| Polymerase chain reaction assays for monitoring antiviral therapy and making therapeutic decisions in the treatment of acouired immunodeficiency syndrome -> Monitor Keywords |
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Polymerase chain reaction assays for monitoring antiviral therapy and making therapeutic decisions in the treatment of acouired immunodeficiency syndromeRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Virus Or BacteriophagePolymerase chain reaction assays for monitoring antiviral therapy and making therapeutic decisions in the treatment of acouired immunodeficiency syndrome description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060110726, Polymerase chain reaction assays for monitoring antiviral therapy and making therapeutic decisions in the treatment of acouired immunodeficiency syndrome. Brief Patent Description - Full Patent Description - Patent Application Claims
1. INTRODUCTION [0001] The present invention relates to methods of monitoring, via polymerase chain reaction, the clinical progression of human immunodeficiency virus infection and its response to antiretroviral therapy. According to the invention, polymerase chain reaction assays may be used to predict immunological decline and to identify, at an early stage, patients whose infection has become resistant to a particular antiretroviral drug regimen. 2. BACKGROUND OF THE INVENTION [0002] Human immunodeficiency virus (HIV) isolated from patients treated with zidovudine (AZT) may demonstrate markedly reduced in vitro susceptibility to AZT (Larder et al., 1989, Science 243:1731-1734; Rooke et al., 1989, AIDS 3:411-415; Land et al., 1990, J. Infect. Dis. 161:326-329; Boucher et al., 1990, Lancet 336:585-590; Japour et al., 1991, Proc. Natl. Acad. Sci. 88:3092-96; Tudor-Williams et al., 1992, Lancet 339:15-19). This reduced susceptibility has been related to the duration of therapy with AZT and the severity of HIV disease at the time AZT therapy is begun (Richman et al., 1990, J. AIDS 3:743-756). Nucleotide sequence analysis of AZT-resistant HIV strains has revealed a number of mutations in the reverse transcriptase (RT) gene associated with decreased AZT susceptibility (Larder et al., 1989, Science 246:1155-1158; Larder et al., 1991, AIDS USA 89:1934-1938; St. Clair et al., 1991, Science 253:1557-1559; Richman et al., 1991, J. Infect. Dis. 164:1075-1081). Molecular cloning experiments have confirmed that these mutations in the RT gene confer AZT resistance (Larder et al., 1989, Science 246:1155-1158; Larder et al., 1991, AIDS 5:137-144; Kellam et al., 1992, Proc. Natl. Acad. Sci. USA 89:1934-1938; St. Clair et al., 1991, Science 253:1557-1559). Of these mutations the one at codon 215 resulting in a single amino acid substitution (Thr.fwdarw.Tyr or Phe) has been shown to be the most common mutation and to have the greatest impact on in vitro susceptibility to AZT (Larder et al., 1991, AIDS 5:137-144; Richman et al., 1991, J. Infect. Dis. 164:1075-1081; Boucher et al., 1992, J. Inf. Dis. 165:105-110). [0003] Several studies have addressed the relationship between in vitro AZT resistance, mutations in the RT gene and clinical disease. Richman and coworkers studied 32 patients with different stages of HIV disease and demonstrated that the development of in vitro AZT resistance was related to the duration of therapy with AZT and to the severity of disease at the time AZT was begun (Richman et al., 1990, J. AIDS 3:743-746). Boucher and coworkers studied HIV P24-antigenemic patients treated with AZT for 2 years. They observed that at 6 months, seven patients with a mutation at codon 215 had a weak, non-statistically significant trend toward lower CD4 counts compared to nine patients who were wild type at codon 215 (Boucher et al., 1990, Lancet 336:585-590). After 2 years nearly all patents had the mutation. Tudor-Williams and coworkers studied HIV isolates from 19 symptomatic children treated with AZT for 9-39 months and showed that in vitro AZT resistance was associated with poor clinical outcome (Tudor-Williams et al., 1992, Lancet 339:15-19). However, adult studies have not shown a precise correlation between the development of in vitro resistance and progression of HIV disease. 3. SUMMARY OF THE INVENTION [0004] The present invention relates to methods of monitoring, via polymerase chain reaction (PCR), the clinical progression of human immunodeficiency virus (HIV) infection and its response to antiviral therapy. It is based, in part, on the discovery that plasma HIV RNA copy number, as measured using PCR, may be used as a sensitive marker of the circulating HIV viral load to assess the therapeutic effect of antiretroviral compounds. In working examples described herein, an increase in plasma HIV RNA copy number was found to correlate with disease progression, and successful antiretroviral therapy was found to correlate with a decline in plasma HIV RNA copy number. [0005] The invention is also based, in part, on the discovery that genetic changes in HIV which confer resistance to antiretroviral therapy may be rapidly determined directly from patient peripheral blood mononuclear cells (PBMC) and/or plasma HIV RNA using a "nested" PCR procedure. In working examples disclosed herein, a mutation at codon 215 of HIV reverse transcriptase (RT) was found to occur in AZT-treated patients which correlated with refractoriness to AZT treatment. The mutation was found in plasma HIV RNA one to eight months before it was detectable in PBMC. The development of the condon 215 mutation in HIV RT was found to be a harbinger of immunological decline, which occurred between six and twelve months after the mutation