Abstract

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.

Highlights

  • Biological therapeutic products derived from living organisms are emerging as a viable method to prevent and treat a variety of human diseases

  • A variant CJD in humans is caused by the consumption of beef from cattle infected with bovine spongiform encephalopathy (BSE) prions[12]

  • We focused on prions that have been shown to be infectious to humans, i.e. prions associated with variant CJD (vCJD) and BSE

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Summary

Introduction

Biological therapeutic products derived from living organisms are emerging as a viable method to prevent and treat a variety of human diseases. Earlier cases of transmission including human immunodeficiency virus (HIV) and hepatitis C virus (HCV) through blood transfusions have guided the implementation of procedures to reduce these risks[7]. These include medical history reviews of donors and sample testing when possible. Prion diseases are characterized by a long incubation period during which PrPSc accumulates in the brain and peripheral tissues while not producing symptoms[15] During this period, it is possible for an individual to make a blood or tissue donation ignorant to his or her own infection. A similar level of sensitivity was obtained in urine samples[21]

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