Protein Misfolding Cyclic Amplification Technology Research Articles

Screening of Anti-Prion Compounds Using the Protein Misfolding Cyclic Amplification Technology.

Prion diseases are 100% fatal infectious neurodegenerative diseases affecting the brains of humans and other mammals. The disease is caused by the formation and replication of prions, composed exclusively of the misfolded prion protein (PrPSc). We invented and developed the protein misfolding cyclic amplification (PMCA) technology for in vitro prion replication, which allow us to replicate the infectious agent and it is commonly used for ultra-sensitive prion detection in biological fluids, tissues and environmental samples. In this article, we studied whether PMCA can be used to screen for chemical compounds that block prion replication. A small set of compounds previously shown to have anti-prion activity in various systems, mostly using cells infected with murine prions, was evaluated for their ability to prevent the replication of prions. Studies were conducted simultaneously with prions derived from 4 species, including human, cattle, cervid and mouse. Our results show that only one of these compounds (methylene blue) was able to completely inhibit prion replication in all species. Estimation of the IC50 for methylene blue inhibition of human prions causing variant Creutzfeldt-Jakob disease (vCJD) was 7.7 μM. Finally, we showed that PMCA can be used for structure-activity relationship studies of anti-prion compounds. Interestingly, some of the less efficient prion inhibitors altered the replication of prions in some species and not others, suggesting that PMCA is useful for studying the differential selectivity of potential drugs.

Application of PMCA to screen for prion infection in a human cell line used to produce biological therapeutics

Advances in biotechnology have led to the development of a number of biological therapies for the treatment of diverse human diseases. Since these products may contain or are made using human or animal (e.g. cattle) derived materials, it is crucial to test their safety by ensuring the absence of infectious agents; specifically prions, which are highly resilient to elimination and produce fatal diseases in humans. Many cases of iatrogenic Creutzfeldt-Jakob disease have been caused by the use of biological materials (e.g. human growth hormone) contaminated with prions. For this reason, it is important to screen cells and biological materials for the presence of prions. Here we show the utility of the Protein Misfolding Cyclic Amplification (PMCA) technology as a screening tool for the presence of human (vCJD) and bovine (BSE) prions in a human cell therapy product candidate. First, we demonstrated the sensitivity of PMCA to detect a single cell infected with prions. For these experiments, we used RKM7 cells chronically infected with murine RML prions. Serial dilutions of an infected cell culture showed that PMCA enabled prion amplification from a sample comprised of only one cell. Next, we determined that PMCA performance was robust and uncompromised by the spiking of large quantities of uninfected cells into the reaction. Finally, to demonstrate the practical application of this technology, we analyzed a human cell line being developed for therapeutic use and found it to be PMCA-negative for vCJD and BSE prions. Our findings demonstrate that the PMCA technology has unparalleled sensitivity and specificity for the detection of prions, making it an ideal quality control procedure in the production of biological therapeutics.

Efficient prion disease transmission through common environmental materials

Prion diseases are a group of fatal neurodegenerative diseases associated with a protein-based infectious agent, termed prion. Compelling evidence suggests that natural transmission of prion diseases is mediated by environmental contamination with infectious prions. We hypothesized that several natural and man-made materials, commonly found in the environments of wild and captive animals, can bind prions and may act as vectors for disease transmission. To test our hypothesis, we exposed surfaces composed of various common environmental materials (i.e. wood, rocks, plastic, glass, cement, stainless steel, aluminum, and brass) to hamster-adapted 263K scrapie prions and studied their attachment and retention of infectivity in vitro and in vivo Our results indicated that these surfaces, with the sole exception of brass, efficiently bind, retain, and release prions. Prion replication was studied in vitro using the protein misfolding cyclic amplification technology, and infectivity of surface-bound prions was analyzed by intracerebrally challenging hamsters with contaminated implants. Our results revealed that virtually all prion-contaminated materials transmitted the disease at high rates. To investigate a more natural form of exposure to environmental contamination, we simply housed animals with large contaminated spheres made of the different materials under study. Strikingly, most of the hamsters developed classical clinical signs of prion disease and typical disease-associated brain changes. Our findings suggest that prion contamination of surfaces commonly present in the environment can be a source of disease transmission, thus expanding our understanding of the mechanisms for prion spreading in nature.

A novel method for preclinical detection of PrPSc in blood

In this study, we demonstrate that a moderate amount of protein misfolding cyclic amplification (PMCA) coupled to a novel surround optical fibre immunoassay (SOFIA) detection scheme can be used to detect the disease-associated form of the prion protein (PrP(Sc)) in protease-untreated plasma from preclinical and clinical scrapie sheep, and white-tailed deer with chronic wasting disease, following natural and experimental infection. PrP(Sc), resulting from a conformational change of the normal (cellular) form of prion protein (PrP(C)), is considered central to neuropathogenesis and serves as the only reliable molecular marker for prion disease diagnosis. While the highest levels of PrP(Sc) are present in the central nervous system, the development of a reasonable diagnostic assay requires the use of body fluids that characteristically contain exceedingly low levels of PrP(Sc). PrP(Sc) has been detected in the blood of sick animals by means of PMCA technology. However, repeated cycling over several days, which is necessary for PMCA of blood material, has been reported to result in decreased specificity (false positives). To generate an assay for PrP(Sc) in blood that is both highly sensitive and specific, we have utilized limited serial PMCA (sPMCA) with SOFIA. We did not find any enhancement of sPMCA with the addition of polyadenylic acid nor was it necessary to match the genotypes of the PrP(C) and PrP(Sc) sources for efficient amplification.

Ultra-efficient Replication of Infectious Prions by Automated Protein Misfolding Cyclic Amplification

Prions are the unconventional infectious agents responsible for transmissible spongiform encephalopathies, which appear to be composed mainly or exclusively of the misfolded prion protein (PrPSc). Prion replication involves the conversion of the normal prion protein (PrPC) into the misfolded isoform, catalyzed by tiny quantities of PrPSc present in the infectious material. We have recently developed the protein misfolding cyclic amplification (PMCA) technology to sustain the autocatalytic replication of infectious prions in vitro. Here we show that PMCA enables the specific and reproducible amplification of exceptionally minute quantities of PrPSc. Indeed, after seven rounds of PMCA, we were able to generate large amounts of PrPSc starting from a 1x10(-12) dilution of scrapie hamster brain, which contains the equivalent of approximately 26 molecules of protein monomers. According to recent data, this quantity is similar to the minimum number of molecules present in a single particle of infectious PrPSc, indicating that PMCA may enable detection of as little as one oligomeric PrPSc infectious particle. Interestingly, the in vitro generated PrPSc was infectious when injected in wild-type hamsters, producing a disease identical to the one generated by inoculation of the brain infectious material. The unprecedented amplification efficiency of PMCA leads to a several billion-fold increase of sensitivity for PrPSc detection as compared with standard tests used to screen prion-infected cattle and at least 4000 times more sensitivity than the animal bioassay. Therefore, PMCA offers great promise for the development of highly sensitive, specific, and early diagnosis of transmissible spongiform encephalopathy and to further understand the molecular basis of prion propagation.