Multiplex Assay for Simultaneous Detection of Mycoplasma genitalium and Macrolide Resistance Using PlexZyme and PlexPrime Technology.

Mycoplasma genitalium is a cause of non-gonoccocal urethritis (NGU) in men and cervicitis and pelvic inflammatory disease in women. Recent international data also indicated that the first line treatment, 1 gram stat azithromycin therapy, for M. genitalium is becoming less effective, with the corresponding emergence of macrolide resistant strains. Increasing failure rates of azithromycin for M. genitalium has significant implications for the presumptive treatment of NGU and international clinical treatment guidelines. Assays able to predict macrolide resistance along with detection of M. genitalium will be useful to enable appropriate selection of antimicrobials to which the organism is susceptible and facilitate high levels of rapid cure. One such assay recently developed is the MG 23S assay, which employs novel PlexZyme™ and PlexPrime™ technology. It is a multiplex assay for detection of M. genitalium and 5 mutations associated with macrolide resistance. The assay was evaluated in 400 samples from 254 (186 males and 68 females) consecutively infected participants, undergoing tests of cure. Using the MG 23S assay, 83% (331/440) of samples were positive, with 56% of positives carrying a macrolide resistance mutation. Comparison of the MG 23S assay to a reference qPCR method for M. genitalium detection and high resolution melt analysis (HRMA) and sequencing for detection of macrolide resistance mutations, resulted in a sensitivity and specificity for M. genitalium detection and for macrolide resistance of 99.1/98.5% and 97.4/100%, respectively. The MG 23S assay provides a considerable advantage in clinical settings through combined diagnosis and detection of macrolide resistance.

EzyAmp signal amplification cascade enables isothermal detection of nucleic acid and protein targets.

Advancements in molecular biology have improved the ability to characterize disease-related nucleic acids and proteins. Recently, there has been an increasing desire for tests that can be performed outside of centralised laboratories. This study describes a novel isothermal signal amplification cascade called EzyAmp (enzymatic signal amplification) that is being developed for detection of targets at the point of care. EzyAmp exploits the ability of some restriction endonucleases to cleave substrates containing nicks within their recognition sites. EzyAmp uses two oligonucleotide duplexes (partial complexes 1 and 2) which are initially cleavage-resistant as they lack a complete recognition site. The recognition site of partial complex 1 can be completed by hybridization of a triggering oligonucleotide (Driver Fragment 1) that is generated by a target-specific initiation event. Binding of Driver Fragment 1 generates a completed complex 1, which upon cleavage, releases Driver Fragment 2. In turn, binding of Driver Fragment 2 to partial complex 2 creates completed complex 2 which when cleaved releases additional Driver Fragment 1. Each cleavage event separates fluorophore quencher pairs resulting in an increase in fluorescence. At this stage a cascade of signal production becomes independent of further target-specific initiation events. This study demonstrated that the EzyAmp cascade can facilitate detection and quantification of nucleic acid targets with sensitivity down to aM concentration. Further, the same cascade detected VEGF protein with a sensitivity of 20nM showing that this universal method for amplifying signal may be linked to the detection of different types of analytes in an isothermal format.

DNAzyme switches for molecular computation and signal amplification.

We have created molecular switches that consist of nucleic-acid cleaving DNAzymes which are temporarily inactivated by hybridization with blocking oligonucleotides. The unique design of the switches offers significant advantages over existing methods. Firstly, the switches are activated by a nucleic acid-cleaving enzyme which can be made to function only in the presence of a specific target analyte. This allows for their use as reporter elements which can be easily adapted for use in computational logical operations. Secondly, the activation of each switch produces an active nucleic acid-cleaving DNAzyme as an output and this allows the switches to be modularly coupled to one another so that the output of one switch functions as the input of another. In addition, the switches are scalable, so that a single input target can produce more than one active DNAzyme output. These features therefore create the means for amplification of signal, which confers significant potential for future biosensing applications where detection of low quantities of target biomarkers is required.

MNAzymes provide a universal mechanism for triggering DNAzyme synthesis cascades.

