Patent Application: US-46636803-A

Abstract:
the present invention relates to a method for transfection of cells using at least one protein capable of forming nucleoprotein filaments , wherein the protein is initially modified with at least one functional component which influences one or more steps of the transfection , the nucleic acid to be transfected is then loaded with the modified protein , whereby the nucleic acid and the protein form a filament - like complex , and this complex is finally added to the cells to be transfected . the invention further relates to a transfection agent consisting of nucleoprotein filaments , with at least one nucleoprotein filament - forming protein being modified with at least one functional component for the transfection . furthermore , the present invention relates to the use of the transfection agent according to the invention for producing a drug for gene therapeutic treatment of humans and animals . the present inventions also includes corresponding pharmaceutical preparations , especially for use in gene therapy as well as the use of such transfection agents as component in kits .

Description:
the following examples are intended to describe the invention in detail , without limiting it to exemplary disclosed substances and methods . the proteins uvsxh6 , uvsxh6 - 2 , h6uvsx und h6uvsx - 2 were used as npf forming proteins ( see fig1 ). amino acids 1 - 391 : uvsx from the phage t4 ( ncbi protein accession no : aad42669 , amino acids 1 - 391 ), amino acids 392 - 394 : linker consisting of the amino acids g 392 gs 394 , amino acid 395 - 400 : h 395 hhhhh 400 for purification by nickel chelate affinity chromatography , amino acid exchange : l 43 → p . amino acids 1 - 391 : uvsx from the phage t4 ( ncbi protein accession no : aad42669 , amino acids 1 - 391 ), amino acids 392 - 394 : linker consisting of the s 392 yg 394 , amino acids 395 - 400 : h 395 hhhhh 400 , amino acids 401 - 403 : c - terminus consisting of the amino acids m 401 ys 403 . amino acids 1 - 4 : n - terminus consisting of the amino acids m 1 sys 4 , amino acids 5 - 10 : h 5 hhhhh 10 , amino acids 11 - 13 : linker consisting of the amino acids s 11 yg 13 , amino acids 14 - 404 : uvsx from the phage t4 ( ncbi protein accession no : aad42669 , amino acids 1 - 391 ), amino acid exchange : q 340 → l . amino acids 1 - 4 : n - terminus consisting of the amino acids m 1 gys 4 , amino acids 5 - 10 : h 5 hhhhh 10 , amino acids 11 - 13 : linker consisting of the amino acids s 11 yg 13 , amino acids 14 - 404 : uvsx from the phage t4 ( ncbi protein accession no : aad42669 , amino acids 1 - 391 ). for the expression of the aforementioned proteins in suitable escherichia coli cells , plasmids were constructed containing a coding sequence for uvsxh6 , uvsxh6 - 2 , h6uvsx or h6uvsx - 2 under the control of the lac promotor ( pexh - uvsxh6 , pexh - uvsxh6 - 2 , pexh - h6uvsx or pexj - i - h6uvsx - 2 , see fig1 ). the plasmids were generated by ligation of two pcr products . the first pcr product was amplified from pmcs5 ( mobitec , g6ttingen , germany ). pmcs5 is constructed in a similar way as pbluescript sk (−) ( stratagene ) and is only different in the 5 ′ region of the coding sequence of the laczα fragment . pmcs5 therefore contains the lac promotor followed by the lac operator , by which the expression of an inserted coding sequence is based on the absence of active lac repressor . in order to achieve constitutive expression in any case , the amplification primers were selected in such way , that the lac operator is not present anymore in the pcr product . the resulting pcr product corresponded to pmcs5 from position 992 to 664 plus restriction overhangs . the nucleotides gaattc ( ecori restriction site ) as well as tgtgtg were added before position 992 ( 3 ′ to the lac promotor ), and the nucleotides actagt ( spe i restriction site ) and cacaca were added behind position 664 to enable the ligation after digestion with the restrictions enzymes ecori and spel . the coding sequence for uvsxh6 , uvsxh6 - 2 or h6uvsx and h6uvsx - 2 was obtained by pcr amplification of t4 dna using primers which contain the desired restriction sites , a ribosomal binding site and the additional codons . at their 5 ′ end before the start codon , the pcr products contained the additional nucleotides 5 ′- cacacagaattcataaaggaagatatcat - 3 ′ ( seq id no : 2 ), as well as the additional nucleotides 5 ′- actagttgtgtg - 3 ′ ( seq id no : 3 ) at their 3 ′ end after the stop codon . an overnight culture of pexh - h6uvsx in dh5 was inoculated with 1 . 5 - 3i dyt / ampicillin ( 200 μg / ml ) 1 : 1000 , and was grown over night at 37 ° c . with 250 upm . the cultures were harvested at 7000 × g , and yielded approx . 5 - 15 g bacteria sediment . this was frozen for 1 - 3 days at − 20 ° c . the sediment was thawed on ice and resuspended in 10 - 20 ml cold starting buffer . the lysis was carried out under slow stirring for 1 h at 4 ° c . using 10 ml lysozym ( serva , 190 . 000 u / mg ) and approx . 