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Title:  Method of treating BK virus nephropathy

United States Patent:  6,605,602

Issued:  August 12, 2003

Inventors:  Vats; Abhay N. (Wexford, PA)

Assignee:  University of Pittsburgh-of the Commonwealth System of Higher Education (Pittsburgh, PA)

Appl. No.:  238483

Filed:  September 10, 2002

Abstract

Provided are oligonucleotides for the quantitation of BK virus (BKV) viral loads in a sample and methods for the diagnosis and management of BKV-associated nephropathy using PCR or QPCR methods. In addition, a PCR method for quantitating the BKV viral load in urine, serum and tissue using the described nucleic acid primers is also disclosed. Furthermore, a method for the management of BKV-associated nephropathy in renal transplant recipients using cidofovir is disclosed. Lastly, a kit is provided containing the described PCR for use in diagnosing and managing 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 nucleic acid test sample; conducting PCR or QPCR on the test sample using an appropriate primer set, as defined herein, to generate an amplicon if BKV target sequences are present in the test sample; and detecting the presence of BKV specific amplicon in the sample. A multiplexed QPCR method also is provided using the above-described primer set in combination with a second PCR primer set.

A method also is provided for the management of BKVN in renal transplant recipients using cidofovir, preferably without co-administration of an anti-diuretic such as probenecid. The method includes quantitating the BKV viral load in a patient specimen and administering to a patient a dosage of cidofovir effective to reduce BKV viral load in the patient. Patients treated according to the method have shown clearance of BKV viruria after 2-4 doses of the drug.

Also provided is a kit containing a PCR primer, or a derivative thereof, for quantitating BKV viral load in a sample using a PCR assay, for use in diagnosing and managing BKVN. The kit may further comprise, without limitation control nucleic acids, primer sets to generate a second amplicon in a multiplexed PCR or QPCR assay; collection containers; a buffer; nucleic acid purification reagents or kits; a mixture of dNTP's; and a thermostable DNA polymerase.

Other details, objects and advantages of the present invention will become apparent with the following description.

DETAILED DESCRIPTION OF THE INVENTION

The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word "about." In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within these ranges. Also, the disclosure of these ranges is intended as a continuous range including every value between the minimum and maximum values.

Described herein is a polymerase chain reaction (PCR) method that is useful in the diagnosis and management of BKVN. The PCR primers described herein are specific for BKV when performed on nucleic acids extracted from blood derivatives, including serum or plasma, urine or tissue samples using QPCR. Finally, the following description shows that cidofovir therapy is useful in the treatment of BKVN in renal transplant recipients.

PCR primers are provided for use in amplifying a region of the BKV genome using PCR assays. The PCR primers are oligonucleotides and include a forward BKVN primer substantially including the sequence 5'-TGATAGCCCAGAGAGAAAAATGC-3' (SEQ ID NO: 1), or a derivative thereof and a reverse BKVN primer substantially including the sequence 5'-TCCACAGGTTAGGTCCTCATTTAAA-3' (SEQ ID NO: 2), or a derivative thereof.

As used herein, the term "oligonucleotide" is intended to embrace not only DNA oligonucleotides, but functional analogs thereof. Functional analogs are oligomeric compounds that are suitable for use as primers in a PCR reaction and therefore have adequate sequence specificity and ability to be elongated by a selected thermophilic polymerase in a PCR reaction. There is no strict size limit to an "oligonucleotide" as defined herein, only that the oligonucleotide can be used in a PCR reaction. As such, there are maximum and minimum functional limits to the size of the oligonucleotide. The minimum size is often dictated by the need for adequate sequence specificity of the oligonucleotide and the maximum size is often dictated by the Tm (melting temperature) of the oligonucleotide, along with other factors important in creating a robust PCR environment. As such, optimized oligonucleotide PCR primers may range from 10 to 35 bases in length, more typically from 15 to 25 bases in length and most typically are about 20 bases long.

The two primers provided above both bind to and amplify a portion (substantially including bases 1797 to 1881 of SEQ ID NO: 3) of the late region of the BKV genome, specifically the VP-1 region of the genome. The forward BKVN primer hybridizes to a target sequence that substantially includes base: 1979 to 1819 of SEQ ID NO: 3. The reverse BKVN primer hybridizes to a target sequence that is bases 1857 to 1881 of SEQ ID NO: 3. The forward BKVN primer may be used in conjunction with a reverse primer that binds downstream of the above reverse BKVN primer. Alternatively, the reverse primer may be used in conjunction with a forward primer that binds upstream of the above forward primer. The terms "upstream" and "downstream" refer to the relative positions along a DNA sequence in the traditional 5' to 3' direction such that an upstream region is 5' of a downstream region.