Novel methods were developed to isothermally detect target nucleic acids and initiate signal amplification cascades. The methods utilize target-specific MNAzymes to activate universal primer molecules. These primers can subsequently promote the autonomous synthesis of DNAzymes capable of cleaving nucleic acid substrates and generating signal, which further creates the potential for circular feedback.

DNA-only cascade: a universal tool for signal amplification, enhancing the detection of target analytes.

Diagnostic tests performed in the field or at the site of patient care would benefit from using a combination of inexpensive, stable chemical reagents and simple instrumentation. Here, we have developed a universal "DNA-only Cascade" (DoC) to quantitatively detect target analytes with increased speed. The DoC utilizes quasi-circular structures consisting of temporarily inactivated deoxyribozymes (DNAzymes). The catalytic activity of the DNAzymes is restored in a universal manner in response to a broad range of environmental and biological targets. The present study demonstrates DNAzyme activation in the presence of metal ions (Pb(2+)), small molecules (deoxyadenosine triphosphate) and nucleic acids homologous to genes from Meningitis-causing bacteria. Furthermore, DoC efficiently discriminates nucleic acid targets differing by a single nucleotide. When detection of analytes is orchestrated by functional nucleic acids, the inclusion of DoC reagents substantially decreases time for detection and allows analyte quantification. The detection of nucleic acids using DoC was further characterized for its capability to be multiplexed and retain its functionality following long-term exposure to ambient temperatures and in a background of complex medium (human serum).

MNAzyme qPCR: a superior tool for multiplex qPCR.

Multicomponent nucleic acid enzymes (MNAzymes) are nucleic acid enzymes composed of multiple oligonucleotide partzymes that only associate to form catalytic complexes in the presence of a target nucleic acid. Once assembled, MNAzymes cleave a separate substrate (probe) between fluorophore and quencher labels to produce a fluorescent signal indicative of the presence of the target. MNAzymes are particularly useful as tools for monitoring the accumulation of amplicons during real-time quantitative PCR (qPCR). The partzyme pairs have sensor domains that are complementary to adjacent regions in the amplicons such that their partial catalytic core domains form a complete active MNAzyme core. The probe-binding domain of the partzymes can be complementary to any one of a series of well-characterized universal probes. Since there is no need to synthesize and optimize new target-specific probes for each new target, MNAzyme qPCR provides a flexible alternative which allows target-specific interrogation with a generic readout. A series of universal probes have been designed that perform with high reliability, yielding consistent and reproducible results for any target, making the development of multiplex qPCR assays faster, cheaper, and simpler. This chapter describes a 5plex MNAzyme RT-qPCR method which simultaneously quantifies five mRNA transcripts with high efficiency and specificity using five unique universal probes, each labeled with a different fluorophore.

MNAzyme qPCR with superior multiplexing capacity.

BACKGROUND: MNAzymes (nucleic acid enzymes formed from multiple partial enzymes) can be linked to PCR to provide a highly specific method for target detection and quantification. We investigated the feasibility of multiplexing MNAzyme quantitative PCR (qPCR) methods. METHODS: We combined MNAzyme components with PCR primers and standard qPCR reagents to perform MNAzyme qPCR and reverse-transcription qPCR (RT-qPCR) assays with a set of universal reporter probes. Assays were performed on single targets and in multiplex formats that combined up to 5 different targets in a single reaction. RESULTS: A comparison of 3 targets amplified in single and triplex formats showed no significant differences with respect to detection limit or amplification efficiency. Likewise, we successfully converted single-target assays for 11 transcripts of interest to triplex assays containing 2 reference transcripts without having to optimize or modify the conditions. A quintuplex RT-qPCR that simultaneously quantified 5 transcripts with 5 universal probes produced high amplification efficiencies and r(2) values for all transcripts. Despite the large numbers of oligonucleotides in the reactions, we observed no false-positive signals, owing to the requirement of 4 target-specific binding events to produce a signal. A quadruplex assay that combined MNAzymes with methylation-specific PCR to measure epigenetic biomarkers of prostate cancer was capable of detecting a single methylated DNA allele in a background of 1000-10 000 unmethylated alleles. The MNAzyme qPCR was compatible with a rapid-cycling protocol. CONCLUSIONS: MNAzymes offer a flexible and unique approach to qPCR that is specific, sensitive, and easily multiplexed. The universal nature of MNAzyme reporter probes removes the need for target-specific probes, thereby making the development of new assays easier and cheaper.

