Patent Application: US-79443101-A

Abstract:
the use of a circular vector dna to produce a pharmaceutical agent for the treatment of mammals or humans by gene therapy wherein the vector dna contains a selection marker gene and a dna sequence that is heterologous for the vector which causes a modulation , correction or activation of the expression of an endogenous gene or the expression of a gene introduced into the cells of the mammal or the human by the vector dna which is characterized in that the vector nucleic acid a ) is amplified under selection pressure and cleaved in such a way that the said selection marker gene and the said heterologous dna are present on separate dna fragments , b ) the dna fragment which contains the heterologous dna or both fragments are recircularized to form vectors , c ) the dna fragments are separated before or after the recircularization d ) the recircularized dna fragment which contains the heterologous dna is isolated and e ) the recircularized dna fragment obtained in this manner is used to produce the pharmaceutical agent .

Description:
determination of the uptake rate of plasmids and production of antibiotic resistances by bacteria of the human lung and intestinal flora it is intended to demonstrate that e . coli plasmid dna , e . g . puc18 or derivatives thereof , can be taken up by bacteria of other genera and that the ampicillin resistance gene can be expressed which leads to ampicillin - resistant clones . the probability of forming ampicillin - resistant microorganisms is determined by the uptake rate of plasmid dna from the substrate and the rate of incorporation of the ampicillin resistance gene by non - homologous recombination into the chromosomal dna of the host bacterium . plasmids with alternative markers or with markers inactivated by stop codons ( see above ) no longer result in the development of resistance . for this various amounts of plasmid dna are added under various conditions to cultures of the above - mentioned organisms . liquid cultures in nutrient medium are carried out using isotonic buffer ( nacl , mg 2 + , ca 2 + ). various concentrations of plasmid dna ( e . g . puc18 / ap r gene ) are added to these cultures . after incubation for several hours while shaking , antibiotic is added . it is incubated further , plated out on agar containing antibiotic and a dilution series is carried out on agar which does not contain antibiotic . the number of living organisms is determined after incubation by counting and identifying or characterising the colonies obtained . in detail for this a bacterial culture incubated overnight is centrifuged , the pellet is washed with phosphate - buffered saline solution ( pbs ), resuspended in 2 % culture volume pbs and admixed with 200 ng plasmid dna / ml . a dilution series is immediately plated out on plates without antibiotic . the suspension is slowly shaken at 37 ° c . and at various intervals ( 1 , 2 and 4 h ) dilution series are plated out with and without antibiotics ( ampicillin 100 μg / ml ) on standard i agar and incubated at 37 ° c . the 10 1 - 10 5 dilutions are plated out on antibiotic plates and the 10 9 - 10 10 dilutions are plated out on plates without selection . the transformed resistant cultures are counted the next day and related to the number of viable germs . alternatively a defined amount of plasmid and a defined germ count of bacteria are plated out on nutrient agar and incubated for several hours , antibiotics are added by spraying and the colonies obtained are counted and identified or characterized . it is also possible to apply a defined amount of plasmid and a defined germ count of bacteria to a nylon filter and to incubate the filters for several hours on nutrient agar . subsequently the filters are transferred onto nutrient agar containing antibiotic , incubated and the colonies obtained are counted and identified or characterized . construction and amplification of safety vectors based on puc18 which no longer possess a selection marker the principle of the method is based on the restriction of the isolated therapeutic plasmid , separation of the small fragments containing the vector part (+ resistance gene ) and religation of the large fragment with the therapeutically active gene in several steps in vitro ( restriction endonucleases + ligase ) or in one step in vivo or in vitro ( site - specific recombinases ). the feasibility of the method was demonstrated using the vector puc18 and the therapeutic plasmid pcmv - cftr ( alton et al . ( 15 )). the plasmid map of pcmv - cftr is shown in fig1 the dna sequence is shown in seq id no : 1 . 1 . in order to facilitate the separation of the insert ( containing the therapeutic gene ) from the vector part , several linkers with restriction cleavage sites were incorporated distributed over the vector . psti and pvui were chosen as the restriction enzymes since they do not cut in the insert ( cmv - cftr ) in the plasmid pcmv - cftr chosen as an example . it is , however , also possible to use other enzymes which have the same property of not cutting in the insert . in addition linkers with more than one restriction cleavage site can also be used . in this example linkers with two restriction cleavage sites namely for psti and pvui were used . thus synthetic dna linkers containing the recognition sequences of pvui + psti were incorporated into puc18 in a defined sequence ( 5 ′ psti - pvui in aatii and afliii ; 5 ′ pvu - psti in alwni ) into the singular aflii , alwni and aatii cleavage sites using common laboratory techniques . the sequence is above all important for the aatii and the alwni cleavage site since this builds in additional safety which , apart from the physical separation , prevents the amp r gene from religating with the insert . therefore the double - stranded linkers must have corresponding overhangs for each of the enzyme cleavage sites in order to clone them in the right orientation or if the linkers are cloned via blunt ends , clones having the correct orientation must be sought after . 