In addition, for any nucleic acid sample to be tested by the PCR assay, a second control PCR assay may be performed using a different (control) set of PCR primers than the BKVN primers (SEQ ID NOS: 1 and 2). The control primers will amplify segments of a broader set of BKV subtypes, with all BKV subtypes or with all polyoma viruses. One example of a primer set that amplifies BKV sequences from a broader range of BKV subtypes than does the BKVN primer set is a VP-1 control primer set including the following primers:

Forward 5'-GCAGCTCCCAAAAAGCC-3' (SEQ ID NO: 5)

Reverse 5'TTCCACAGGTTAGGTCCTCA-3' (SEQ ID NO: 6),

or analogs or derivatives thereof. As shown below, this primer set identifies both BKVN-associated and non-BKVN-associated BKV subtypes in urine, while the BKVN primer set identifies viruria only in BKVN patients.

Other suitable control PCR assays utilize primer sets that are specific to a broader range of BKV subtypes or polyoma viruses than the BKVN primer set. An example of a primer set that would identify all polyoma viruses is a T antigen-specific primer set, such as those disclosed in Biel et al., (2000) "Rapid Quantification and Differentiation of Human Polyomavirus DNA in Undiluted Urine from Patients after Bone Marrow Transplantation," J. Clin. Microbiology 38(10): 3689-95 and Limaye et al., (2000) Quantitation of BK Virus Load in Serum for the Diagnosis of BK Virus-associated Nephropathy in Renal Transplant Recipients," J. Infectious Diseases 183:1669-72, both of which are incorporated herein by reference in their entirety. Additional suitable control primer sets amplify non-viral sequences, such as, without limitation, human GAPDH and human 18S ribosomal RNA, which are available commercially from Applied Biosystems.

As stated above, the primers may have one of the nucleic acid sequences above or may be a derivative of one of the above sequences. The term "derivative," as used herein, is any oligonucleotide that has similar binding characteristics to a target DNA sequence as one of the above two primers, SEQ ID NO: 1 or SEQ ID NO: 2, and, therefore, is useful as a PCR or QPCR primer as described herein. For example, and without limitation, a derivative may be formed by attaching, inserting or deleting one or more nucleic acids to or from one of the above primer sequences, for instance by the addition of a restriction site to the 5' end of one or both of the primers. As an additional non-limiting example, SEQ ID NOS: 2 and 6, or versions thereof with the addition or deletion of certain 5' or 3' flanking nucleotides, may be considered derivatives of each other. Also, a derivative may include one or more nucleotides that are derivatized to contain functional groups, or altered nucleotides, such as inosine. Generally, it is preferred that the derivative contains from about 10 to about 35 nucleic acids, but it may contain more or less nucleotides, depending on a number of empirical factors that impact the usefulness of any given primer in a PCR reaction. The only limitation to the analogs or derivatives is that they hybridize to the desired target sequence of the BKV genome (for instance, for the BKVN primer set, the sequences of SEQ ID NOS: 1 and 2) and can serve as a PCR or QPCR primer, as can be determined empirically by a person of skill in the art.

A PCR assay or method also is provided for rapidly and accurately detecting the presence of BKVN in tissues or bodily fluids and thereby providing a tool for the management of BKV patients. A PCR assay or method is a procedure whereby a segment of a nucleic acid molecule, is amplified repetitively and exponentially to produce a large amount of DNA molecules (amplicons) which consist only of the amplified segment. The procedure requires a large number of replication cycles, usually 20 to 40 cycles. In each cycle, two oligonucleotide primers bind to complementary strands of the nucleic acid molecule and define the limits of the amplified portion. A primer-dependent polymerase then replicates the strands to which the primers have bound. Thus, in each cycle, the number of DNA duplexes is doubled. This general procedure and numerous variations thereof are well known to those skilled in the art.

