Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2001-02-08
2003-04-01
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S005000, C435S006120, C435S039000, C536S024300, C536S023100
Reexamination Certificate
active
06541246
ABSTRACT:
The present invention relates to a method for detecting and for quantifying, in diverse biological media, adenovirus nucleic acids by real-time polymerase chain reaction (PCR) measurement. A subject of the invention is also oligonucleotides for carrying out this method.
BACKGROUND OF INVENTION
Gene therapy is currently undergoing considerable development. Adenoviruses, which are naturally responsible for generally benign infections, are among the vectors used because of their many advantages.
The extension of the clinical development of these adenoviral vectors to late phases II and III for the purposes of registration and of authorization for marketing makes it necessary to develop, validate and implement specific and sensitive techniques which allow the measurement of the quantitative and qualitative biodistribution of adenovectors in diverse biological compartments, and the detection of the emergence, at low viral load, of infection with contaminants from the production of adenovectors (in particular replication-competent recombinant adenovectors). It is also necessary to detect intercurrent adenoviral infections which are due to wild-type viruses of various serotypes, which are responsible for the vast majority of the incidence of these natural infections, whether these infections are clinically florid or latent, and to determine the virological kinetics of these wild-type infections and the clinical significance in diverse situations, in the general population or in patients who are at risk (patients suffering from cancer, transplant patients, immunosuppressed or immunodepressed patients, etc.).
The presence of an adenovirus in permissive cell culture is visualized, in the detection techniques routinely used to date, by a cytopathic effect in the cell layer. In order to demonstrate that this cytopathic effect is indeed due to an adenovirus, an antigen which is specific for the adenovirus is detected by the ELISA method.
Techniques for amplifying extracted nucleic acids of adenoviruses by PCR have also been described. Thus, Japanese patent application JP 07327700 describes a PCR-type method in which portions of the DNA encoding hexon, which is an adenovirus capsid protein, are amplified. The amplification products are “dot-blotted”, then hybridized with probes which are specific for each serotype. This method is thus intended to identify the subgroups and serotypes, and not to detect and quantify all of the adenoviruses present in a sample. In addition, the sensitivity of this method is low and thus generally unsuitable for precise measurements, such as those required during the monitoring of patients treated by gene therapy.
Pring-Akerblom et al. (Journal of Medical Virology, 58:87-92, (1999), have also described a method which uses six pairs of primers in the same PCR reaction (so-called multiplex PCR). The identification of the subgroup is carried out by loading the PCR products onto electrophoresis gel and then determining the size of the PCR products.
Crawford-Miksza et al. (Journal of Clinical Microbiology, 37:1107-1112, (1999), have described a hexon PCR which specifically detects type 4, 7 and 21 adenoviruses using consensus primers comprising inosine as the nucleic base in the event of mismatching in certain variable position. In that document, nine distinct primers are described.
These prior techniques use a standard PCR and a conventional analysis method which is generally on electrophoresis gel with or without hybridization.
None of these techniques make it possible both to broadly detect adenoviruses originating from the majority of the known serotypes, and to quantify the viral loads thereof with a sensitivity which is suitable for the needs of gene therapy in particular.
The aim of the present invention is thus to resolve these problems by providing a technique which is sensitive and which makes it possible to detect the majority of the adenovirus serotypes.
The applicants have surprisingly shown that such advantages are obtained by amplifying in real time a given sequence of the DNA encoding hexon with the aid of primers having limited degeneracy, and revealing the amplification product with the aid of a nondegenerate probe.
SUMMARY OF THE INVENTION
The present invention first of all relates to a method for detecting and/or for quantifying adenovirus nucleic acids in a biological sample wherein:
an adenovirus nucleotide sequence is amplified by real-time PCR using degenerate sense primers and degenerate antisense primers, wherein said primers are chosen from oligonucleotides having at least 80% homology with a sequence between nucleotides 21000 and 22000 of type 5 adenovirus sequence, and which corresponds to sequence SEQ ID No. 4, and oligonucleotides comprising a complementary sequence thereof, and
an amplification reaction product may be detected using a nondegenerate probe comprising an oligonucleotide having at least 80% homology with a sequence between nucleotides 21000 and 22000 of type 5 adenovirus sequence, which corresponds to sequence SEQ ID No. 4, and oligonucleotides comprising a complementary sequence thereof, wherein said product may be detected during a number of amplification reaction cycles which is sufficient to allow the production of a measurable amount of amplification product.
The term “real-time PCR” is intended to mean any amplification technique which makes it possible to monitor the evolution of an ongoing amplification reaction. Polymerase Chain Reaction is abbreviated as “PCR”.
Generally, sequences of the primers and/or of the probe may comprise at least 80% homology with at least one sequence between nucleotides 540 and 780 of sequence SEQ ID No. 4, or with a complementary sequence thereof. The two primers are chosen on the sense and antisense strands, respectively, and in such a way as to allow the amplification of a DNA fragment. With regard to the probe, it is chosen so as to hybridize with the DNA fragment resulting from the amplification. The PCR primers in accordance with the present invention which are used to amplify the target adenovirus nucleic acid in a sample are located in a region which is constant for the hexon gene of human adenoviruses, and the hybridization site of the probe in accordance with the present invention is located between the two primers. In one embodiment, the adenoviruses are of human tropism.
Generally, said oligonucleotides may comprise at least 15 nucleotides, and the probe has a theoretical melting temperature Tm which is higher than the theoretical Tm of the primers by approximately 10° C.±0.5.
A method of the invention is termed hexon PCR method in abbreviated form in the remainder of the text. It comprises a repetition of a cycle comprising:
separation of the strands to be amplified by heating the DNA extracted from the sample,
hybridization of the probe,
hybridization with primers as defined above, and
elongation with a polymerase.
In one embodiment, the amplification method comprises the sense and antisense primers HEX1 and HEX2 described below. In a further embodiment, the amplification product is hybridized with the HEX probe described below.
One subject of the present invention is an oligonucleotide, comprising at least 10 consecutive nucleotides of the following sequence SEQ ID No. 1 or of a sequence having at least 80% sequence homology with said sequence:
5′-YCC CAT GGA YGA GCC CAC MCT-3′
in which Y represents C or T, and M represents A or C.
In one embodiment of the invention, said oligonucleotide has at least 90% sequence homology with said sequence. In another embodiment, said oligonucleotide has at least 95% sequence homology with said sequence.
In yet another embodiment, said oligonucleotide comprises between 15 and 30 nucleotides.
One subject of the present invention is a HEX1 sense primer comprising a mixture of oligonucleotides which satisfy this definition. The HEX1 primer has three degeneracies at positions 1, 10 and 19 which make it possible to cover the majority of the serotypes.
Another subject of the present invention is a second oligonucleotide, c
Gautier Eric
Saulnier Patrick
Vidaud Michel
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Gencell S.A.
Park Hankyel T.
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