4 g glass beads ( sigma , g - 8893 ). then , 50 μl dnase i ( serva , 2 mg / ml ) were added and further incubated for another 30 min . after centrifugation of the lysate ( 45 min , 11 . 000 × g , 4 ° c . ), the supernatant was filtered through a sterile filter ( pore size 0 . 45 μm ) and loaded on an equilibrated 1 ml hitrap ™ chelating column ( pharmacia ) preloaded with ni ++ ions . the further purification steps were carried out according to the respective pharmacia protocol for proteins containing a histidin hexamer . aliquots of the various elution fractions were added to sds / coomassie gels . the purest fractions were combined and further concentrated in centriplus ym30 columns ( millipore ) according to the respective protocol . then , it was dialyzed twice ( dialysis tube : spectra / por , mwco : 25 . 000 ) against an at least one thousand - fold volume of zi buffer for at least one hour at 4 ° c ., then over night at 4 ° c . against zi buffer / 50 % glycerine . the dialysate was aliquoted in 30 - 50 μl fractions and stored at − 80 ° c . zi buffer : 76 mm k 2 hpo 4 , 17 mm kh 2 po 4 , 14 mm nah 2 po 4 , ph = 7 . 2 starting buffer : 20 mm pi , 0 . 5 m nacl , 10 mm imidazole , ph = 7 . 4 washing buffer : 20 mm pi , 0 . 5 m nacl , 20 - 50 mm imidazole , ph = 7 . 4 elution buffer : 20 mm pi , 0 . 5 m nacl , 100 - 500 mm imidazole , ph = 7 . 4 the concentration of the uvsx proteins was determined by measuring the od 280 using the extinction coefficient calculated with the gene inspector ™ ( textco , inc .) software . for h6uvsx it was 2 . 5 - 3 . 5 μg / μl . h6uvsx , purified over ni ++ sepharose , was incubated with 200 ng of a 1 kb pcr fragment ( with and without 1 mm atp - γ - s ). a shift of the dna in the agarose gel caused by protein binding shows , that h6uvsx binds double - stranded dna based on the concentration , and that this binding is enhanced by atp - γ - s . with atp - γ - s , protein - dna complexes are formed which can be stained more intensely with ethidium bromide than without atp - γ - s , which indicates a topological change of the nucleoprotein filament by the nucleotide analog . generation of a transfecting agent , based on uvsx as npf forming protein with a nuclear localization signal as a functional component . as in example 1 , plasmids were generated which permit the expression of the fusion proteins uvsxh6n2 , uvsxh6n2 - 2 , n2h6uvsx and n2h6uvsx - 2 , which are based on the proteins described in example 1 , and in addition contain a nuclear localization signal ( see fig1 ). amino acids 1 - 391 : uvsx from the phage t4 ( ncbi protein accession no . : aad42669 , amino acids 1 - 391 ), amino acids 392 - 394 : linker consisting of the amino acids g 392 g s 394 , amino acids 395 - 400 : h 395 hhhhh 400 , amino acids 401 - 403 : linker consisting of the amino acids g 401 gs 403 , amino acids 404 - 417 : nuclear localization signal nls - 2 ( amino acids 2 - 15 , seq id no : 9 from wo 00 / 40742 ), amino acids 418 - 421 : c - terminus of uvsx from the phage t4 ( ncbi protein accession no : aad42669 , amino acids 388 - 391 ), amino acids 422 - 426 : c - terminus consisting of the amino acids k 422 lvtg 426 , amino acid exchange : y 238 → v . amino acids 1 - 391 : uvsx from the phage t4 ( ncbi protein accession no : aad42669 , amino acids 1 - 391 ), amino acids 392 - 394 : linker consisting of the amino acids s 392 yg 394 , amino acids 395 - 400 : h 395 hhhhh 400 , amino acids 401 - 403 : linker consisting of the amino acids m 401 ys 403 , amino acids 404 - 417 : nuclear localization signal nls - 2 ( amino acids 2 - 15 , seq id no : 9 from wo 00 / 40742 ), amino acids 418 - 420 : c - terminus consisting of the amino acids g 418 yp 420 . amino acids 1 - 3 : n - terminus consisting of the amino acids m 1 sy 3 , amino acids 4 - 17 : nuclear localization signal nls - 2 ( amino acids 2 - 15 , seq id no : 9 from wo 00 / 40742 ), amino acids 18 - 20 : linker consisting of the amino acids l 18 ys 20 , amino acids 21 - 26 : h 21 hhhhh 26 , amino acids 27 - 29 : linker consisting of the amino acids s 27 yg 29 , amino acids 30 - 420 : uvsx from the phage t4 ( ncbi protein accession no : aad42669 , amino acids 1 - 391 ), amino acid exchange : q 356 → l . amino acids 1 - 4 : n - terminus consisting of the amino acids m 1 gyp 4 , amino acids 5 - 18 : nuclear localization signal nls - 2 ( amino acids 2 - 15 , seq id no : 9 from wo 00 / 40742 ), amino acids 19 - 21 : linker consisting of the amino acids s 19 ys 21 , amino acids 22 - 27 : h 22 hhhhh 27 , amino acids 28 - 30 : linker consisting of the amino acids s 28 yg 30 , amino acids 31 - 421 : uvsx from the phage t4 ( ncbi protein accession no : aad42669 , amino acids 1 - 391 ). for the expression in suitable escherichia coli cells , plasmids were constructed containing a coding sequence for uvsxh6n2 , uvsxh6n2 - 2 , n2h6uvsx or n2h6uvsx - 2 under the control of the lac promotor ( pexh - uvsxh6n2 , pexh - uvsxh6n2 - 2 , pexh - n2h6uvsx or pexh - n2h6uvsx - 2 , see fig1 ). the plasmids were generated as described in example 1 by ligation of two pcr products ( see fig1 ). the purification of uvsxh6n2 was carried out as described in example 1 ) for h6uvsx . uvsxh6n2 binds to double - stranded dna . the binding is stabilized by atp - γ - s : in order to examine the influence of various atp analogues on the binding performance of uvsxh6n2 to dna , the protein was first incubated with a 1 kb dna fragment and various atp analogues . then , a 1 . 