MNAzymes, a versatile new class of nucleic acid enzymes that can function as biosensors and molecular switches.

To increase the versatility and utility of nucleic acid enzymes, we developed multicomponent complexes, known as MNAzymes, which produce amplified "output" signals in response to specific "input" signals. Multiple oligonucleotide partzymes assemble into active MNAzymes only in the presence of an input assembly facilitator such as a target nucleic acid. Once formed, MNAzymes catalytically modify a generic substrate, generating an amplified output signal that heralds the presence of the target while leaving the target intact. We demonstrated several applications including sensitive, isothermal target detection; discrimination of polymorphisms; and highly specific monitoring of real-time polymerase chain reaction (PCR). Furthermore, we showed their capacity to function as molecular switches and to work in series to create a molecular cascade. The modular nature of MNAzymes, together with the separation of input and output functionalities, provides potential for their integration into diverse devices such as diagnostic biosensors, molecular computers, and/or nanoscale machines.

Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34+ cells.

Gene transfer has potential as a once-only treatment that reduces viral load, preserves the immune system and avoids lifetime highly active antiretroviral therapy. This study, which is to our knowledge the first randomized, double-blind, placebo-controlled, phase 2 cell-delivered gene transfer clinical trial, was conducted in 74 HIV-1-infected adults who received a tat-vpr-specific anti-HIV ribozyme (OZ1) or placebo delivered in autologous CD34+ hematopoietic progenitor cells. There were no OZ1-related adverse events. There was no statistically significant difference in viral load between the OZ1 and placebo group at the primary end point (average at weeks 47 and 48), but time-weighted areas under the curve from weeks 40-48 and 40-100 were significantly lower in the OZ1 group. Throughout the 100 weeks, CD4+ lymphocyte counts were higher in the OZ1 group. This study indicates that cell-delivered gene transfer is safe and biologically active in individuals with HIV and can be developed as a conventional therapeutic product.

Diagnosis and monitoring of PML-RARalpha-positive acute promyelocytic leukemia by quantitative RT-PCR.

The last 15 yr have produced dramatic improvements in the survival rate of patients with acute promyelocytic leukemia (APL). These improvements have been due mainly to the introduction of targeted therapies and improved methods for diagnosing and monitoring this disease. The underlying molecular lesion in APL involves a t(15:17) translocation which leads to the generation of PML-RARalpha fusion transcripts and proteins. The PML-RARalpha fusion transcripts have been shown to be useful markers for establishing the diagnosis and for monitoring the response to treatment. This manuscript describes the application of QZyme reverse-transcription polymerase chain reaction (RT-PCR) to the quantification of PML-RARalpha transcripts as a marker of APL. QZyme is a method for real time detection and quantification of target genes or transcripts. The principle of QZyme analysis is similar to other quantitative PCR systems; however, the mechanism is quite different. QZyme exploits the catalytic activity of DNAzymes (deoxyribozymes), which are oligonucleotides that can bind and cleave nucleic acid substrates. The approach is well suited to monitoring minimal residual disease (MRD) in patients with APL, as a result of its ability to detect low numbers of transcripts and accurately measure differences in concentration over a broad dynamic range. Further, its capacity for duplex analysis has multiple advantages for analysis of clinical specimens. Protocols for duplex, single-tube QZyme RT-PCR assays, which allow simultaneous quantification of PML-RARalpha fusion transcripts (either L-type and V-type, or S-type) and the internal control BCR transcript, are provided. These protocols can be used for analyzing patient RNA specimens and are suitable for clinical trial monitoring. For this type of work, it is recommended that investigators validate the assays to ensure reproducible, accurate, and specific results on the equipment in their own laboratories. Assay validation is critical for real-time quantitative RT-PCR (RQ-PCR) and is often overlooked. A guide to the steps involved in validation and recommendations for acceptance criteria is included in this chapter.

Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients.

BACKGROUND: An anti-HIV-1 tat ribozyme, termed Rz2, has been shown to inhibit HIV-1 infection/replication and to decrease HIV-1-induced pathogenicity in T-lymphocyte cell lines and normal peripheral blood T-lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2. METHODS: Apheresis was used to obtain a peripheral blood cell population from each of four HIV-negative donors. After enrichment for CD4+ T-lymphocytes, ex vivo expansion and genetic manipulation (approximately equal aliquots of the cells were transduced with the ribozyme-containing (RRz2) and the control (LNL6) retroviral vector), these cells were infused into the corresponding HIV-1-positive twin recipient. Marking was assessed over an initial 24-week period and in total over an approximate 4-year period. RESULTS: The gene transfer procedure was shown to be safe, and technically feasible. Both RRz2- and LNL6-gene-containing peripheral blood mononuclear cells (PBMC) were detected at all time points examined to 4 years. There was concomitant gene construct expression in the absence of the need for ex vivo peripheral blood cell stimulation and there was no evidence of immune elimination of the neoR T-lymphocytes nor of silencing of the Moloney murine leukemia virus long terminal repeat. CONCLUSIONS: The proof of principle results reported here demonstrate safety and feasibility of this type of gene transfer approach. While not specifically tested, T-lymphocytes containing an anti-HIV gene construct may impact on HIV-1 viral load and CD4+ T-lymphocyte count, potentially representing a new therapeutic modality for HIV-1 infection.

Critical steps in the implementation of hematopoietic progenitor-cell gene therapy using ribozyme vectors: a laboratory protocol.

The implementation of a hematopoietic progenitor-cell gene-therapy program involves the performance of laboratory procedures and compliance with the current code of Good Manufacturing Practices. This chapter explains the multiple laboratory steps used in our recent Phase I gene transfer study for HIV. This study employed a retroviral vector to deliver an anti-HIV ribozyme to CD34+ hematopoietic progenitor cells.

Anti-human immunodeficiency virus hematopoietic progenitor cell-delivered ribozyme in a phase I study: myeloid and lymphoid reconstitution in human immunodeficiency virus type-1-infected patients.

A phase I gene transfer clinical study was undertaken to examine the ability to introduce a potential anti-human immunodeficiency virus (HIV) gene therapeutic into hematopoietic progenitor cells (HPC), thereby contributing to multilineage engraftment. The potential therapeutic effect of genetically modifying HPC with protective genes in HIV-infected adults depends in part on the presence of adult thymic activity and myeloid capacity in the setting of HIV replication. Herein we report the presence and expression of a retroviral vector encoding an anti-HIV-1 ribozyme in mature hematopoietic cells of different lineages, and de novo T-lymphocyte development ensuing from genetically engineered CD34(+) HPC. Sustained output of vector-containing mature myeloid and T-lymphoid cells was detected even in patients with multidrug-resistant infection. In addition, the study showed that the degree of persistence of gene-containing cells was dependent on transduced HPC dose. These novel findings support the concept of gene therapy as a modality to effect immune reconstitution with cells engineered to inhibit HIV replication and this report represents the first demonstration of long-term maintenance of a potential therapeutic transgene in HIV disease.

Inactivation of p16INK4a by CpG hypermethylation is not a frequent event in colorectal cancer.

BACKGROUND AND OBJECTIVES: Gene promoter hypermethylation is common in colorectal cancer and is associated with transcriptional silencing. However, the clinicopathological significance of p16(INK4a) gene silencing with hypermethylation is unknown. Therefore, the aim of this study was to analyze loss of p16 expression and its relationship to hypermethylation in sporadic colorectal cancer. METHODS: Tissue from 426 colorectal cancers underwent histological analysis. Immunohistochemistry was performed for p16 expression. Fresh tumor DNA was analyzed for microsatellite instability (MSI) and the presence of K-ras mutations. In addition, DNA was bisulphite-modified and analyzed for p16(INK4a) promoter methylation by methylation-specific PCR. RESULTS: There were 25% of tumors with p16(INK4a) promoter hypermethylation. These tumors were associated with older patients, right-sidedness, MSI and were poorly differentiated, mucinous, and had intraepithelial and peritumoral lymphocytes and a Crohn's-type lymphocytic reaction (P < 0.05). However, only right-sidedness was significant on multivariate analysis (P < 0.001). Only 8.1% of tumors did not express p16, and this was associated with hypermethylation (P < 0.05). CONCLUSION: p16(INK4a) promoter methylation, although common in colorectal cancer, does not result in a clinicopathologically distinct subgroup of tumors and infrequently results in transcriptional silencing. This suggests that p16(INK4a) gene inactivation does not have an important role in the pathogenesis of sporadic colorectal cancer.

