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Objective: The binding and encapsulation of [³H] pGL3 luciferase reporter plasmid DNA by red blood cell (RBC) ghosts, intended as a vehicle for transfection and ultimately for gene therapy, were studied using two methods for DNA compaction. Methods and Results: In the first approach, DNA was compacted through binding electrostatically to poly-L-lysine. Complexes were constructed to have a slight negative charge. Experimentally, it was found that a high percentage of binding was to the outside of the resealed RBC ghosts. An alternative approach using polyethylene glycol6000 at a final concentration of 15% (weight/volume) was used to collapse [³H] pGL3 DNA in the presence of 0.025M MgCl2. Addition of the reagents, premixed with DNA, to a pelleted suspension of RBC ghosts followed by a short incubation and then addition of 1.5M NaCl to restore tonicity, resulted in resealing of the ghosts. Uptake of [³H] pGL3 DNA by the ghosts was approximately 20% of the input amount of DNA. Further work showed that 60–70% of the DNA was inside the resealed ghosts and largely present in the supercoiled form. At no stage was any freezing and thawing used. Conclusion: Transfection studies have demonstrated that pGL3 DNA carrying the luciferase gene is successfully transferred from RBC ghosts to recipient HeLa cells in culture under mild fusion conditions. [ABSTRACT FROM AUTHOR]

A conjugate consisting of streptavidin (biotinylated transferrin)-biotinylated polylysine for DNA delivery to cells was modified by partial nicotinylation of the polylysine component of the conjugate and used for transfection studies. A conjugate of biotin[sup 10]- nicotinyl[sub 60]-polylysine[sub 250] containing 60 weakly basic nicotinyl (pyridine-3-carboxyl) residues was prepared. The design of the modified polylysine was directed to the possible binding of H[sup +] ions in the endosome-lysosomal vesicles (pH 5-6) by the nicotinyl groups, thus circumventing the use of chloroquine. The results obtained, however, while showing a 5- to 6-fold increase in luciferase transfection activity still necessitated an absolute requirement for chloroquine. A further polylysine conjugate containing a larger number of nicotinyl residues, biotin[sup 10]-nicotinyl[sub 120]-polylysine[sub 250], also was prepared and studied. This macromolecule stimulated luciferase activity to a small extent and was also dependent on chloroquine. Smaller biotinylated polylysine[sub 100] conjugates containing nicotinyl groups were also prepared. These were biotin[sup 10]-nicotinyl[sub 30]-polylysine , and biotin[sub 10]-nicotinyl[sub 60]-polylysine[sub 100], respectively. Both substances, however, gave opaque, hazy aqueous solutions with precipitates on standing and could not be used for further experimental work. The results indicate that the introduction of weakly basic nicotinyl (pyridine-3-carboxyl) groups onto polylysine[sub 250] give conjugates that are unable to replace the lysosomotrophic agent chloroquine in the HeLa cell sysem studied. A 5- to 6-fold increase in luciferase activity, however, was found with biotin[sup 10]-nicotinyl[sub 60]- polylysine[sub 250]. [ABSTRACT FROM AUTHOR]

Poly-L-lysines of chain lengths varying from 70 to 300 residues are shown to bring about luciferase pRSVL DNA uptake and expression in HeLa cells. Transfection was approximately 50% that of the cationic liposome DOTAB. Expression was higher in the presence of chloroquine. Of interest was the fact that luciferase activity depended on the polysine/DNA charge ratio (+/-). Maximum activity occurred at a charge ratio (+/-) of 3, while at a charge ratio of 1 (conjugate electrically neutral) activity was much lower. At the higher charge ratios (+/-) of 4 and 5, luciferase activity decreased. The results obtained are discussed. [ABSTRACT FROM AUTHOR]