7 kb dna fragment was added for competition . if the binding of the protein to the dna is stabilized by addition of an atp analog , it can be expected that uvsxh6n2 less likely binds a competing dna fragment as long as no equilibrium is present . as can be seen in fig3 , the protein - dna complex which was generated with the 1 kb fragment and uvsxh6n2 , remains stable in absence of atp - γ - s and the 1 . 7 kb fragment , compared to all other used atp analogues , i . e . the 1 . 7 kb fragment apparently is not or only marginally occupied by liberated uvsxh6n2 molecules within the observation time . generation of a transfection agent with a mixture of modified and unmodified npf - forming protein various ratios of h6uvsx and uvsxh6n2 were incubated with a 1 kb dna fragment . the two proteins retarded the dna in different degrees , due to their different molecular weight ( see column 2 and 3 of fig4 ). if the proteins are mixed before addition of dna , intermediate complexes are formed based on the ratio of h6uvsx to uvsxh6n2 , which yield a sharp band and therefore have an equal mean molecular weight . this shows that the dna is statistically equally occupied by both proteins . transfection of a cell line ( nih3t3 ) with uvsx - nls in combination with electroporation nih3t3 - zellen ( adherent , cultivated until 70 - 80 % confluent ) were transfected with a vector coding for the heavy chain of the murine mhc class i proteins h - 2k k . 1 × 10 6 cells were electroporated with 25 ng vector dna which has been preincubated in binding buffer ( 76 mm k 2 hpo 4 , 17 mm kh 2 po 4 , 14 mm nah 2 po 4 , 5 mm mgcl 2 ph 7 . 21 ) with 14 μg uvsx or uvsx - nls as well as with or without 1 mm atp - γ - s for 30 minutes at room temperature . for this , the cells were added to a total volume of 100 μl electroporation buffer ( 103 mm nacl , 5 . 36 mm kcl , 0 . 41 mm mgcl 2 , 23 . 8 mm nahco 3 , 5 . 64 mm na 2 hpo 4 , 11 . 2 mm glucose , 0 . 42 mm ca ( no 3 ) 2 , 20 mm hepes , 3 . 25 μm gluthathione ) and electroporated in a cuvette with 2 mm electrode spacing . the electroporation was carried out by an exponential discharge at a voltage of 240 v and a capacity of 450 μf . the half - life of the voltage drop was typically 12 msec . immediately after the electroporation , the cells were flushed out of the cuvettes with culture medium ( rpmi with 10 % fcs ), incubated for 10 min at 37 ° c ., and then transferred to a culture dish with prewarmed culture medium . after 6 h incubation , the cells were harvested and washed twice with pbs , and then incubated with a cy5 - coupled anti - h - 2k k antibody and analyzed flow - cytometrically ( facscan ). the number of dead cells was determined by staining with propidium iodine . six hours after the electroporation , 7 . 4 % or 8 . 7 % of the cells transfected with free vector dna express the h - 2k k protein ( minus the background of 0 . 25 % in average ). in comparison , the expression rate of the cells that have been transfected with vector uvsx was 2 . 9 % or 3 . 8 %. the expression rate after a transfection with vector uvsx - nls was 19 . 2 % or 18 . 9 % ( see fig5 a - 5 d ). for examination of the nuclear transport , the physical procedure of electroporation was chosen so that no other biochemical components except uvsx influence the transfection . the tight binding of uvsx with atp - γ - s to dna , however , impairs the mobility of the complex in the electric field and therefore reduces the efficiency of the electroporation by approx . 60 %. the attachment of a nuclear transport signal alone results in an increase of the expression shortly after the transfection by a factor of averagely 5 . 7 in this system . the increase of the expression rate by uvsx - nls compared to uvsx therefore demonstrated that using a nuclear transport signal as functional component , dna is transported into the nucleus by uvsx . an analysis performed shortly after the transfection is significantly improved since even cells become accessible that have not divided between transfection and analysis . transfection of a cell line ( nih3t3 ) with uvsx - scrambled - nls or uvsx - nls in combination with electroporation in order to test the influence of the nuclear localization signal on the transfection , an uvsx derivative was generated for comparison purposes which corresponds in its net charge to an uvsx protein with a nls , but itself does not contain a functional nls . using partly homologous oligonucleotide primers , the uvsx gene was amplified from plasmid pexh - uvsxh6n2 - 2 ( compare fig1 ), so that the amino acid sequence seq id no : 1 (“ scrambled ”, i . e . a mixed nls sequence ) is expressed at the c - terminus of the resulting protein uvsxh6n2sc instead of the amino acid sequence eedtppkkkrkved ( seq id no : 4 (“ nls -”, corresponding to the amino acids 2 - 15 from seq id no : 9 from wo 00 / 40742 ). the scrambled amino acids correspond to the described nls - 2 with respect to their composition but not with respect to their order . the net charges of uvsxh6n2 - 2 and uvsxh6n2sc are therefore equal , but only uvsxh6n2 - 2 contains an intact nuclear localization signal . the protein uvsxh6n2sc was purified as described for the proteins in example 1 . nih3t3 cells ( adherent , cultivated until 70 - 80 % confluent ) were transfected with this vector coding for a fluorescent marker protein . for this purpose , 25 ng vector dna in binding buffer ( see example 1 ) were initially incubated with 1 . 