The Message is Sufficient: Towards Nontoxic Delivery of Endonucleases
Authors: Christopher Baum
Journal: Molecular Therapy (2006)

The development of leukemogenesis following insertional mutagenesis in a recent gene therapy trial treating X-SCID patients with a retroviral vector has led to increased interest in nonviral systems that can mediate stable integration of transgenes into the chromosomes of mammalian cells. As is the case with retroviral delivery systems, nonviral alternatives require an endonuclease to bind and paste the donor DNA into an accessible site of the cellular genome. Nonviral systems offer two major promises. The first is that the use of physicochemical methods for gene delivery are less likely than viral vectors to cause immune reactions. The second is that nonviral methods allow us to circumvent the restrictions on our ability to design alternative endonucleases that are able to be efficiently packaged into viral particles and interact with the retroviral genome. Because the constraints of viral particle formation no longer apply, it might be relatively easy to use designer endonucleases that mediate rather neutral or, if possible, sequence-specific integration [1Ginsburg D.S. Calos M.P. Site-specific integration with phiC31 integrase for prolonged expression of therapeutic genes.Adv. Genet. 2005; 54: 179-187Crossref PubMed Scopus (47) Google Scholar, 2Cathomen T. Weitzman M.D. Gene Repair: Pointing the finger at genetic disease.Gene Ther. 2005; 12: 1415-1416Crossref PubMed Scopus (13) Google Scholar]. However, long-term expression of the endonuclease needs to be avoided so as to prevent genotoxic side effects resulting from its persistent or off-target activity. Studies employing the site-specific Cre recombinase or recombinant Zinc-finger nucleases have provided evidence for toxic side effects resulting from ectopic expression of these endonucleases [3Loonstra A. et al.Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells.Proc. Natl. Acad. Sci. USA. 2001; 98: 9209-9214Crossref PubMed Scopus (458) Google Scholar, 4Pfeifer A. et al.Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo..Proc. Natl. Acad. Sci. USA. 2001; 98: 11450-11455Crossref PubMed Scopus (193) Google Scholar, 5Silber D.P. Livingstone D.M. Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity.Mol. Cell. 2001; 8: 233-243Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 6Galla M. et al.Retroviral pseudotransduction for targeted cell manipulation.Mol. Cell. 2004; 16: 309-315Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 7Alwin S. et al.Custom zinc-finger nucleases for use in human cells.Mol. Ther. 2005; 12: 610-617Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar]. Retroviruses escape the issue of persistent endonuclease expression by mediating proteolytic maturation of their integrase enzyme only after budding of the virion particles from the producer cells. Replication-deficient retroviral vectors thus carry integrase activity in the form of a protein that associates with a specific recognition motif in the proviral DNA prior to entry of the vector into the nucleus. In contrast, physicochemical methods for stable transgene insertion typically express the endonuclease from plasmids that are transiently cotransfected or codelivered along with the donor plasmids that contain recognition motifs for the endonuclease. A productive ménage a trois of endonuclease, donor plasmid and cellular DNA requires a stochastic rendez-vous in the rather complex nuclear environment of the transduced cell. For this to occur with any efficiency, it is necessary to reach a particular threshold level of both endonuclease protein and donor plasmid in the target cells in order for highly efficient integration to take place [7Alwin S. et al.Custom zinc-finger nucleases for use in human cells.Mol. Ther. 2005; 12: 610-617Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 8Izsvak 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 (186) Google Scholar]. This requirement underlies the intense interest in the development of improved methods of endonuclease delivery in the field of gene transfer technology. In this issue of Molecular Therapy, McIvor and colleagues show that in order to promote integration of a transposon DNA, it is sufficient to deliver the corresponding transposase, in this case Sleeping Beauty, in the form of a recombinant mRNA [9Wilber A. et al.RNA as a source of transposase for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues.Mol. Ther. 2006; 13 (this issue.)Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar]. That the message is sufficient for endonuclease delivery has previously been demonstrated using the Cre and Flp recombinases, which in contrast to Sleeping Beauty require relatively low levels of expression to function [6Galla M. et al.Retroviral pseudotransduction for targeted cell manipulation.Mol. Cell. 2004; 16: 309-315Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Ponsaerts P. et al.Messenger RNA electroporation is highly efficient in mouse embryonic stem cells: successful FLPe- and Cre-mediated recombination.Gene Ther. 2004; 11: 1606-1610Crossref PubMed Scopus (16) Google Scholar]. The study by McIvor and colleagues is the first to use this approach for a transposase, and the first to demonstrate the potential utility of mRNA-mediated endonuclease delivery in vivo. Clearly a promising experimental result, but can we envisage its translation for clinical application? The data described in this issue's report point to some potential limitations that should be the focus of future studies. In vitro, the highest efficiency obtained with mRNA-mediated transposase delivery was only nine times greater than background integration [9Wilber A. et al.RNA as a source of transposase for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues.Mol. Ther. 2006; 13 (this issue.)Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar], which remains several orders of magnitude below the efficiency observed when using retroviral (including lentiviral) vectors. How can the method be improved? Delivering more mRNA might help when treating cultured cells, but appears unrealistic in vivo. In the current study, the authors injected 100 µg of purified mRNA per mouse, which would correspond to several hundred grams in an adult patient. To my knowledge, production of purified mRNA at such a scale is not feasible at this time. Moreover, mRNA delivery could impact the biology of the target cell, as discussed by McIvor and colleagues, and as has been observed in studies using dendritic cells [11Van Meirvenne S. et al.In vivo depletion of CD4+CD25+ regulatory T cells enhances the antigen-specific primary and memory CTL response elicited by mature mRNA-electroporated dendritic cells.Mol. Ther. 2005; 12: 922-932Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar], such that a further increase in dose does not appear to be a wise solution. Messenger dose reduction might be achievable using an endonuclease/transposase with higher specific activity. Moreover, particle-mediated mRNA delivery systems could increase the effective concentration in the target cells by more efficient targeting of the relevant cell type. Retroviral particles deficient in reverse transcription are a paradigm for this approach, but certainly not the only solution we might envisage [6Galla M. et al.Retroviral pseudotransduction for targeted cell manipulation.Mol. Cell. 2004; 16: 309-315Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar]. Finally, one might wish to explore the utility of recombinant proteins as an alternative to mRNA, as previously shown for Cre recombinase [12Jo D. et al.Epigenetic regulation of gene structure and function with a cell-permeable Cre recombinase.Nat. Biotechnol. 2001; 19: 929-933Crossref PubMed Scopus (128) Google Scholar, 13Peitz M. et al.Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes.Proc. Natl. Acad. Sci. USA. 2002; 99: 4489-4494Crossref PubMed Scopus (270) Google Scholar, 14Will E. et al.Unmodified Cre recombinase crosses the membrane.Nucleic Acids Res. 2002; 30: e59Crossref PubMed Scopus (58) Google Scholar]. The added value of protein-mediated delivery might be the potential to associate donor plasmid and endonuclease prior to cell entry, inspired by the retroviral principle. The study presented by McIvor and colleagues represents an important step towards nontoxic transgene insertion, and the toolbox remains stocked with a number of attractive instruments that should eventually help us to reach this goal in the clinical setting.