Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-02-14
2002-09-17
Zitomer, Stephanie (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S041000, C435S069100, C435S091100, C435S091520, C435S194000, C435S196000, C435S091530, C435S091200, C435S320100, C536S023100, C536S024100, C536S024200, C536S024330, C536S025400
Reexamination Certificate
active
06451563
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the synthesis of a linear, double-stranded, covalently closed DNA molecule that can be used as a vector for gene therapy. More generally, the present invention also relates to a method of obtaining preparations of DNA molecules essentially free of contamination by genomic DNA originating from organisms employed in the making of the DNA molecules.
2. Background Information
Gene therapy and genetic vaccination are modern molecular approaches promising to change the way of future medical practice. Despite the great expectations inspired by these methods, however, some basic problems will have to be solved before these methods find general clinical acceptance.
Both gene therapy and genetic vaccination need methods to transfer genetic information into cells or tissues of a patient and to subsequently express the transferred information within those cells. There are safety, efficacy, and specificity issues associated with this transfer process and the transfer means employed. Generally speaking, viral transfer means are very efficient and specific but may be a cause for concern regarding both epidemiological and immunological issues. Non-viral transfer means such as naked DNA are considered to be much safer regarding possible reversion to pathogenicity, but may offer less efficacy and specificity. These issues are discussed, and some possible solutions offered, in our application WO 98/21322, the disclosure of which is incorporated herein by reference. The main aspect of Application No. WO 98/21322 is a minimalistic vector construct consisting mainly of linear double-stranded DNA, which is covalently closed by short oligodesoxyribonucleotide loops to essentially prevent exonucleolytic degradation and to allow attachment of specificity-inducing moieties. This construct will be referred to within the present specification also as, for example, a dumbbell expression construct, a dumbbell construct, and/or a dumbbell-shaped construct.
Several methods are known or can be fashioned without inventive activity in order to make such constructs. One can amplify the expression cassette forming the main, double-stranded part of the dumbbell construct by polymerase chain reaction (PCR), subsequently digest the amplified fragment by means of restriction enzymes, leaving overlapping ends, and ligate short hairpin oligonucleotides to the ends of the digested amplification fragments. Thereby, one will obtain dumbbell-shaped constructs that are essentially easily purified by HPLC on a large scale. The use of heat-stable or thermostable polymerases, however, makes the process uneconomical and gives rise to impurities in the product due to the high error rate of the polymerase. Alternatively, the main part of the construct can be amplified as part of a bacterial plasmid by fermentation, cut out from the plasmid backbone by restriction digest, and be ligated to hairpin oligonucleotides as described above. This leaves the desired product contaminated by backbone sequences, the removal of which is one main objective of making the minimalistic dumbbell constructs described in the present application. The backbone contamination can be removed by chromatography, electrophoresis, or other methods based on size. Since both vector backbone and expression cassette may be, and in practice often are, in the same size range—that is, between approximately 1.5 kilobasepairs and approximately 5 kilobasepairs, this separation based on size differences can be difficult and often renders suboptimal results. Therefore, methods are needed to obtain the desired constructs in an essentially easy, economical process.
OBJECT OF THE INVENTION
One object of the present invention may be to provide a process to obtain linear double-stranded covalently closed DNA “dumbbell” constructs from plasmids by restriction digest, subsequent ligation with hairpin oligodesoxyribonucleotides, optionally in the presence of restriction enzyme, and a final digest with endo- and exonucleolytic enzymes that degrade essentially all contaminating polymeric DNA molecules but the desired construct. Another object of the present invention may be to provide a process to obtain said dumbbell constructs employing endonuclease class II enzymes, recognizing non-palindromic sequences, and having the restriction site generate overlapping ends away from the enzyme recognition site. Furthermore, yet another object of the invention may be to provide a process to obtain linear, covalently closed DNA molecules, such as plasmids, essentially free from contamination by genomic DNA, by submitting the DNA preparation to a facultative endonucleolytic degradation step and an obligatory exonucleolytic degradation step.
SUMMARY OF THE INVENTION
According to the present invention, the DNA molecule forming the main, double-stranded part of the desired expression cassette dumbbell construct may be amplified by fermentation as part of a bacterial plasmid. The DNA molecule is isolated from the plasmid, on or in which it can be contained as a single or multiple copy within the plasmid sequence. Subsequently, the DNA molecule may be cut from the vector backbone by restriction endonucleases that may leave essentially short single overlaps at the restriction ends, preferably of three or more nucleotides in length.
In the next step, the resulting mix of expression cassette construct and vector backbone may be reacted in the presence of a DNA ligase with essentially short, hairpin-forming oligodesoxyribonucleotides that may comprise a single-stranded overlap hybridizing to the overlap generated by the restriction enzyme, resulting in a covalently closed single-stranded molecule with oligonucleotide loops at both ends. According to the invention, the resulting mix of dumbbell-shaped expression construct and backbone sequences, at least some of which backbone sequences may be in dumbbell form also, may be reacted with a restriction endonuclease that cuts only the backbone sequence and the recognition sequence that is not provided on the dumbbell expression construct. This endonuclease digest renders a mix of covalently closed molecules of the desired product, as well as digested backbone molecules and contaminating sequences with open 5′ hydroxyl ends and 3′ hydroxyl ends. This mixture is subsequently submitted to extensive exonuclease digestion. The use of the exonuclease activity of bacteriophage T4 or T7 DNA polymerases was found, in at least one embodiment of the present invention, to be the best mode of executing this step of exonuclease digestion because of the essentially high specificity and processivity of these enzymes. However, any other specifically exonucleolytic activity can be employed to practice at least one possible embodiment of the present invention. The resulting digestion product is a mixture of the desired dumbbell construct, enzymes and buffer components, and desoxynucleotide monomers. From this mixture, the desired product can be purified essentially easy in a single and simple chromatographic step.
The main class of restriction enzymes used in molecular biology is endonuclease class I. These enzymes recognize short palindromic sequences and cut within the recognition site. These enzymes give very practical results when the products are to be used compatibly in cloning experiments. In the ligation described above, however, they may offer a serious drawback. The palindromic overlap generated by the enzyme can lead to reactions between restriction fragments, which reactions are referred to in the present specification as intra- or inter-polymeric reactions. The desired reaction, however, is a polymer-to-oligomer reaction. The former type of reaction—that is, intra- or inter-polymeric reactions—can be suppressed by an essentially large excess of hairpin oligomer, which in turn leads to the formation of hairpin dimers.
According to another aspect of the invention, this problem—that is, the problem of intra- or inter-polymeric reactions between or among restriction fr
Junghans Claas
Schroff Matthias
Wittig Burghardt
Mologen Forschungs Entwicklungs und Vertriebs GmbH
Nils H. Ljungman & Associates
Zitomer Stephanie
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