was detectable in plasma HIV RNA. [0006] In particular embodiments of the invention, PCR assay may be used to monitor the clinical progression of HIV infection in patients receiving antiretroviral therapy. An increase in plasma HIV copy number detected by such an assay would correlate with refractoriness to treatment. If a patient being treated with an antiretroviral therapeutic agent exhibits an increase in plasma HIV RNA copy number, a physician should consider altering the patients treatment regimen. It should be noted that the present invention offers the advantage of being more sensitive in measuring HIV virus than standard methods which measure plasma p24 antigen or infectious virus detectable by culture techniques. [0007] In further embodiments of the invention, PCR assay may be used to detect mutations at codon 215 of HIV RT which correlate with resistance to antiretroviral therapy and which precede immunologic decline by 6-12 months. Once mutation at codon 215 has been detected in a patient undergoing antiretroviral therapy, an alteration in the therapeutic regimen must be considered. The speed at which a modified regimen should be instituted may depend on whether the mutation is present in plasma HIV RNA or PBMC. If the mutation is present in PBMC, a rapid alteration in therapy may be warranted. [0008] In patients suffering from HIV infection, opportunistic infections arising as a result of a compromised immune system can be rapidly fatal. It is therefore extremely important to strive to avoid deterioration of the immune system in these patients. Because the present invention enables the early prediction of immunological decline, it allows alteration of a patient's therapeutic regimen so as to avoid opportunistic infections, and therefore may be used to promote survival and improve the quality of life of HIV-infected patients. 4. DESCRIPTION OF THE FIGURES [0009] FIG. 1. Human immunodeficiency virus RNA copy number in 200 .mu.l of plasma from 72 subjects as determined by cDNA gene amplification. Of 39 patients who were not currently receiving antiretroviral therapy, 20 had a CD4 count<200/mm.sup.3 (HIV copy number 1,369.+-.707) and 19 had a CD4 count>200/mm.sup.3 (HIV copy number 44.+-.10). Of 33 subjects who were currently on AZT, 14 had a CD4 count<200/mm.sup.3 (HIV copy number 295.+-.5) and 19 had a CD4 count>200/ml.sup.3 (HIV copy number 16.+-.5). Mean copy number (open circles) of subjects not on therapy was 690.+-.360 as compared to 134.+-.219 for patients currently on AZT (P<0.05, independent sample t test). [0010] FIG. 2. Human immunodeficiency virus RNA copy number in plasma from 27 subjects before (pre) and 1 mo after (post) dideoxynucleoside therapy. (.smallcircle.) AZT; (.circle-solid.) AZT+ddI; and (.tangle-solidup.) ddI alone. Mean copy number decreased from 540.+-.175 to 77.+-.35 after therapy (P<0.05 paired t test). [0011] FIG. 3. Human immunodeficiency virus RNA copy number in plasma from 9 subjects with two samples obtained before initiation of therapy (pre 1 and pre 2) and two samples obtained 1 and 2 mo after commencing therapy (post 1 and post 2). (.tangle-solidup.) ddI alone; (.circle-solid.) ddI+AZT. [0012] FIG. 4. Serial CD4 counts in PBMC (cells/.mu.l) of 17 patients in which HIV reverse transcriptase carried a mutation at codon 215 (top) and of 21 patients in which HIV reverse transcriptase was wild type at codon 215 (bottom). [0013] FIG. 5. CD4 cell counts in PBMC from serial time points in 37 patients. .smallcircle.=wild type sequence in serum specimen, =mutant sequence in serum, .uparw.=wild type sequence in PBMC, .dwnarw.=mutant sequence in PBMC; A: 16 patients mutant at codon 215 in both serum HIV RNA and PBMC (proviral DNA). B: 10 patients mutant at codon 215 in serum HIV RNA but wild type in their PBMC. C: 11 patients whom remained wild type at codon 215 in their serum HIV RNA and PBMC. [0014] FIG. 6. Relationship of PBMC to serum genotypes in the 38 patients at study endpoint. [0015] FIG. 7. Nucleotide sequences of SK38, SK39, and SK19. 5. DETAILED DESCRIPTION OF THE INVENTION [0016] The present invention relates to methods of monitoring, via PCR, the clinical progression of HIV infection in patients receiving antiretroviral therapy. For purposes of clarity and not by way of limitation, the detailed description of the invention is divided into the following subsections: [0017] (i) PCR assay of plasma HIV RNA; [0018] (ii) PCR assay of peripheral blood mononuclear cells; [0019] (iii) PCR assay for mutation at codon 215 of HIV reverse transcriptase; and [0020] (iv) utility of the invention. [0021] It should be noted that heparin appears to have an inhibitory effect on gene amplification via PCR. It is therefore desireable to avoid using heparin as an anticoagulant of patient blood samples. If herapin has been used in a sample, the sample may be purified of heparin, for example, by collecting virus by ultracentrifugation. [0022] 5.1 PCR Assay of Plasma HIV RNA [0023] According to the invention, it is desireable to avoid degradation of RNA in plasma samples prior to measurement of HIV RNA copy number. Therefore, in preferred embodiments of the invention, guanidinium is added to plasma or serum samples prior to storage at a concentration of about 2.5M and samples are kept frozen at -70.degree. C., with no samples stored for longer than about 3 months. Serum may be used interchangeably with plasma according to the invention. Continue reading about Polymerase chain reaction assays for monitoring antiviral therapy and making therapeutic decisions in the treatment of acouired immunodeficiency syndrome... 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