5 mm atp - γ - s and 16 - 18 μg of the described proteins for 30 min at room temperature . the protein - dna complexes were each added to 3 × 10 5 nih3t3 - zellen , resuspended in 80 μl electroporation buffer ( 140 mm na 2 hp 4 / nah 2 po 4 , 10 mm mgcl 2 , 5 mm kcl , ph 7 . 2 ). the electroporation was carried out in a cuvette with 2 mm electrode spacing by an exponential discharge at a voltage of 240 v and a capacity of 450 μf , the half life of the voltage drop was typically 12 msec . after addition of 400 μl medium , composed of rpmi 1640 , gibco company , 5 % fcs , 2 mm glutamax ( l - alanyl - l - glutamine , invitrogen ), 100 u / ml penicillin / streptomycin , 0 . 5 mm β - mercaptoethanol , the cells were added to culture dishes ( 6 - well plates ) with 1 ml prewarmed medium and were incubated at 37 ° c . and 5 % co 2 . after 6 h , the flow - cytometric analysis was carried out ( facscan ). the result is graphically shown in fig6 : 9 % of the cells transfected with free vector dna express the marker protein . the expression rate of the cells that have been transfected with vector - uvsx - nls ( dna + uvsxh6n2 - 2 ), however , was 21 %. in comparison , the expression rate of the cells after transfection with vector - uvsx - scrambled - nls ( dna + uvsxh6n2sc ) was only 4 %. uvsx , modified with a nuclear localization signal therefore results in a markedly increased efficiency compared to free dna and also compared to the modification by a non - functional nuclear localization signal . since the latter transfection represents the control , this means that the transfection efficiency could be increased to 5 - fold by modification of the uvsx . therefore , the transfection efficiency can be markedly increased by the method according to the invention or the transfection agent . furthermore a targeted control of the transfection method , here , for example , a directing into the nucleus , is possible in an advantageous way when the npf - forming protein is modified ( see also example 6 ). 140 ng of a 1 . 7 kb expression vector dna fragment were incubated with 9 μg modified uvsx protein as described above in binding buffer and 1 mm atp - γ - s in a final volume of 20 μl for 30 min at rt . bsa - cy5 was used as injection marker immediately before the injection in a concentration of approx . 1 μg / μl . nih3t3 cells that had been seeded to subconfluency the day before on cellocate coverslips ( eppendorf ) were microinjected through samples loaded onto femtotipps ( eppendorf ) using a micromanipulator and transjector ( eppendort ) under an inverse fluorescence microscope ( leica dmil ). the analysis was carried out in a fluorescence microscope ( olympus bx 60 fluorescence microscope , digital b / w camera spot - rt from diagnostic instruments inc ., analysis software : metaview imaging system from universal imaging corporation ) after 5 hours of further incubation of the cells at 37 ° c . and 5 % co2 . fig7 shows microinjected nih3t3 cells . the images 1 and 2 show cells that were injected with dna and uvsxh6n2sc into the cytoplasm , the images 3 and 4 show cells that were injected with dna and uvsxh6n2 - 2 , and images 5 and 6 show cells that were injected only with dna . expression was only observed when the protein - dna complexes contained uvsxh6n2 - 2 , i . e . an uvsx protein modified with a nuclear localization signal ( fig7 , image 3 ). in the cells of the controls ( fig7 , image 5 : only dna , or fig7 , image 1 : dna with uvsx - scrambled - nls ) that had been clearly injected only in the cytoplasm and not in the nucleus during microinjection , no expression was observed , not even using a very long exposure . cloning , expression and purification of the sasp protein from b . subtilis the sspc gene from bacillus subtilis , which codes for a sasp (, small acid - soluble spore protein ”), was synthesized from 8 oligonucleotides according to the khorana method ( described in bertram and gassen : gentechnische methoden , gustav fischer verlag , 1991 , p . 212 - 213 ) and ligated between the ncoi and bgiii restriction sites of the plasmid para13 ( cagnon et al ., 1991 ; protein engng . 4 : 843 - 847 ). this was based on the protein sequence with the ncbi protein accession no : np — 389876 . the reverse transcription to dna was carried out using the codon preferences of genes , strongly expressed in e . coli and described in ( andersson and kurland 1990 ; microbiol . rev . 