Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2001-02-27
2003-06-03
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S462000, C435S463000
Reexamination Certificate
active
06573100
ABSTRACT:
FIELD OF THE INVENTION
The invention concerns the use of vector DNA without a selection marker gene in gene therapy as well as the use of these vectors for the production of pharmaceutical agents for gene therapy.
BACKGROUND OF THE INVENTION
The gene therapy of somatic cells can be carried out for example using retroviral vectors, other viral vectors or by non-viral gene transfer (for review cf. T. Friedmann (1989)(1), Morgan (1993)(2)).
Delivery systems that are suitable for gene therapy are for example retroviruses (Mulligan, R. C. (1991)(3)), adeno associated virus (McLughlin (1988)(4)), vaccinia virus, (Moss et al. (1987)(5)), bovine papilloma virus, (Rasmussen et al. (1987)(6)) or viruses from the herpes virus group such as the Epstein Barr virus (Margolskee et al. (1988)(7)) or herpes simplex virus.
Non-viral delivery systems are also known. “Naked” nucleic acid, preferably DNA, is usually used for this or nucleic acid together with an auxiliary substance such as e.g. with transfer reagents (liposomes, dendromers, polylysine-transferrin conjugates (Wagner et al. (1990)(14), Felgner et al. (1987)(8)).
In order to provide the nucleic acid that can be used for gene therapy in a therapeutic amount, it is necessary to multiply these nucleic acids before the therapeutic application. This involves at least one selection step which utilizes a marker gene located the nucleic acid and its gene product. Common selected markers are for example ampicillin, chloramphenicol erythromycin, kanamycin, neomycin and tetracycline (Davies et al. (1978)(9)).
Several protocols for gene therapy are already know which are either still at the stage of animal experiments (Alton et al. (1993) (15); WO 93/1224 (10); (Hyde et al. (1993) (16), Debs et al. (1991) (17)) or already in clinical trials on patients (Nabel (1993) (18), (1994) (19)). A vector based on pBR322 or pUC18/19 is usually used in these protocols which carries an ampicillin resistance gene as the bacterial selection marker.
When nucleic acids are administered in a gene therapy treatment bacteria present in the respiratory and digestive tract and on the skin may take up the number of acids. However, when the marker is an active antibiotic resistance gene (AB
R
gene) this may produce an antibiotic resistance in the patient as an undesired side effect. This is particularly disadvantageous with cystic fibrosis is treated by gene therapy. In this case large amounts of vector nucleic acid are administered to the patient as plasmid DNA or as an aerosol using liposomes as a DNA transfer reagent (Alton et al. (1993)(15)).
Patients with a cystic fibrosis illness usually additionally suffer from bacterial lung infections for example
Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae
which are usually to by administering antibiotics such as penicillin. Hence a resistance of the patients to these antibiotics is disadvantageous.
The previously described protocols for CF gene therapy by means of CFTR plasmid/liposome conjugates and publications of in vitro or animal experiments use vectors based on pUC18/19 or pBR322 which contain the ampicillin resistance gene as the bacterial selection marker (Alton et al. (1993) (15); WO 93/1224 (10); Hyde et al. (1993)(16)).
SUMMARY OF THE INVENTION
The common
E. coli
vectors based on pUC or pBR with the ampicillin resistance gene (Nabel et al. (1993)(18); Lori et al. (1994) (20); Cotten et al. (1994) (21); Lew et al. (1994) (22) etc.) are also used in the other in vivo gene therapy protocols and publications of in vitro or animal model studies with naked DNA or DNA/transfer system conjugates.
The invention concerns the use of a circular vector DNA to produce a pharmaceutical agent for the treatment of mammals or humans by gene therapy in which the vector 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 said heterologous DNA or both fragments are recircularized to form vectors,
c) the said DNA fragments are separated before or after the recircularization
d) the recircularized DNA fragment which contains the said heterologous DNA is isolated and
e) the recircularized DNA fragment obtained in this manner is used to produce the pharmaceutical agent.
The cleavage in step (a) is preferably carried out by means of restriction endonucleases. In this case it is recircularized by adding ligase (step b). It is also preferred to carry out the cleavage and recircularization in one step by recombination with site-specific recombinase systems (SSR).
The use of site-specific recombinase systems (SSR systems) enables the AB
R
gene to be separated in an elegant manner from the remaining part of the vector (plasmid origin of replication and insert) if the specific recombination sites are placed correctly. For this purpose two specific recombination sites must be incorporated upstream and downstream of the AB
R
gene. If an SSR is added, this leads to a specific recombination between both recombination sites by which means the DNA pieces between the recombination sites are separated. In this manner two ring-like molecules are formed (one with the AB
R
gene and one with the insert and the plasmid origin of replication) each of which carries one recombination site.
It is essential that the DNA is recircularized again after deletion of the vector part (by ligase or recombinase), since circular DNA can be transfected with a higher efficiency than linear DNA (Chen and Okayama (1987) (37)) and has a longer half-life in the blood or in the target cell i.e. is less susceptible to nuclease action.
The two circular molecules formed in this manner can be separated from one another by chromatographic methods. The larger the difference in the size between the two molecules, the more effective is the separation. The circular therapeutic DNA obtained now only contains the. therapeutically active gene plus necessary regulatory elements in order to ensure a gene expression in the human target cells as well as, for technical reasons, the
E. coli
plasmid origin of replication which, however, does not interfere at all. The interfering AB
R
gene is deleted.
The site-specific recombination can be carried out in vivo as well as in vitro. In the case of the in vivo site-specific recombination an SSR gene integrated into the host cell DNA (or F episome) is induced, the gene product formed, the SSR, carries out the specific recombination reaction in vivo on the therapeutic plasmid which is additionally present in the cell. The recombination products are isolated from the cell and separated in chromatographic processing steps. In order to carry out the site-specific recombination in vitro, purified SSR is added to the therapeutic plasmid isolated by conventional methods. After the recombination is completed the circular final products are separated from one another by chromatographic process steps.
In principle three systems are available as SSR:
1. The SSR systems of lysogenic phages:
e.g. the cre/lox system of the bacteriophage P1 (Sauer and Henderson (1988) (44); Baubonis (1993) (46)) the &lgr;int system of the bacteriophage &lgr; (Landy et al. (1989) (42)) or the Gin system of the bacteriophage Mu (Klippel et al. (1993) (41)).
2. The SSR systems of the yeast plasmid 2&mgr; and analogous plasmids from other yeast strains:
e.g. the “FLP/FRT” system of the 2&mgr; episome from
Saccharomyces cerevisiae
(Cox et al. (1983) (40)), the “R” SSR system of the episome pSR1 from
Zygosaccharomyces rouxi
(Matsuzaki et al. (1990) (43)), the SSR system of the episome pKD1 from
Kluyveromyces drosophilarium
(Chen et al. (1986) (38)) or the SSR system
Ruger Rudiger
Seeber Stefan
Crouch Deborah
Fulbright & Jaworski LLP.
Roche Diagnostics GmbH
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