Nucleic acid derivatives

Details Nucleic acid derivatives Nucleic acid derivatives

1997-04-23

2002-12-03

Sisson, Bradley L. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S006120, C435S091100, C435S091200, C435S091330, C435S091510, C435S974000, C536S023100, C536S024300, C536S024330, C536S025300, C536S023720, C536S024320

Reissue Patent

active

RE037918

ABSTRACT:

The present invention relates to a method for assessing the sensitivity of an HIV-
1
sample to zidovudine and to diagnostic assays for use in such assessment.
One group of viruses which has recently assumed a particular importance are the retroviruses. Retroviruses form a sub-group of RNA viruses which, in order to replicate, must first ‘reverse transcribe’ the RNA of their genome into DNA (‘transcription’ conventionally describes the synthesis of RNA from DNA). Once in the form of DNA, the viral genome is incorporated into the host cell genome, allowing it to take full advantage of the host cell's transcription translation machinery for the purposes of replication. Once incorporated, the viral DNA is virtually indistinguishable from the host's DNA and, in this state, the virus may persist for as long as the cell lives. As it is believed to be invulnerable to attack in this form, any treatment must be directed at another stage of the virus life cycle and would, have to be continued until all virus-infected cells have died.
A species of retrovirus has also been reproducibly isolated from patients with AIDS and is now named as human immunodeficiency virus (HIV-
1
) and is also known as human T-cell lymphotropic virus III (HTLV III), AIDS associated retrovirus (ARV), or lymphadenopathy associated virus (LAV).
This virus has been shown preferentially to infect and destroy T-cells bearing the OKT
4
surface marker and is now generally accepted as the aetiologic agent of AIDS. The patient progressively loses his set of T-cells, upsetting the overall balance of the immune system, reducing his ability to combat other infections and pre-disposing him to opportunistic infections which frequently prove fatal. Thus, the usual cause of death is AIDS victims is by opportunistic infection, such as pneumonia or virally induced cancers, and not as a direct result of HIV infection.
The complete nucleotide sequence of the AIDS virus HIV-
1
or as it was previously known HTLV-III has been elucidated (Ratner, L., et al. Nature, Vol. 313, p 277, 24 Jan. 1985).
Recently, HIV-I has also been recovered from other tissue types, including B-cells expressing the T
4
marker, macrophages and non-blood associated tissue in the central nervous system. This infection of the central nervous system has been discovered in patients expressing classical AIDS symptoms and is associated with progressive demyelination, leading to wasting and such symptoms as encephalopathy, progressive dysarthria, ataxia and disorientation. Further conditions associated with HIV infection are the asymptomatic carrier state, progressive generalised lymphadenopathy (PGL) and AIDS-related complex (ARC). HIV-
1
can also be present in other tissues or physiological fluids such as urine, plasma, blood, serum, semen, tears, saliva or cerebrospinal fluid.
3′-Azido-3′-deoxythymidine-(hereafter called “zidivudine”) is used in the control of HIV infections including AIDS and ARC. Zidovudine is a thymidine analogue whose triphosphate form inhibits the replication of the human immunodeficiency virus (HIV) by competitive binding to viral reverse transcriptase (RT) and DNA chain termination after phosphorylation by cellular enzymes (Furman, P. A et al., Pric. Natl. Acad. Sci. USA, 1986, 83: 8333). It is an effective antiviral agent both in vitro and in vivo against a variety of retroviruses (Mitsuya H et al., Cancer Res., 1987, 47: 3140; Ruprecht R. M et al., Nature, 1986, 323: 476.) and has been demonstrated to improve the quality and length of life of patients with AIDS and advanced ARC (Fischl M. A et al., N. Engl. J. Med., 1987, 317: 185; Schmitt F. A. et al, N. Engl. J. Med 1988, 31 9: 1573; Greagh-Kirk T et al, J. Am. Med. Assoc., 1988, 260.: 3009) and also in asymptomatics with low CD
4
+
cell