54 : 98 - 210 ). the resulting plasmid para13 - sasp served as a matrix for the cloning of two other plasmids , which code for sasp proteins , carrying a polyhistidine sequence of 6 histidines either at the n - terminus ( h6 - sasp ) or at the c - terminus ( sasp - h6 ) ( fig8 ). the plasmids were transformed into the e . coli strain bl21 ( de3 ) plyss ( novagen , madison ) and plated out on lb / ampicillin / glucose ( 0 . 2 %). 20 ml m9 minimal medium ( 0 . 2 % glucose ) each was inoculated with single colonies and grown over night at 37 ° c . and 220 rpm . the following day , the cultures were induced with 0 . 2 % arabinose at an od 600 of approx . 1 . 0 and were grown for another 6 hours . raw extracts ( 0 . 5 ml pelleted culture in pbs / loading buffer ) were applied to high resolution sds gels ( according to schagger and von jagow 1987 ; anal . biochem . 166 : 368 - 79 ) and stained with coomassie blue ( fig9 a ). for preparative purification , 2 l m9 / glucose were inoculated 1 : 200 with an overnight culture . again , the culture was induced with 0 . 2 % arabinose at an od 600 of approx . 1 , 0 , and further grown for 6 h . the bacteria were then pelleted and frozen at − 20 ° c . in the following , the purification of sasp - h6 is described exemplary . approx . 7 g of the pellet was thawed and resuspended in 14 ml starting buffer ( see example 1 ), supplemented with a tablet of complete edta - free protease inhibitor cocktail , roche , mannheim , and sonificated on ice for 3 min at 280 watts ( labsonic u , braun biotech , melsungen repeating duty puls ; 0 . 5 sec ). the extract was centrifuged at 4 ° c . apart from this , the purification was carried out as described for the uvsx proteins over hitrap chelating columns ( amersham pharmacia , uppsala ). the fractions between 200 mm and 500 mm imidazole , containing the protein in high concentration , were combined and concentrated to approx . 3 ml using centriplus columns ( ym - 3 , millipore , eschborn ). the sasp protein was then dialyzed three times against 1 × zi buffer ( see example 1 ), and aliquots were frozen at − 80 ° c . in a concentration of approx . 5 μg / μl . 125 ng ( in each case ) of a 1 . 7 kb dna fragment were preincubated with different amounts of sasp - h6 protein in 1 × zi buffer or 1 / 10 × zi buffer for 30 min at room temperature , and then applied to an 0 . 8 % tae - agarose gel . fig9 b shows that the dna is completely bound by the protein . the diffuse appearance of the dna - protein bands may be due to a dissociation of the proteins from the dna during the gel run . generation of a transfection agent based on sasp as npf - forming protein with a nuclear localization signal as modification for generation of an sasp with a nuclear localization signal ( nls ) as functional component , a dna sequence coding for a nuclear localization signal (, nls - 2 ”, amino acids 2 - 15 , seq id no : 9 from wo 00 / 40742 ) was added to the c - terminus of the already available clone para13 - sasp - h6 ( see fig8 ) by pcr amplification . the aforementioned plasmid served as pcr template . the resulting plasmid para13 - sasp - h6n2 ( see fig8 ) was transformed as described in example 7 , and the protein sasp - h6n2 was purified accordingly . 125 ng each of a 1 . 7 kb dna fragment were preincubated with different amounts of sasp - h6n2 protein in 1 × zi - puffer for 30 min at room temperature , and then applied to a 0 . 8 % tae - agarose gel . fig1 b shows that the dna is held back by the nls - modified sasp as well . the dna protein bands appear diffuse . generation of a transfection agent with a integrin binding motif for the association of the complex to the cell surface as functional component integrins are membrane - anchored adhesion proteins on the cell surface some of which recognize a peptide motif of three amino acids ( arginine - glycine - aspartic acid or , rgd ” motif ) as binding partner . the binding results in clustering of several integrin molecules on the cell surface and in endocytosis ( plow et al ., 2001 ). by modification of uvsx as npf - binding protein with an rgd motif , it can be achieved that the transfection agens can be taken up specifically via integrins into the endosomal compartments of the cells . the used proteins h6uvsx and uvsxh6n2 - 2 are identical to the ones shown in fig1 and described in example 1 and 2 . the protein h6uvsx *( rgd2 ) was generated by chemical coupling . the protein named rgd2 is a nonapeptide ( nitrgdtyi ) consisting of the penton base protein of adenovirus type 7 ( bal et al ., 2000 ), which has been synthesized in such way that a chemically active group is present at the n - terminal amino group ( smcc , succinimidyl 4 [ n - maleimidomethyl ]- cyclohexane - 1 - carboxylat ) which permits the coupling to free cysteine sh groups in the uvsx protein . for the coupling , 6 nmol h6uvsx were incubated with 60 nmol peptide in 76 mm k 2 hpo 4 , 17 mm kh 2 po 4 , 14 mm nah 2 po 4 , ph = 7 . 