Patent Application: US-98715601-A

Abstract:
the present invention relates to a synergistic method of gene transfection , including gene therapy of human diseases . the invention provides use and preparation of specific combinations of cationic lipids and cationic polymers for the transfection of nucleic acids into host cells . this invention includes also the use of described combinations in specific systems for gene transfection , expression , repair , activation , inhibition and regulation .

Description:
the invention will be described in more detail in the following experimental section of this specification , referring to the enclosed drawings , wherein [ 0019 ] fig1 shows the effect of addition of dosper to the pei - dna complexes on the β - galactosidase activity . the cells were transfected with tkbpvlacz plasmid ( 1 μg ) complexed with three different peis ( average mw 700 , 2000 and 25 000 ) with different n / p ratios ( 1 - 50 ). the transfections were carried out without ( a ) or with ( b ) addition of dosper ( dosper / dna ratio of 1 ) to the pei - dna complexes . the cells were incubated in the transfection solution for 6 h and then in the growth medium for 42 h . the β - galactosidase activity was measured by onpg assay . the values are means of β - galactosidase activity / mg protein ± se , n = 6 . when only dosper was used , β - galactosidase activity was 0 . 36 + 0 . 53 mu / mg protein . [ 0020 ] fig2 shows the effect of different dosper / dna ratios on the β - galactosidase activity . the cells were transfected with 1 μg tkbpvlacz plasmid at a dosper / dna ratios 0 - 7 . 5 ( a ) or dna was condensed with peis at n / p ratios 1 ( b ) and 30 ( c ) before the addition of dosper . the cells were incubated in the transfection solution for 6 hours and then in the growth medium for 42 h . the β - galactosidase activity was measured by onpg assay . the values are the means of β - galactosidase activity / mg protein ± se , n = 6 . [ 0021 ] fig3 shows the analysis of transfection efficiency by x - gal staining . the cells were transfected with 1 μg tkbpvlacz plasmid complexed with pei700 and pei2k at the n / p ratio of 30 and / or dosper at a dosper / dna ratios of 0 , 2 . 5 and 5 . the cells were incubated in the transfection solution for 6 h and then incubated in the growth medium for 42 h . after the incubation , the cells were washed with pbs and fixed with 4 % paraformaldehyde for 15 min . then , the cells were stained with x - gal ( 1 mg / ml ) for 3 h at 37 ° c . and washed with pbs . [ 0022 ] fig4 shows the analysis of complex formation by agarose gel electrophoresis . tkbpvlacz plasmids were complexed with peis ( mw 700 , 2000 and 25 000 ) at the n / p ratios 1 , 2 . 5 and 5 . with n / p ratios above 1 , all the dna was complexed and remained in the well . tkbpvlacz plasmid ( 2 μg ) was diluted with 150 mm nacl to total volume of 30 μl . different amounts of pei were also diluted with nacl to the total volume of 30 μl . after a 10 min incubation , the solutions were mixed and the pei - dna complexes were allowed to form during another 10 min incubation . then , 14 μl of loading buffer was added and 17 , 5 μl ( 0 , 5 μg dna ) of each solution was loaded in a well for electrophoretic separation ( 0 , 6 % agarose in 1x tris - acetate - edta buffer ; electrophoresis at 28 v for 6 h ). dna was visualized with ethidium bromide . dulbecco &# 39 ; s modified eagle medium ( dmem ), foetal bovine serum and penicillin - streptomycin were purchased from gibco brl ( u . k . ), dosper liposomal transfection reagent and x - gal ( 5 - bromo - 4 - chloro - 3 - indolyl - β - d - galactoside ) from boehringer mannheim ( germany ) and polyethylenimines and onpg ( o - nitrophenol - β - d - galactopyranoside ) from sigma - aldrich ( usa ). the ptkbpvlacz plasmid was synthesized by prof . mart ustav at university of tartu , estonia . the ptkbpvlacz plasmid was produced in e . coli ( dh5α ) and purified using the commercial kits ( qiagen , germany ). all other chemicals were of cell culture and molecular biological quantity . the ptkbpvlacz expression plasmids ( 1 μg / well ) were transfected in the subconfluent cv1 - p cell cultures ( a monkey fibroblastoma cell line ). the cells were cultured in 24 - well plates in the atmosphere containing 5 % co 2 at 37 ° c . the bacterial lacz gene encoding the β - galactosidase enzyme was used as a reporter gene . preparation of transfection mixtures were made separately for pei , dosper and pei / dosper combinations . first , 10 μg of ptkbpvlacz plasmid was diluted to a final volume of 150 μl of 150 mm nacl . for the pei transfection mixture , 10 mm pei was diluted to a final volume of 150 mm nacl , incubated for 10 min , added to plasmid dna dilution and incubated for another 10 min before the gene transfection . for the dosper transfection mixture , dosper was diluted at the ratio of 115 in 150 mm nacl and incubated for 15 min before the use for gene transfection . in the case of pei / dosper combination , dosper solution was added to the dna - pei mixture which was then incubated for an additional 15 min . for gene transfections , the transfection mixtures were pipetted dropwise to cell cultures with 1 ml of freshly added dmem without serum and antibiotics . after 6 h exposure , the transfection mixture was replaced with 1 ml of fresh dmem ( with 9 % foetal bovine serum and 90 u penicillin - streptomycin ). the cells were incubated further up to 42 h before analyses of the β - galactosidase activity . at the end of experiments , the cells were washed with pbs , lysed with 150 μl lysis reagent ( 25 mm glycylglycine , 15 mm mgso 4 , 4 mm egta , 1 % triton x - 100 , 1 mm dtt , 1 mm pmsf ), and centrifuged at 13 000 rpm for 5 min ( eppendorf centrifuge 5415c , eppendorf - netheler - hinz , germany ). the activity of β - galactosidase was measured with onpg assay from the supernatant : 20 μl of the supernatant , 80 μl h 2 o and 100 μl of 2x β - gal solution ( 2 mm mgcl 2 , 1 mm β - mercaptoethanol , 1 . 33 mg / ml onpg in sodium phosphate buffer ( 0 . 2m )) were put in a 96 - well plate and incubated up to 1 h at rt . in the onpg assay , the detection is based on the cleavage of β - bond from onpg by the β - galactosidase enzyme resulting in the yellow o - nitrophenol molecule . the reaction was stopped with 1m na 2 co 3 , when o - nitrophenol anionizes and absorbs lightwaves at 405 nm wavelength . samples were analysed by measuring absorbance at 405 nm wavelength with the bio - tek elx - 800 microplate reader ( bio - tek instruments , usa ) and kc - 3 pc - program . protein concentrations were measured with bio - rad protein assay ( coomassie brilliant blue , bio - rad laboratories , usa ). 15 μl of supernatant was diluted to 800 μl of h 2 o , and 200 μl of protein assay dye reagent concentrate was added . absorbance was read at 595 nm wavelength using hitachi u - 2000 spectrophotometer . the x - gal staining was used for histochemical analysis of β - galactosidase enzyme . therefore , the cells were washed with pbs , fixed with 4 % paraformaldehyde ( 15 min , rt ) and washed again twice with pbs . then , the cells were incubated in the x - gal staining solution ( x - gal 1 mg / ml , mgcl 2 2 , 5 mm k 3 fe ( cn ) 6 , 5 mm k 4 fe ( cn 6 ) x 3 h 2 o , 0 . 01 % sodiumdeoxycholate , 0 . 02 % nonidet p - 40 ) for 3 h at + 37 ° c . the activity of β - galactosidase was detected as a blue color of 3 , 5 ′- dichromo - 4 , 4 ′- dichloroindigo molecule resulting from the cleavage of x - gal substrate by the β - galactosidase in the transfected cells . after x - gal staining , the cells were washed with pbs and photographed with nikon diaphot 300 microscope and nikon f - 601 camera . the pei - dna complexes were prepared as described earlier . for electrophoresis , 0 . 5 μg dna was pipetted into an agarose gel ( 0 . 6 % agarose in 1x tris - acetate - edta buffer ; electrophoresis at 28 v for 6 h ). the gels were stained in ethidium bromide solution ( 0 . 