Fluorescence Methods Reveal Intracellular Trafficking of Gene Transfer Vectors: The Light toward the End of the Tunnel
Authors: Philip L. Leopold
Journal: Molecular Therapy (2000)

My father has a way of tempering undue optimism by reminding me that the proverbial “light at the end of the tunnel” could be the headlamp of an oncoming locomotive. At times, the quest to find a perfect vector for gene therapy might well be viewed with a touch of my father's trepidation … and good humor. A major goal in the field of nonviral vectors for gene therapy is improved intracellular targeting efficiency, a property inherent in viral vectors. However, it is difficult to make rational decisions on vector design unless one knows something of the fate of the vector once it enters the cell. To aid in the characterization of gene transfer vectors in cells, several groups have employed analytical methods based on fluorescence. We are all familiar with methods such as indirect immunofluorescence, fluorescence in situ hybridization, and conjugation of fluorophores to vectors to evaluate the intracellular disposition of the vector. However, other fluorescence-based methods that have been well established in other fields of basic research are now being brought to bear on problems in this field. The attractions of fluorescence methods are manifold: (i) fluorescence methods have high signal-to-noise ratios allowing quantitative detection of subtle changes in the sample, (ii) data acquisition based on optical methods is rapid, (iii) the ability to introduce fluorescent compounds into a variety of molecules enables versatility, and (iv) optical methods do not necessarily require harsh conditions for detection and thus permit data acquisition from living samples. One example of the novel application of a fluorescence method for analyzing gene transfer vectors is found in this issue of Molecular Therapy. James and Giorgio (1James M.B. Giorgio T.D. Nuclear-associated plasmid, but not cell-associated plasmid, is correlated with transgene expression in cultured mammalian cells.Mol. Ther. 2000; 1: 339-346Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) have adapted flow cytometry to extract data on the intracellular trafficking of a lipid–DNA complex. Flow cytometry is normally utilized to resolve data at the level of the whole cell. By combining subcellular fractionation of nuclei with flow cytometry, the authors were able to make relevant observations regarding the intracellular fate of a fluorescently labeled plasmid, finding that the relative amount of nuclear-associated fluorescence in two cell lines corresponded to the comparative transfection efficiency. The authors pressed their technology further by performing an absolute quantitation of both cell-associated and nuclear-associated plasmid such that the physical number of plasmids per cell or per nucleus could be determined. It is precisely this quantitative, detailed picture of the fate of the vector that will permit evaluation of subsequent iterations in vector design. Other prominent examples of novel applications of fluorescence technology to vector evaluation in cells have recently emerged. The first step in evaluating the fate of an internalized vector is to ensure that the vector is, in fact, inside the cell. Chu et al. (2Chu Q. Tousignant J.D. Fang S. Jiang C. Chen L.H. Cheng S.H. Scheule R.K. Eastman S.J. Binding and uptake of cationic lipid:pDNA complexes by polarized epithelial airway cells.Hum. Gene Ther. 1999; 10: 25-36Crossref PubMed Scopus (40) Google Scholar) took advantage of a fluorescence quenching assay to demonstrate internalization of a lipid–DNA complex and showed that vector internalization was exceedingly rare at the apical surface of an intact monolayer of airway epithelial cells. Once inside of cells, vectors must escape from endosomes to the cytosol. Our own work has employed the principle of pH-sensitive fluorescence emission in combination with adenovirus vectors that have been covalently modified by addition of a fluorescein derivative (3Leopold P.L. Kreitzer G. Miyazawa N. Rempel S. Pfister K.K. Rodriguez-Boulan E. Crystal R.G. Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis.Hum. Gene Ther. 2000; 11: 151-165Crossref PubMed Scopus (217) Google Scholar). Using the ratio of fluorescence emission at two different excitation wavelengths, it was possible to evaluate whether adenoviral vectors were located within endosomes (acidic pH) or in the cytosol (neutral pH). After gaining access to the cytosol, a principal difficulty facing vectors is translocation of the plasmid to the nucleus. Lukacs et al. (4Lukacs G.L. Haggie P. Seksek O. Lechardeur D. Freedman N. Verkman A.S. Size-dependent DNA mobility in cytoplasm and nucleus.J. Biol. Chem. 2000; 275: 1625-1629Crossref PubMed Scopus (628) Google Scholar) recently quantified the cytoplasmic diffusion kinetics of plasmids of defined size using a technique termed fluorescence recovery after photobleaching. Using a laser to destroy plasmid-associated fluorophore in a defined area of the cell, the group measured the time required for other fluorescent plasmids to fill that space. Their finding that plasmids in excess of 2 kb were essentially stationary in the cytoplasm underscores the need to incorporate a packaging system to improve DNA mobility in the cytosol. If packaging of DNA is important, then unpackaging of DNA is doubly important to permit nuclear import and transcription of the viral genome or plasmid. Fluorescence-based methods are now being used in vitro to address both packaging and unpackaging of lipid–DNA complexes. The process of lipid–DNA complex formation was studied by Zuidam et al. (5Zuidam N.J. Hirsch-Lerner D. Margulies S. Barenholz Y. Lamellarity of cationic liposomes and mode of preparation of lipoplexes affect transfection efficiency.Biochim. Biophys. Acta. 1999; 1419: 207-220Crossref PubMed Scopus (105) Google Scholar) who used fluorophores that sense their environment to show that molecular interactions between lipids and DNA were dependent on the molar ratios of the components and that the molecular interactions changed in a reversible manner when the ratios of components changed. During complex formation, DNA becomes condensed, a property quantitatively assessed by Trubetskoy et al. (6Trubetskoy V.S. Slattum P.M. Hagstrom J.E. Wolff J.A. Budker V.G. Quantitative assessment of DNA condensation.Anal. Biochem. 1999; 267: 309-313Crossref PubMed Scopus (26) Google Scholar) who utilized a fluorophore's ability to quench fluorescence from neighboring fluorophores when present at high concentration. Two other studies investigated dissociation of vector complexes using fluorescence resonance energy transfer (FRET), a method in which a fluorescent signal from one fluorophore (the donor) becomes dequenched as it is diluted away from a second fluorophore (the acceptor). Using FRET, Harvie et al. (7Harvie P. Wong F.M.P. Bally M.B. Characterization of lipid DNA interactions. I. Destabilization of bound lipids and DNA dissociation.Biophys. J. 1998; 75: 1040-1051Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) demonstrated that lipids leave lipid–DNA complexes more slowly if the lipids entered the complex as preformed liposomes as opposed to monomers. While some components of a nonviral vector may need to dissociate from the complex to permit gene expression, other elements such as nuclear localization sequences may need to remain associated with DNA during intracellular trafficking. Zelphati et al. (8Zelphati O. Liang X. Hobart P. Felgner P.L. Gene chemistry: Functionally and conformationally intact fluorescent plasmid DNA.Hum. Gene Ther. 1999; 10: 15-24Crossref PubMed Scopus (111) Google Scholar) used FRET to demonstrate the extreme avidity of the noncovalent association between a peptide nucleic acid (PNA) and a plasmid specifically engineered with target sequences for the PNA. The authors pointed out that stable PNA–DNA interactions could be used to link targeting motifs to plasmid DNA. While the in vitro data from these assays are compelling, application of these fluorescence methods to vectors during intracellular trafficking will lead to particularly exciting advances in the field. We are likely to see further applications of fluorescence methods in vector design and evaluation. In addition to the utilization of fluorescent dyes described above, vectormediated expression of fluorescent proteins and fluorescence-based assays of protein function are likely to be important players in rapid screening processes for new molecular therapeutics. Perhaps a bit of optimism regarding continuing advances in gene therapy is in order. After all, our understanding of the mechanisms involved in vector function is rapidly advancing, the family of vectors available for gene transfer continues to grow, and the number of therapies currently undergoing evaluation in the clinic bodes well for investigators and patients alike. As we proceed in our work, fluorescence methods are likely to provide a light toward the end of the tunnel.