2 for one hour at 37 ° c ., and was then purified by several washing steps with incubation buffer over a microcon filter ( 10 kda cut off ) to remove excess peptide . the successful coupling was detected by an altered running performance in an sds polyacrylamide gel electrophoresis . fig1 shows two independently generated preparations of h6uvsx *( rgd2 ) on an sds polyacrylamide gel . the coupled peptide results in an increase of the molecular weight and thus in an alteration of the running performance in sds gels . binding of uvsx - nls and uvsx - rgd to double - stranded dna and formation of mixed npfs : reactions with 140 ng ( in each case ) of a purified 1 . 6 kb pcr dna fragment with a mixture of 15 μg h6uvsx *( rgd2 ) and 4 μg uvsxh6n2 - 2 , with 4 μg uvsxh6n2 - 2 alone and with 15 μg h6uvsx *( rgd2 ) alone were incubated in 76 mm k 2 hpo 4 , 17 mm kh 2 po 4 , 14 mm nah 2 po 4 , ph = 7 . 2 , 5 mm mgcl 2 and 1 mm atp - γ - s in a final volume of 20 μl for 30 min at room temperature and were then transferred to a 0 . 8 % tae / agarose gel which was afterwards stained with ethidium bromide . the electrophoresis was carried out at 100 v for 1 h . fig1 shows that the two differently modified proteins form npfs with the dna which markedly differ in their running performance . npfs consisting of a double - stranded dna fragment and a mixture of h6uvsx *( rgd2 ) and uvsxh6n2 - 2 ( lane 1 ), or of uvsxh6n2 - 2 alone ( lane 2 ) or of h6uvsx *( rgd2 ) alone ( lane 3 ), were separated electrophoretically in an agarose gel . since protein is present in low amounts , free dna fragment is present as well . both proteins in a reaction bind together to the dna fragment and result in a mixed npf that has a molecular weight that lies between that of the npfs of the pure proteins ( lane 1 ). from this it can be concluded that uvsx - nls and uvsx - rgd bind to double - stranded dna and can form mixed npfs . as in example 1 , plasmid uvsxh6n2nit - 2 ( fig1 ) which permits the expression of a fusion protein of uvsx and an integrin - binding rgd motif , was generated recombinantly . amino acids 1 - 391 : uvsx from the phage t4 ( ncbi protein accession no . : aad42669 , amino acids 1 - 391 ), amino acids 392 - 394 : linker consisting of the amino acids s 392 yg 394 , amino acids 395 - 400 : h 395 hhhhh 400 , amino acids 401 - 403 : linker consisting of the amino acids m 401 ys 403 , amino acids 404 - 417 : nuclear localization signal nls - 2 ( amino acids 2 - 15 , seq id no : 9 from wo 00 / 40742 ), amino acids 418 - 420 : c - terminus consisting of the amino acids g 418 yp 420 and amino acids 421 - 432 : rgd motif , nit ″: n 421 itrgdtyipyp 432 ( seq no : 5 ). 1 μg each of a purified 1 . 6 kb pcr dna fragment containing alexafluor488 - labeled dutp ( molecular probes , eugene , oreg ., usa ) instead of dttp , was incubated in 76 mm k 2 hpo 4 , 17 mm kh 2 po 4 , 14 mm nah 2 po 4 , ph = 7 . 2 , 5 mm mgcl 2 and 1 mm atp - γ - s withe 100 μg purified uvsxh6n2 or uvsxh6n2nit - 2 in a final volume of 200 μl for 30 min at room temparature . then , 10 μl of each npf reaction was applied to a 0 . 8 % tae / agarose gel which was subsequenty stained with ethidium bromide . the electrophoresis was carried out for 1 hour at 100 volts . fig1 shows that the dna was completely retarded . thus , the dna binding of the proteins is not hampered by the fluorescein labelling of the dna . nih3t3 cells were plated in 6 well plates ( 3 × 10 5 per well ), incubated over night at 37 ° c . and 5 % co 2 , and were washed the next morning with prewarmed fcs - free medium ; afterwards 2 ml fcs - free medium was added . 190 μl of the npf reactions were added ( see above ), incubated for 30 min at room temperature , the supernatant was removed , the cells were washed and covered with 3 ml medium ( with 10 % fcs ). after another incubation for 1 hour at 37 ° c . in the incubator , the analysis was carried out under the fluorescence microscope . in fig1 a and 14 b , one picture each is shown in bright field ( lower ) and reflected light fluorescence ( upper ) in the bright field , vesicular intracellular compartments are visible which glow in the fluorescent light due to the endocytosed dna ( fig1 a , b , upper ). from each well , several pictures were taken . the cells were counted and the proportion of cells containing at least one fluorescent vesicular compartment was determined in percent ( shown in fig1 ). each picture shows a mean of 35 cells . of the reactions with uvsxh6n2nit - 2 , nine pictures were analyzed , and of the reactions with uvsxh6n2 - 2 , five pictures were analyzed . the result shows that the modification of uvsx with an integrin - binding motif as a functional component ( uvsxh6n2 - nit - 2 ) results in a markedly increased endocytotic uptake of the transfection agents in the cells compared to the control ( uvsxh6n2 - 2 ). generation of a transfection agent based on hrad51 as npf - forming protein the proteins hrad51h6 and hrad51h6n2 were used as npf - forming proteins ( see fig1 ). amino acids 1 - 339 : human rad51 ( ncbi protein accession no : q06609 , amino acids 1 - 339 ), amino acids 340 - 343 : linker consisting of the amino acids y 340 syg 343 , amino acids 344 - 349 : h 344 hhhhh 349 for purification by nickel chelate affinity chromatography , amino acids 350 - 352 : c - terminus consisting of the amino acids m 350 ys 352 . amino acids 1 - 339 : human rad51 ( ncbi protein accession no : q06609 , amino acids 1 - 339 ), amino acids 340 - 343 : linker consisting of the amino acids y 340 syg 343 , amino acids 344 - 349 : h 344 hhhhh 349 for purification by nickel chelate affinity chromatography , amino acids 350 - 352 : linker consisting of the amino acids m 350 ys 352 , amino acids 353 - 366 : nuclear localization signal nls - 2 ( amino acids 2 - 15 , seq id no : 9 from wo 00 / 40742 ), amino acids 367 - 369 : c - terminus consisting of the amino acids g 367 yp 369 . for expression of the above mentioned proteins in suitable escherichia coli cells , plasmids were constructed that contain a coding sequence for hrad51h6 or hrad51 h6n2 under the control of the lac promoter ( pexh - hrad51h6 or pexh - hrad51h6n2 , see fig1 ). pexh - uvsxh6 - 2 and pexh - uvsxh6n2 - 2 were used as source plasmids ( see fig1 ). the coding region for uvsx was cut out with ecor v and bsiw i , and replaced by a pcr fragment with the coding region for hrad51 which had been cut out in the same way . this was amplified from a human cdna library using hrad51 - specific primers that contain the desired restriction sites . at the 5 ′- end before the start codon , the pcr products contained the additional nucleotides 5 ′- cacacatctagacgtacggatatcat - 3 ′ ( seq id no : 6 ′), and at their 3 ′- end they contained the additional nucleotides 5 ′- tactcgtacggaggtggcggccgctgtgtg - 3 ′ ( seq id no : 7 ) instead of the stop codon . a preculture of 5 ml dyt / ampicillin ( 100 μg / ml ) was inoculated with a colony of pexh - rad51h6 or pexh - rad51h6n2 in dh5 , and was grown for 5 hours at 37 ° c . with 250 rpm . 10 i dyt / ampicillin ( 100 μg / ml ) was inoculated with this preculture , and was allowed to grow for another 24 hours at 37 ° c . with 210 upm . the cultures were harvested at 7000 × g , and yielded apprpx . 30 - 50 g bacterial pellet . this was frozen for 1 - 3 days at − 20 ° c . the pellet was thawed on ice , and resuspended in 100 ml cold starting buffer . the cells were then solubilized by ultrasound using a b . braun labsonic u ( large probe , parameters : 300 watts , 0 . 5 sec pulse duration per second , 8 min sonification ). then it was incubated with 10 mg lysozyme ( serva , 190 . 000 u / mg ) for 1 hours at 4 ° c . and for another 30 min after addition of 50 μl dnase i ( serva , 2 mg / ml ) with slow stirring . after removing the lysate by centrifugation ( 45 min , 18000 × g , 4 ° c . ), the supernatant was filtered through sterile filters ( pore sizes 0 . 45 μm and 0 . 2 μm ) and loaded on an equilibrated 1 ml hitraptm chelating column ( pharmacia ) preloaded with ni ++ ions . the further purification steps were carried out according to the respective pharmacia protocol for proteins that have been provided with a histidine hexamer . aliquots of the various elution fractions were applied to sds / coomassie gels . the purest fractions were combined and were further concentrated over centriplus ym30 columns ( millipore ) according to the respective protocol . then it was dialyzed twice ( dialysis tubing : spectra / por , mwco : 25 . 000 ) against an at least one thousand - fold volume of zi buffer for 1 hour each at 4 ° c ., then over night at 4 ° c . against zi buffer / 50 % glycerine . the dialysis product was aliquoted in 30 - 50 μl fractions and stored at − 80 ° c . fig1 a shows the purified hrad51 - h6 and hrad51 - h6n2 proteins . as in example 1 , but different elution buffer : 20 mm pi , 0 . 5 m nacl , 100 - 1000 mm imidazole , ph = 7 . 4 the concentrations of the hrad51 proteins was determined by measurement of the od 280 using the extinction coefficient calculated with the gene inspector ™ software ( textco , inc .). binding of nls - modified hrad51 to double - stranded dna : hrad51h6n2purified over ni ++ - sepharose was incubated with 100 ng each of a 0 . 9 kb pcr fragment . a dna shift in the agarose gel caused by the protein binding shows that hrad51h6n2 cooperatively binds double - stranded dna based on the concentration ( fig1 b ). even with low amounts of hrad51h6n2 , single dna molecules are completely bound by hrad51h6n2 , and are therefore retarded maximally , so that the retardation of the dna does not increase any more by increasing the amount of protein . it is concluded that hrad51h6n2 binds dsdna . generation of a transfection agent based on uvsx with a signal for the non - endosomal membrane permeation and a nuclear localization signal as functional component as in example 1 , a plasmid was generated that permits the expression of the fusion protein uvsxh6n2vp22c50 ( see fig1 ), which additionally contains a part of the tegument protein vp22 ( gene ul49 ) of the human herpes virus 1 and is based on the protein uvsxh6n2 - 2 described in example 2 . the here used vp22 peptide acts as a signal for the non - endosomal permeation through the cell membrane . thus , the fusion protein uvsxh6n2vp22c50 