The first step of the PCR method involves obtaining a nucleic acid from a patient specimen. The patient specimen can be any tissue or bodily specimen. However, it is preferred that the specimen be one of a urine specimen, a serum specimen, or a kidney tissue specimen, obtained from a patient by standard methods. A urine specimen can be collected in a container. A plasma specimen may be obtained from whole blood collected in a heparin coated test tube and then spun in a centrifuge at a speed sufficient to separate the blood into two layers: a cellular layer and a plasma layer. The plasma layer is then collected. A kidney tissue specimen may be obtained by biopsy.

After obtaining a specimen, DNA in the specimen, if present, is purified to the extent necessary for detection of target nucleic acid by PCR and/or QPCR. Such purification methods are well known in the art and include a variety of techniques including extractions, precipitations and chromatographic methods. Kits for the purification of nucleic acids from tissue specimens, including bodily fluids, are commercially available from many sources, including Quiagen, Inc., of Valencia, Calif. Once purified, the DNA can be diluted in water or a buffer system suitable for conducting a PCR reaction. At any time, part of the specimen may be used for analysis while the remaining part of the sample may be stored. For example, part of the specimen can be stored in a freezer at -70oC.

When the sample is a tissue specimen, DNA must be extracted from the specimen. A variety of methods are known to be useful for liberating nucleic acid from cells and virus in the specimen. As above, kits for isolation of viral nucleic acid from tissue are commercially available, for instance from Quiagen, Inc.

In the DNA purification step, certain specimens may not contain sufficient amounts of nucleic acid to reproducibly purify DNA from the specimen. In such a case, it may be desirable to add a carrier to the specimen before purifying the DNA. A variety of carriers are known in the art, including, without limitation, DNA, RNA and glycogen or combinations thereof, but can be any compound or composition that can enhance the sensitivity and/or reproducibility of the DNA purification step. The choice of carrier, and how much carrier is added to a given specimen would depend on the choice of DNA purification assay and empirical factors that readily can be optimized by a person of skill in the art.

A carrier might contain a control DNA, preferably not containing a non-human or polyoma virus sequence that can be amplified by PCR. The control DNA would include a sequence that would provide an internal reference to account for variations in the efficiency of the DNA purification procedure. In a multiplex QPCR procedure, an additional primer set and TaqMan probe can be used that specifically amplifies and detects sequences found specifically in the control DNA. If a specific amount of control DNA is added to every specimen, the results of the BKNV-specific QPCR could be adjusted to account for variations in the DNA purification step. The control DNA might be a part of the carrier itself, for instance, and without limitation, if non-human genomic DNA is used as a carrier. However, in that case the control DNA might be too prevalent in a given sample. If that is the case, the carrier might include both a carrier portion, such as genomic DNA or glycogen, and an appropriately diluted control DNA, such as, without limitation, plasmid DNA including a sequence to be amplified.

The next step involves conducting PCR or QPCR on any purified isolated from the specimen (the test sample) using a BKNV primer, or a derivative thereof, to generate an amplicon. The theories and mechanisms of PCR and QPCR are well known in the art, along with variations in these techniques. Similarly, a large variety of suitable PCR and QPCR protocols are broadly available and are useful in the assays described herein with no modification or with modification that can be readily determined and optimized empirically. These primers are able to bind to and amplify a specific portion of the BKV genome, which is useful in the diagnosis and management of BKVN.

Suitable reaction mixtures or compositions for conducting PCR and/or QPCR on a nucleic acid sample contain at least a portion of a purified nucleic acid test sample prepared as described above; one or more primers, at least one of which is selected from SEQ ID NO: 1 and SEQ ID NO: 2, or analogs or derivatives thereof; a mixture of deoxynucleotide triphosphates (dNTP's); and a thermostable DNA polymerase in a buffered solution, typically containing magnesium. A thermostable DNA polymerase is a DNA polymerase, such as Taq, Pfu or Tfl polymerases, that is capable of elongation at elevated temperatures, typically greater than 40oC. Magnesium, in the form of magnesium ion, typically is an element of a PCR reaction.

Next, the PCR and/or QPCR reaction solution is cycled. Typical PCR reactions include three steps, a denaturation step, an annealing step and an elongation step. The annealing steps may be performed as part of the same step at the same temperature, as shown in the examples below.

