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 1.5 M 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.