contains a membrane transduction signal in addition to a nuclear localization signal ( nsl ). amino acids 1 - 391 : uvsx from the phage t4 ( ncbi protein accession no : aad42669 , amino acids 1 - 391 ), amino acids 392 - 394 : linker consisting of the amino acids s 392 yg 394 , amino acids 395 - 400 : h 395 hhhhh 400 , amino acids 401 - 403 : linker consisting of the amino acids m 401 ys 403 , amino acids 404 - 417 : nuclear localization signal nls - 2 ( amino acids 2 - 15 , seq id no : 9 aus wo 00 / 40742 ), amino acids 418 - 422 : linker consisting of the amino acids g 418 ypgs 422 , amino acids 423472 : part of the tegument protein vp22 ( gen ul49 ) of the human herpes virus 1 ( ncbi protein accession no : np — 044651 , amino acids 252 - 301 ), amino acids 473 - 474 : c - terminus consisting of the amino acids p 473 r 474 . for the expression in suitable escherichia coli cells , pexhuvsxh6n2 - 2 ( see example 1 and fig1 ) was opened by restriction enzyme digestion with acc65 i and spe i , and was ligated with a pct product cut with acc65 i and nhe i , which contains at the 5 ′- end the additional nucleotides 5 ′- cacacaggtacccgggatcc - 3 ′ ( seq id no : 8 ) and at its 3 ′- end the additional nucleotides 5 ′- cctaggtaataataagcggccgcgctagctgtgtg - 3 ′ ( seq id no : 9 ), in addition to the coding sequence for the last 50 amino acids of the tegument protein vp22 ( gene ul49 ) of the human herpes virus 1 ( ncbi nucleotide accession no : nc — 001806 , complementary sequence of the nucleotides 105486 - 106391 ) ( see fig1 ). the purification of uvsxh6n2vp22c50 was carried out as described in example 1 for h6uvsx ( see fig1 a ). binding of a mixture of various modified uvsx ( uvsx - nls - vp22 and uvsx - nls ) to double - stranded dna : 140 ng ( in each case ) of a purified 1 . 7 kb pcr fragments were incubated in 96 mm k 2 hpo 4 , 21 . 5 mm kh 2 po 4 , 18 mm nah 2 po 4 , ph = 7 . 2 , 5 mm mgcl 2 and 1 . 3 mm atp - γ - s with the amounts according to fig1 b of purified uvsxh6n2vp22c50 or uvsxh6n2 - 2 for 30 min at room temperature , then , all reactions were applied to a 0 . 8 % tae / agarose gel which was afterwards stained with ethidium bromide . the two proteins were different regarding their molecular weight and net charge , and therefore retarded the dna differently during electrophoresis , with the complex with uvsxh6n2vp22c50 remaining stuck in the gel pocket and not migrating any more ( see lanes 1 and 7 of fig1 b ). when the proteins are mixed before they are added to the dna , intermediate complexes are formed depending on the ratio of uvsxh6n2 - 2 and uvsxh6n2vp22c50 , the migration performance of which is between those of the unmixed complexes ( fig1 b ). this shows that the dna is occupied by both proteins . also possible is a mixture of differently modified or one - or two - times modified npf - forming proteins . transfection of a cell line ( nih3t3 ) with complexes of dna and a mixture of uvsx - nls - vp22 and uvsx - nls 2 . 5 × 10 5 cells ( nih3t3 ) were plated out in each well of a 6 - well plate , and transfected on the following day with a vector containing a gene for the expression of a fluorescent reporter protein . for this , 0 μg - 1 μg linear or 1 μg circular dna was preincubated with 36 μg uvsxh6n2vp22c50 or a mixture of 19 μg uvsxh6n2vp22c50 and 39 μg uvsxh6n2 - 2 in binding buffer ( 76 mm k 2 hpo 4 , 17 mm kh 2 po 4 , 14 mm nah 2 po 4 , 5 mm mgcl 2 , 1 mm atp - γ - s , ph 7 . 21 ) for 30 min at room temperature , and together with 1 ml rpmi was added to cells that have before been washed once with pbs / bsa . after a 1 h incubation at 37 ° c ., 5 % co 2 in the incubator , 1 ml rpmi / 20 % fcs was added respectively and further incubated in the incubator . 4 h or 24 h later , the cells were analyzed in the fluorescence microscope . cells were observed that expressed the reporter gene after treatment with complexes of linear or circular dna and the mixture of uvsxh6n2vp22c50 and uvsxh6n2 - 2 ( see fig1 ). the number of the transfected cells increased with the amount of used dna or dna - protein complexes . however , no expression of the reporter gene was achieved with dna complexes containing only uvsxh6n2vp22c50 , or in absence of dna . therefore it appears that by modification of an npf - forming protein , here uvsx , with a membrane - active peptide , here vp22 , the transfection of cells and here especially the membrane permeation is facilitated . the modular character of the method or transfection agent according to the invention is underlined by the combination of differently modified proteins , here modification with vp22 and nls ( see also fig2 ). whereas the vp22 - modified uvsx permits the non - endosomal membrane permeation , the nls - modified uvsx directs the transfected dna from the cytoplasma to the nucleus ; the transfected dna can then be expressed there . thus , individual steps of the complex transfection procedure can be controlled specifically , flexible and with high efficiency in especially advantageous manner . andersson , k . 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