The next step of one method described herein is to detect the production of a BKV-specific amplicon. In a non-quantitative assay, the BKV-specific amplicon may be detected in an ethidium bromide stained agarose gel according to standard methods. The amount of BKV nucleic acid in a test sample may be quantitated by comparing the amount of BKV-specific amplicon produced versus a control. The probe can be any nucleic acid that can bind to the BKV-specific amplicon. For instance, the probe may have the nucleic acid sequence 5'-TTACAGCACAGCAAGAATTCCCCTCCC-3' (SEQ ID NO: 4). The probe can be labeled so that binding of the probe to the amplicon may be detected. The label can be any sort of label known in the art, for instance, fluorescent, radioactive, enzyme or antigenic labels. In one example, the 5' end of the probe may be labeled with a FAM dye and/or the 3' end of the probe be labeled with a TAMRA dye.

A QPCR method involves quantitating the amount of BKV nucleic acid in the nucleic acid sample. This is done by comparing the production rate of the BKV-specific amplicon product versus the production rate of a control DNA sequence or amplicon. A control DNA sequence or amplicon is a non-target nucleic acid sequence of known quantity that is used to calculate the amount of target BKV nucleic acid in the sample. The products of the QPCR process may be compared to control samples after a fixed number of PCR cycles to determine the relative virus load in the tissue or bodily fluid. One method of comparing the relative quantities of the products of the QPCR process is by gel electrophoresis, for instance, by running the samples on a gel and detecting those samples by one of a number of known methods including, without limitation, Southern blotting and subsequent detection with a labeled probe, staining with ethidium bromide and incorporating fluorescent or radioactive tags in the amplicons.

However, the progress of the PCR reaction typically is monitored by analyzing the relative rates of amplicon production for each PCR primer set in real time. Monitoring amplicon production may be achieved by a number of processes, including without limitation, fluorescent primers, fluorogenic probes and fluorescent dyes that bind double-stranded DNA. A common method is the fluorescent 5' nuclease assay. This method exploits the 5' nuclease activity of certain thermostable DNA polymerases (such as Taq or Tfl DNA polymerases) to cleave an oligomeric probe during the PCR process. The oligomer is selected to anneal to amplified sequences under elongation conditions. The probe typically has a fluorescent reporter on its 5' end and a fluorescent quencher of the reporter at the 3' end. A probe useful in this process is preferably the probe having the nucleic acid sequence

5'-TTACAGCACAGCAAGAATTCCCCTCCC-3' (SEQ ID NO: 4). In addition, the probe can be labeled so that binding of the probe to the amplicon may be detected. The label can be any sort of label known in the art. In one embodiment, the 5' end of the probe is labeled with a FAM dye and the 3' end of the probe is labeled with a TAMRA dye. So long as the oligomer is intact, the fluorescent signal from the reporter is quenched. However, when the oligomer is digested during the elongation process, the fluorescent reporter is no longer in proximity to the quencher. The relative accumulation of free fluorescent reporter for a given amplicon may be compared to the accumulation of the same amplicons for a control sample and/or to that of a control gene, such as .beta.-actin or 18S rRNA to determine the relative abundance of a given cDNA product of a given RNA in a RNA population. Products and reagents for the fluorescent 5' nuclease assay are readily available commercially, for instance from Applied Biosystems of Foster City, Calif.

Equipment and software also are readily available for monitoring amplicon accumulation in PCR and QPCR according to the fluorescent 5' nuclease assay and other QPCR procedures, including the ABI Prism 7700 Sequence Detection System (TaqMan), commercially available from Applied Biosystems.

The procedures described herein also may be used in multiplex QPCR processes. In its broadest sense, a multiplex PCR process involves production of two or more amplicons in the same reaction vessel. Multiplex amplicons may be analyzed by gel electrophoresis and detection of the amplicons by one of a variety of methods, such as, without limitation ethidium bromide staining, Southern blotting and hybridization to probes, or by incorporating fluorescent or radioactive moieties into the amplicons and subsequently viewing the product on a gel. However, real-time monitoring of the production of two or more amplicons is preferred. The fluorescent 5' nuclease assay is the most common monitoring method. Equipment is now available (for example, the above-described Amplified Biosystems product) that permits the real-time monitoring of accumulation of two or more fluorescent reporters in the same tube. For multiplex monitoring of the fluorescent 5' nuclease assay, oligomers are provided corresponding to each amplicon species to be detected. The oligomer probe for each amplicon species has a fluorescent reporter with a different peak emission wavelength than the oligomer probe(s) for each other amplicon species. The accumulation of each unquenched fluorescent reporter can be monitored to determine the relative amounts of the target sequence corresponding to each amplicon.

