PubMed · 16998971
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Gene therapy.. https://pubmed.ncbi.nlm.nih.gov/16998971/
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The purpose of this research was to develop and characterize a gene delivery vehicle with a poly(ethylene glycol) (PEG) backbone with the aim of overcoming limitations, such as cytotoxicity and rapid clearance, associated with current commonly used non-viral carriers. PEG was functionalized with DNA-binding peptides (DBPs) to make a vehicle (DBP-PEG) capable of condensing DNA. Complexes of plasmid DNA and DBP-PEG were formed and characterized by measuring particle size, zeta potential, and transfection efficiency as a function of N:P charge ratios (DBP-PEG amino groups:DNA phosphate). Dynamic light scattering showed that DBP-PEG was able to condense DNA efficiently resulting in a population of particles in the range of 250-300 nm. Neutral or slightly positive zeta potentials were measured for charge ratios of 3.5:1 and greater. DBP-PEG/DNA complexes, made with plasmids encoding the green fluorescent protein (GFP) and beta-Galactosidase (beta-Gal) genes, were used to transfect Chinese hamster ovary (CHO) cells. DBP-PEG/DNA was capable of transfecting cells and maximum transfection efficiency was observed for N:P ratios from 4:1 to 5:1, corresponding to zeta potentials from -4 to +1.6 mV. The effect of the DBP-PEG vehicle on cell viability was assayed. DBP-PEG was associated with a higher percentage of viable cells ( approximately 95%) than either polyethylenimine (PEI) or poly-L-lysine (PLL), and with transfection efficiency greater than PLL, but with somewhat lower than PEI. The results of this work demonstrate that PEG can be used as the backbone for gene delivery vehicles.
N116Y, H-RAS mutant, has dominant negative activity in the RAS function and a suppressive effect on the growth of various types of cancer cells. However, a replication error of N116Y is of potential concern for carcinogenesis in clinical application. To decease the concern, we constructed modified N116Y by deleting the carboxyl terminus, which is necessary for the oncogenic function of Ras. One of the C-terminal deletion mutants of N116Y, N116Y-Cdel2 showed a growth-suppressing effect on various human cancer cell lines in vitro: the cervical cancer cell line HeLa, the pancreatic cancer cell line PCI43, the colon cancer cell lines SW480 and LoVo, and the tongue cancer cell line SAS. In addition, the suppressive effect of N116Y-Cdel2 on LoVo cells was also observed in vivo using a nonviral gene transfer vector, HVJ envelope. Our experiments suggest that the modified N116Y is a potential candidate gene for human cancer gene therapy.
Ultrasound-mediated gene transfection (sonotransfection) is a promising physical method for gene therapy, especially for cancer gene therapy. To investigate the optimal sonotransfection conditions and to determine whether the optimal transfection rate using sonotransfection is comparable to that of electrotransfection or liposome-mediated transfection, we sonicated different cancer cell lines (U937, HeLa, PC-3, Meth A and T-24) using a 1-MHz unfocused ultrasound at different intensities, pulse repetition frequencies and exposure times. The ideal ultrasound conditions were noted to be at 1.5 Watt/cm(2) pulsed at 0.5 Hz with a duty factor of 50%. The results showed that transfection rate increased with the number of pulses, and peaked between 10 and 15 pulses before it started to decline. Using such optimal conditions, we have shown that sonotransfection is superior to electrotransfection and liposome-mediated transfection at the fixed conditions used in the present study. These findings suggest that sonotransfection could be a better alternative to other non-viral methods (e.g. electroporation and liposome-mediated transfection) of gene transfection, particularly in cancer gene therapy.