture, thereby leading to sustained transgene expression. They then assessed the SB-hybrid amplicon system in utero by injecting the vectors into the lateral ventricle of mouse brains at embryonic day 14.5 (E14.5). Strikingly, on postnatal day 90 (P90), widespread transgene expression was observed throughout the brain, with particularly pronounced expression appearing in the subventricular zone, septofimbrial nucleus, dentate gyrus, hippocampus, and the primary and secondary motor cortices. Examination of the transduced tissue with immunofluorescence microscopy revealed that nearly 100% of the NeuN-positive neurons expressed the β-galactosidase–neomycin (βgeo) fusion reporter gene. Surprisingly, the transgene expression was confined only to doublecortin- or class III β-tublin (TuJ1)-positive immature neurons and to mature NeuN-positive neurons. This suggests that the target of vector transduction at E14.5 must have primarily been multipotent neural stem/progenitor cells residing along the ventricular zone. The SB-mediated transgene integration into the genomes of such neural progenitor cells could well explain the widespread βgeo expression in the P90 brains. Although the molecular mechanisms of the neuronal lineage-restricted transduction of the vectors need to be further investigated, the new integration-competent HSV amplicon vector system holds great potential for a number of applications in neuroscience research. The system may find application for gene marking and genetic engineering of neural stem/progenitor cells, the generation of animal disease models for genetic neurological disorders, and experimental therapies of animal disease models. Although the current study investigated transduction by the vector only in the developing mouse brain, similar analyses will be possible in different organs of developing embryos. With a vector capacity as large as 150 kb now possible with HSV amplicon vectors [8Wade-Martins R. et al.An infectious transfer and expression system for genomic DNA loci in human and mouse cells.Nat. Biotechnol. 2001; 19: 1067-1070Crossref PubMed Scopus (155) Google Scholar, 9Wade-Martins R. Saeki Y. Chiocca E.A. Infectious delivery of a 135-kb LDLR genomic locus leads to regulated complementation of low-density lipoprotein receptor deficiency in human cells.Mol. Ther. 2003; 7: 604-612Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar], it will be very interesting to determine the size limit of the IR/DR-flanked transgene cassette that can be efficiently transposed into host chromosomes by the SB-mediated system. It should be possible to consolidate all the SB transposon components into a single vector by expressing the SB transposase under the control of a tightly regulatable, rapamycin-based dimerizer system [10Wang S. Petravicz J. Breakefield X.O. Single HSV-amplicon vector mediates drug-induced gene expression via dimerizer system.Mol. Ther. 2003; 7: 790-800Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar]. Such consolidation would significantly improve the efficacy of stable transduction and should decrease the risk of nonspecific transposase-mediated DNA rearrangements and extended DNA transpositions. Recently, several papers have reported SB-mediated DNA transposition as a powerful tool for cancer genomics [11Collier L.S. et al.Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse.Nature. 2005; 436: 272-276Crossref PubMed Scopus (341) Google Scholar, 12Dupuy A.J. et al.Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system.Nature. 2005; 436: 221-226Crossref PubMed Scopus (416) Google Scholar]. The ability to deliver the SB transposon system using HSV infection may provide a methodological basis for even better methods of random mutagenesis that can identify oncogenes and tumor suppressor genes. In particular, the technology may be useful for the identification and characterization of genes involved in gliomagenesis. The hybrid SB-HSV amplicon vector system also clearly holds promise for clinical application. However, before this can be realized, several hurdles need to be overcome, including the possible risk of carcinogenesis from the random integration of therapeutic transgenes and, as stated earlier, the lack of a large-scale vector production system. It is hoped that a new methodology can be developed and that SB-mediated transgene integration can be limited to defined genomic loci. To this end, further characterization and development of the SB-hybrid amplicon vector system will be key to realizing this goal.