5 mg etbr in 1 l h 2 o ) for 30 min at rt to visualize dna after electrophoresis . the role of pei / dna ratios on the transfection efficiency of the pei / dosper combination in the first experiments , the effect of different n / p ratios of pei / dna complexes was studied on the transfection efficiency of the pei / dosper combination . here , the transfection efficiency was measured as a total β - galactosidase activity in the cell extracts using the calorimetric onpg assay . initially , we studied the effect of dosper alone , three peis ( 700 , 2k , 25k ) alone and their different combinations on the transfection efficiency in the cv1 - p cell cultures ( fig1 ). therefore , each pei was studied at varying n / p ratios with and without dosper using a constant dosper / dna ratio , to find out if there would be any combinatory potentiation of transfection efficiency . we used dosper alone at the dosper / dna ratio of 1 , each of three peis alone at six n / p ratios varying from 1 - 50 or different combinations of dosper and peis . in these experiments , dosper , pei 700 and pei 2k were all ineffective when they were used alone for gene transfection , whereas pei 25k had a significant transfection efficiency . however , the transfection efficiency of pei 700 was slightly potentiated with dosper at the n / p ratios of 30and 50 . furthermore , when pei 2k was used together with dosper , the transfection efficiency was significantly improved especially at the n / p ratio of 30 , while the smaller n / p ratios of 1 and 5 produced no or only a weak potentiation of β - galactosidase activity . this effect was even higher than that achieved by pei 25k which itself produced a significant rise in β - galactosidase activity especially at the n / p ratio of 10 . when dosper was added to pe125k / dna complex , the transfection efficiency was still enhanced , and the highest effect was seen at the n / p ratio of 5 . the role of dosper / dna ratios on the transfection efficiency of the pei / dosper combination in the second experiment , the effect of different dosper / dna ratios was studied on the transfection efficiency of the pei / dosper combination ( fig2 ). therefore , the n / p ratios of 1 and 30 were selected for each pei , and different amounts of dosper ( dosper / dna ratios of 0 - 7 . 5 ) were added to pei / dna complexes . when the cells were transfected using dosper alone ( i . e . without peis ), the best β - galactosidase activity was achieved at the dosper / dna ratio of 7 . 5 . when the combination of pei / dosper was used at the n / p ratio of 1 for pei / dna complexes , no significant changes were seen in the transfection efficiency with any of peis ( 700 , 2k and 25k ) in comparison to dosper alone . however , for pei 700 at the n / p ratio of 30 , the transfection efficiency was significantly higher with the combination of pei / dosper . the highest potentiation was obtained at the dosper / dna ratio of 5 , and was more than 3 - fold compared to the best results achieved with dosper alone . on the other hand , for pei 2k at the nip ratio of 30 , the transfection efficiency was not altered with the combination of pei / dosper when compared to the plain dosper . however , now the maximum β - galactosidase activity was achieved with somewhat lower dosper / dna ratios ( 2 . 5 and 5 ) than with dosper alone ( 7 . 5 ). the use of pei 25k at the n / p ratio of 30 did not enhance the transfection efficiency of pei / dosper combination compared to dosper or pei 25k alone . the number of transfected cells was measured using the histochemical x - gal staining . when dosper was added to peiidna complexes , the number of blue - colored cells ( indicating successful lacz gene transfections and expressions ) was increased in comparison to pei or dosper alone ( fig3 ). there was no staining seen in any cells which were transfected only with pei 700 or pei 2k at the n / p ratio 30 . in the dosper - mediated gene transfection at the dosper / dna ratio of 5 , there was less than 1 % of the cells were stained . the number of stained cells increased significantly , when pei 700 or 2k at the n / p ratio of 30 , was used to condense the plasmid dna prior addition of dosper , showing the averages of with 4 . 8 ± 0 . 8 % for pei 700 and 4 . 3 ± 1 % for pei 2k ( data not shown ). analysis on the formation of the pei / dna complexes at different n / p ratios agarose gel electrophoresis was used to study the ability of peis to condense dna , and the relationship of complex formation to the potentiated transfection efficiency bof the pei / dosper combinations . each pei at the n / p ratios & gt ; 2 . 5 could complex all dna since all the dna was staying in the wells , whereas at the n / p ratio of 1 , dna was not totally complexed which was seen as the gel electrophoretic movement of free dna ( fig4 ). 1 murray k d et al . cationic liposome - 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