Diagnosis and molecular monitoring of acute promyelocytic leukemia using DzyNA reverse transcription-PCR to quantify PML/RARalpha fusion transcripts.

BACKGROUND: PML/RARalpha fusion transcripts provide a readily accessible marker for diagnosis of acute promyelocytic leukemia (APL) and for monitoring response to therapy. Survival rates are improved by therapies guided by such monitoring. We assessed the potential of DzyNA reverse transcription-PCR (RT-PCR) for measurement of PML/RARalpha fusion transcripts. METHODS: Parallel single-tube DzyNA RT-PCR protocols were developed to allow real-time fluorescent quantification of PML/RARalpha fusion transcripts and a low abundance control transcript, normal BCR. Calibration curves, generated using cell line RNA, allowed estimation of these transcripts in RNA from patients with APL at various stages of the disease. RESULTS: DzyNA RT-PCR calibration curves were linear for both transcripts over a broad range and demonstrated interassay variations of 12% (mean, 658 ng) and 10% (mean, 263 ng), respectively. The protocols detected low concentrations of transcripts and resolved twofold dilutions. PML/RARalpha mRNA was quantified in 10 patients at diagnosis and in 1 patient over a 7-year period. Monitoring of transcript concentrations effectively reflected the disease course in one patient and demonstrated that an increase in PML/RARalpha transcripts can be detected 4-6 months before hematologic relapse, with no false-positive results. CONCLUSION: DzyNA RT-PCR has potential for use in clinical practice as a tool for diagnosis of APL and forsubsequent monitoring of minimal residual disease and detection of molecular relapse.

The role of hMLH1 methylation in the development of synchronous sporadic colorectal carcinomas.

PURPOSE: AB. B. subset of sporadic colorectal carcinomas show microsatellite instability, usually as a result of biallelic hMLH1 gene promoter methylation. Synchronous tumors occur in up to 5 percent of patients with colorectal cancer, but their cause is poorly understood. We hypothesized that in the setting of sporadic microsatellite instability cancers, synchronicity may reflect a global predisposition of colorectal epithelium toward tumor development because of gene hypermethylation. METHODS: We identified 14 individuals with 33 synchronous cancers from a series of 362 patients with 381 sporadic colorectal cancers. We then analyzed the synchronous lesions for microsatellite status, hMLH1 protein expression, and hMLH1 promoter methylation. RESULTS: Seven of 33 synchronous tumors (21 percent) showed microsatellite instability, compared with 36 of 348 solitary tumors (10.3 percent, P = 0.06). The 14 patients with synchronous tumors were significantly older than those with solitary tumors (mean age 79.4 vs. 68.2 years, P = 0.01), and 5 of these patients had at least one microsatellite instability tumor. However, only one patient harbored synchronous tumors that were all of the microsatellite instability type. Methylation of the hMLH1 promoter was seen in 9 synchronous cancers from 27 assessable lesions in 7 patients and was associated with microsatellite instability (P = 0.01), right-sidedness (P = 0.01), and loss of expression of hMLH1 (P = 0.03). Only one case showed methylation in all synchronous tumors, whereas in five cases synchronous tumors showed different methylation status within the one individual. CONCLUSION: Our data suggest that synchronous tumors arise as independent events and that the slightly greater frequency of synchronous tumors in individuals with microsatellite instability cancers is likely to be a chance event reflecting the older age of these individuals rather than arising from a predisposition toward cancer as a result of global hypermethylation of colorectal epithelium.