In traditional multiplex QPCR procedures, the selection of PCR primer sets having similar annealing and elongation kinetics and similar sized amplicons are desirable. The design and selection of appropriate PCR primer sets is a process that is well known to a person skilled in the art. The process for identifying optimal PCR primer sets, and respective ratios thereof to achieve a balanced multiplex reaction also is known. By "balanced," it is meant that certain amplicon(s) do not out-compete the other amplicon(s) for resources, such as dNTPs. For instance, by limiting the abundance of the PCR primers for the more abundant RNA species in an RT-PCR (reverse transcriptase polymerase chain reaction) experiment will allow the detection of less abundant species. Equalization of the Tm (melting temperature) for all PCR primer sets also is encouraged. See, for instance, ABI PRISM 7700 Sequence Detection System User Bulletin #5, "Multiplex PCR with TaqMan VIC Probes," Applied Biosystems (1998/2001), incorporated herein by reference. For example, a second primer set that could be used is a set of primers including SEQ ID NOS: 5 and 6 or a primer set that produces a polyoma T antigen-specific amplicon, as described above.

In the commercialization of the above-described methods for PCR, QPCR and multiplexed QPCR, certain kits for detection of BKV nucleic acids will be particularly useful. One example of such a kit would include reagents necessary for the one-tube multiplexed QPCR process described above. In one example, the kit would include the above-described reagents, including a corresponding PCR primer set, a thermostable DNA polymerase, such as Taq polymerase, and a suitable fluorescent reporter, such as, without limitation, a probe for a fluorescent 5' nuclease assay, a molecular beacon probe, a single dye primer or a fluorescent dye specific to double-stranded DNA, such as ethidium bromide. Thermostable DNA polymerases are commonly and commercially available from a variety of manufacturers. Additional materials in the kit may include: suitable reaction tubes or vials; a barrier composition, typically a wax bead, optionally including magnesium; reaction mixtures (typically 10x) for the PCR reaction, including, without limitation, necessary buffers and reagents, such as dNTP's; nuclease free water; control nucleic acid(s) and/or any additional buffers, compounds, co-factors, ionic constituents, proteins and enzymes, polymers, and the like that may be used in PCR or QPCR reactions.

The constituents of the kits may be packaged together or separately, and each constituent may be presented in, without limitation, one or more tubes or vials, or in cartridge form (a modular unit containing one or more reagents for use in an automated device), as is appropriate. Such tubes, vials or cartridges are referred to herein generically as "containers." The constituents, independently or together, may be packaged in a variety of states, including without limitation, in lyophilized, glassified, aqueous or other forms as is appropriate.

Also provided herein is a method for the management of BKVN in renal transplant recipients using cidofovir. The method includes quantitating the BKV viral load in a patient specimen and administering to a patient a dosage of cidofovir effective to reduce BKV viral loads in the patient. The first step of quantitating the BKV viral load in the patient specimen can be accomplished through the use of the above-described primers and methods for quantitating the BKV viral load in a specimen.

The second step is administering to the patient a dosage of cidofovir effective to reduce BKV viral loads in the patient. Cidofovir typically is administered in a dosage of at least about 5 mg/kg. In addition, cidofovir is usually administered in conjunction with an anti-diuretic, probenecid. However, in the present method, cidofovir is administered in a specific low dosage, typically without probenecid. A dosage effective to reduce BKV viral loads in a patient, as used in this application, means a dosage of from about 0.25 mg/kg to about 1.0 mg/kg, 2-3 times weekly. The dosage level typically is based upon a number of factors including, without limitation, the patient's BKV viral load, the responsiveness of the BKV viral load to previous doses of cidofovir and external indicators of kidney function, such as creatinine levels.

Treatment with cidofovir can improve a patient's graft prognosis because the dosage can be adjusted to ensure that the viral infection is controlled, without reducing immunosuppression and possibly causing rejection of the graft.

Claim 1 of 10 Claims

I claim:

1. A method of managing BKV-associated nephropathy in a renal transplant patient comprising administering to the renal transplant patient a dose of cidofovir effective to reduce BKV viral load in the patient.




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