Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Phosphorus containing other than solely as part of an...
Reexamination Certificate
2002-09-10
2003-08-12
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Phosphorus containing other than solely as part of an...
C514S086000, C514S256000
Reexamination Certificate
active
06605602
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
A PCR method is provided for detecting BK virus, and particularly for managing BK virus-associated nephropathy in renal transplant patients along with related PCR primers and primer sets. A method for reducing BK virus viral load also is provided.
2. Description of the Related Art
BK virus (BKV) is a human polyoma virus that was originally isolated from the urine of immunocompromised patients. Since then, a number of BKV variants (subtypes) have been isolated. BKV causes a subclinical (asymptomatic) infection in the majority of the general population within the first 10 years of life. Subsequent to infection, the virus normally remains latent in the kidney. However, the virus may become reactivated at a later point in time as a result of immunosuppression, for example, following renal transplantation.
BKV contains a double stranded DNA (dsDNA) genome. The complete DNA sequence of BKV is approximately 5,100 base pairs, however this varies with each variant of BKV. For example, the Dunlop strain of BKV contains 5,153 base pairs (see, for example, Self et al. (1979), “The Genome of Human Papovavirus BKV,”
Cell
18:963-77, incorporated herein by reference in its entirety). The BKV genome contains a coding region and a non-coding control region, but is functionally divided into three regions. The coding region can be further divided into the early region and the late region. The early region contains the coding sequence for two non-structural proteins: the T-antigen protein and the t-antigen protein. The late region contains the coding sequence for four structural proteins: VP-1, VP-2, VP-3 and the agno protein. The non-coding control region contains the transcriptional control elements for both early and late gene expression, as well as containing the viral origin of replication.
The role of BKV infection in renal allograft dysfunction has been controversial. Studies have reported a range of clinical outcomes, varying from asymptomatic to significant renal dysfunction. However, recent studies have shown that BKV causes nephropathy in up to 5% of renal allografts. In addition, BKV infection in renal transplant recipients can be associated with significant morbidity. The controversy surrounding the role of BKV in renal dysfunction is due to the difficulty in diagnosing and monitoring BKV infection. One problem is that serological and traditional viral culture techniques are either not specific or not readily available. In addition, these techniques may not be practical in situations where rapid diagnosis is required in order to make timely patient management decisions.
The diagnosis of BKV-associated nephropathy (BKVN) usually is made by allograft biopsy. A positive biopsy shows viral inclusion bodies, often associated with variable mononuclear infiltrates, and tubulitis that may resemble acute rejection. Immunohistochemical techniques are not sensitive enough to detect latent virus in biopsies that lack viral inclusion bodies that are otherwise detectable by standard light microscopy. A complicating factor in the diagnosis of BKVN nephropathy is the fact that BKVN can present in many different ways. BKV infection has presented with features that were variably diagnosed as acute rejection, interstitial nephritis, drug toxicity, ureteric stenosis, and also asymptomatic. Thus a great degree of caution and a high index of suspicion are needed for the proper diagnosis and management of BKVN.
Even after BKVN has been properly diagnosed, clinical management remains a significant challenge. No clinically proven anti-polyomavirus drugs are currently available for the management of BKVN even though in vitro studies have demonstrated that several drugs, including retinoic acid derivatives, DNA gyrase inhibitors, cytosine arabinoside and cidofovir, inhibit polyoma viral DNA replication. Although various therapeutic strategies have been suggested and tried, the results are often variable and dismal. As a result, therapy for BKVN is usually based on renal allograft biopsy findings. The difficulty in clinical management is compounded by the fact that even when biopsies show tubulitis, suggesting the possibility of underlying rejection, there is little or no transient response to corticosteroids in most cases. Another possibility for clinical management is to reduce immunosuppression. Although reducing immunosuppression decreases the viral load, it increases the risk of rejection.
Given the challenges posed by the diagnosis and management of BKVN, the development of non-invasive quantitative techniques to monitor viral load can have a significant impact on the clinical management of these cases. For example, quantitation of the viral load would allow a physician to monitor a patient's response to specific anti-viral therapy. In addition, it would allow a physician to lower a patient's level of immunosuppression sufficiently to permit stimulation of anti-viral immunity without reducing it to a point that would precipitate acute rejection.
Several references discuss PCR assays for the detection of BKV in urine and/or blood. However, those references teach primers that bind to and amplify BKV viral DNA in the early (T and t antigen) region. Other references disclose PCR primers that bind to and amplify sequences located in the late region of the BKV genome, and particularly in the VP-1 region (Li Jin, “Molecular Methods for Identification and Genotyping of BK Virus,”
Methods in Molecular Biology
, vol. 165, pp. 33-48 (2001), Li Jin, “Rapid Genomic Typing of BK Virus Directly from Clinical Specimens,”
Molecular and Cellular Probes
, vol. 7, pp. 331-334 (1993), Li Jin, “Genomic Typing of BK Virus in Clinical Specimens by Direct Sequencing of Polymerase Chain Reaction Products,”
J. Medical Virology
, vol. 41, pp. 11-17 (1993), and Baksh et al., “Molecular Genotyping of BK and JC Viruses in Human Polyomavirus-Associated Interstitial Nephritis After Renal Transplantation,”
Amer. J. Kidney Disease
, vol. 38, no. 2, pp. 354-365 (August 2001) each of which are incorporated herein by reference in their entirety). These primers function to generate PCR amplicons that were used to distinguish BKV subtypes by PCR combined with restriction enzyme analysis or sequencing analysis.
Cidofovir (HPMPC, Vistide, (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine) is an acyclic nucleoside phosphonate with broad-spectrum activity against a wide variety of DNA viruses. It is reportedly effective in patients diagnosed with progressive multifocal leukoencephalopathy, an infection caused by JC virus. Cidofovir is usually administered in a dosage of 5 mg/kg, with approximately 75-80% of the cidofovir dose excreted in the urine unchanged within 24 hours of administration. In addition, cidofovir is usually given in conjunction with the anti-diuretic probenecid. Cidofovir is nephrotoxic and, therefore, is contraindicated in patients with impaired renal function. Nevertheless, cidofovir was studied herein for its effect on BKV load in kidney transplant recipients diagnosed with BKV associated nephropathy.
BRIEF SUMMARY OF THE INVENTION
Provided are PCR primers for the quantitation of BKV viral loads in a sample using a PCR or quantitative PCR (QPCR) assay and methods for the diagnosis and management of BKVN. PCR primers are provided for binding to and amplifying a region of the BKV genome using PCR or QPCR. The PCR primers include a forward BKVN primer, preferably including substantially the sequence 5′-TGATAGCCCAGAGAGAAAAATGC-3′ (SEQ ID NO: 1), and/or a derivative thereof, and a reverse BKVN primer, preferably including substantially the sequence 5′-TCCACAGGTTAGGTCCTCATTTAAA-3′ (SEQ ID NO: 2), or a derivative thereof.
Also provided is a method for quantitating the BKV viral load in a viruria, serum or plasma (viremia) tissue sample using one or more of the BKNV primers, or derivatives thereof, in a PCR or QPCR assay. The method includes the steps of: performing a nucleic acid purification method on a patient specimen to obtain a nuclei
Kirkpatrick & Lockhart LLP
Mosher Mary E.
University of Pittsburgh-of the Commonwealth System of Higher Ed
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