2 . the polylinker of puc18 was exchanged for a newly synthesized polylinker in order to firstly facilitate the cloning of the cmv - cftr gene cassette and secondly to enable a clean separation of the vector and insert (= cmv - cftr gene cassette ). the synthetic dna polylinker contains the restriction cleavage sites in the following sequence : *) only one strand of the synthetic linker is shown . the parentheses mean that the polylinker has overhangs for the corresponding enzymes and can in this orientation be ligated in the correct orientation into puc18 which had been cleaved with hindiii and ecori . 3 . the 5 . 8 kb dna fragment containing the cmv - cftr gene cassette which was also cut out by means of a xhoi / noti double digestion from pcmv - cftr was inserted in the correct orientation between the xhoi and the noti cleavage sites of puc - pp — 1 ( addition of ligase ). the plasmid that was formed was named pcmv - cftr_pp2 . when restricted with pvui + psti it disintegrates into fragments of sizes : 5 . 8 and 0 . 85 , 0 . 55 , 0 . 42 , 0 . 36 , 0 . 34 , 0 . 12 kb . pcmv - cftr_pp2 was transformed in e . coli . e . coli ( pcmv - cftr_pp2 ) was fermented , disrupted and the plasmid dna was purified by means of a qiagen ® column according to the manufacturer &# 39 ; s specification ( quiagen ; germany ). the purified plasmid dna was cleaved with pvui ( 10 u / μg ; 1 μg in 20 μl volume ; according to the specification of the pvui manufacturer ). the dna was then recleaved with pvui + psti ( 5 u / μg pvui + 10 u / μg psti ). the fragments were separated by a molecular sieve column and the much larger 5 . 8 kb fragment was separated by this means . the eluted 5 . 8 kb fragment was religated by means of t4 ligase ( according to the specification of the manufacturer ). the 5 . 8 kb recircularized dna ( 1 mg dna in 1 ml buffer ) containing the cmv - cftr gene cassette was again purified by means of a qiagen column and taken up in a small amount of buffer tris hcl / edta buffer ( 10 mmol / tris hcl , 0 . 1 mmol / l edta ( ph = 7 . 0 ). ba ) in vivo use of the cre / lox system for the recombinant deletion of the ab r gene : the procedure was carried out similarly to the methods described by sauer ( 1988 ) ( 44 ) via a cre - recombinase - mediated release of circular plasmids which were present integrated into large linear dna molecules flanked by loxp sites . however , in these methods e . coli strains were used in which the cre - recombinase gene was present constitutively expressed . for the example described here the cre - recombinase gene must be present under the control of a strictly regulatable promoter . for this purpose two 34 bp loxp recombination sites were incorporated upstream and downstream of the ampicillin resistance gene into the therapeutic plasmid pcmv - cftr . the plasmid was named pcmv - cftr — 2loxp . the plasmid obtained pcmv - cftr — 2loxp was cloned in e . coli xl1 - blue_λimm434nin5_xi - t5 - cre ( construction analogous to sauer ( 1988 ) ( 44 )) which contains the lysogenic phage λ containing the cre - recombinase gene under the control of the strictly regulatable t5 promoter ( bujard et al . ( 1987 ) ( 36 )) as well as a lac1 q - gene ( f - episomally coded ). e . coli xl1 - blue_λimm434nin5_x1 - t5 - cre ( pcmv - cftr — 2loxp ) was cultured in lb medium containing 100 μg / ml ampicillin . the t5 promoter was induced by addition of 5 mm iptg and thus the expression of the cre - recombinase gene was switched on . the cre - recombinase carried out an in vivo recombination of the two loxp sites in pcmv - cftr — 2loxp . as a result the plasmid pcmv - cftr_loxp was converted into two circular plasmids of greatly differing size . subsequently the e . coli cells were harvested , disrupted and the mixture of circular plasmids was isolated . the mixture was composed of unchanged pcmv - cftr - 2loxp , the circular amp r gene containing a loxp site and the desired final product ( the circular dna containing the therapeutic insert + a loxp site + the puc origin of replication ). subsequently the circular dna molecules of various sizes were separated by means of chromatographic methods and the desired final product was taken up in citrate buffer ( ph = 7 . 0 ). since the cre / lox recombination can proceed in both directions , it is important that the reaction period or the time of harvest after iptg induction is optimized . since the t5 promoter system is present not completely repressed even in lac1 q strains , there is always a low expression of the cre - recombinase gene and thus a low level of recombinations . by placing one of the two loxp sites between the amp r gene and the origin of replication it can , however , be ensured that the cre - mediated recombinations only lead to the formation of constructs which cannot replicate and no longer mediate ampicillin resistance . they are rapidly lost during the replication cycles under a selection pressure by ampicillin addition . 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