As with any anti-infective agent, concern about the potential development of resistance has engendered extensive investigations evaluating factors which might potentially alter the sensitivity of retroviruses to zidovudine.
A study carried out to measure zidovudine sensitivity of HIV isolates from patients with AIDS or ARC after zidovudine treatment has in fact revealed that a number of isolates from patients treated for six months or more showed reduced sensitivity to zidovudine whereas isolates from untreated individuals and those treated for less than six months showed uniform sensitivity to the drug (Larder, B. A., Darby, G, and Richman, D. D., Science, Vol. 243, 1731, 31st Mar. 1989).
At present the way to determine the sensitivity of HIV-
1
strains to “zidivudine”) is to isolate HIV-
1
from a patient's peripheral blood lymphocytes. HIV-
1
isolates can be made by co-cultivation of peripheral blood lymphocytes (PBL's) with cells of the continuous line MT-
2
. (Harada, S., Koyanagt, Y., Yamamoto, N., Science 229, 563 (1985). This procedure can take anything from between four sad fourteen days before HIV-
1
can be isolated. The diagnosis of resistant strains of HIV-
1
relies firstly on the isolation of virus and then on sensitivity testing by a tissue culture method and so is consequently extremely slow.
The present inventors have discovered the basis for resistance of HIV-
1
to zidovutne at the nucleic acid level. Five nucleotide substitutions have been identified in the HIV-
1
genome which result in a change of four amino acids in the RT protein. This discovery has important implications for the detection of resistant HIV isolates because of the highly conserved nature of these nucleotide mutations in RT conferring resistance to zidovudine and opens the way to the routine detection of such resistant isolates.
It is possible to carry out a diagnostic assay for the screening of bodily samples from patients for an assessment of the sensitivity of HIV-I to zidovudine. Using the knowledge of the mutations identified as important in the development of highly resistant strains of HIV-
1
such as assay can be developed.
An analysis of a group of resistant mutants was carried out by nucleotide sequencing which allowed the identification of mutations in the HIV RT gene that confer resistance to zidovudine. The complete RT coding region (1.7 kb) was obtained far each isolate using polymerase chain reaction (PCR) amplification of infected cell DNA. The nucleotide changes at the five positions in HIV-
1
RT that confer resistance to zidovudine are illustrated in FIG.
1
. Numbering of the nucleotides of the HIV-
1
RT gene is as reported by Ratner et al (Ratner et al., Nature. 313, 277, (1985)).
It is demonstrated that these specific mutations confer zidovudine-resistance, as an infectious molecular HIV clone containing only these nucleotide changes is resistant to zidovudine. (See Example 4).
From analysis of clinical samples it appears that the sensitivity of HIV-
1
to zidovudtne changes over a period of time. It appears that mutations may occur at any of one or more of the five identified sites as the time from the onset of treatment with zidovudtne advances and it is clear than as HIV-
1
sample which carries all five mutations is highly resistant to zidovudine.
At this time it is not possible to predict any order of occurence of these mutations, although particular attention is being focussed on the two nucleotides of the wild-type DNA sequence (or its corresponding RNA) or to the two nucleotides of the mutant DNA sequence (or its corresponding RNA) set forth in
FIG. 1
at the
2772
- and
2773
- positions.
Accordingly in a fast aspect of the invention there is provided a method for assessing the sensitivity of an HIV-
1
sample to zidovudine, which comprises:
(i) isolating nucleic acid from the sample,
(ii) hybridising an oligonucleotide to the nucleic acid, the oligonucleotide being complementary to a region of the wild-type DNA sequence (or its corresponding RNA) or to a region of the mutant DNA sequence set forth in
FIG. 1
(or its corresponding RNA) and terminating at the 3′-end with the nucleotide in the
2328
-,
2338
-,
2772
-,
2773
- or
2784
-position,
(iii) attempting polymerization of

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