Characterization of proteins, mRNAs, and miRNAs of circulating extracellular vesicles from prostate cancer patients compared to healthy subjects.

Prostate cancer (PC) is the fifth leading cause of death in men globally. Measurement of the blood PSA level is still considered the gold-standard biomarker test for PC despite its high rate of delivering false positives and negatives that result in an inappropriate medical response, including overtreatment. We collected extracellular vesicles (EVs) from the blood plasma of PC patients with organ-confined, extracapsular-invading, and seminal vesicle-invading tumors and from healthy subjects. We examined the protein, mRNA, and miRNA content of these EVs using mass spectrometry (MS), a human PC PCR array, and a miScript miRNA PCR array, respectively. The proteomic analysis showed distinct groups of proteins that are differently expressed in each group of patients, as well as in healthy subjects. Samples from healthy subjects and each tumor type were used for both mRNA and miRNA arrays. The mRNA analysis showed distinct groups of mRNAs that were overexpressed in healthy or in one of the three tumor types but not in the EVs of the other groups. The miRNA analysis showed distinct groups of miRNAs as well. The fold of regulation in the expression of the identified mRNA and miRNA of each stage of the disease from healthy subjects showed that various mRNAs and miRNAs could discriminate the disease stage. Overall, our data suggest many molecular marker candidates for distinguishing between healthy subjects and PC patients using the cargo of circulating vesicles, as well as markers to discriminate between the different tumor types. Once verified, these markers might have a diagnostic value for PC.

Current state of Health Canada regulation for cellular and gene therapy products: potential cures on the horizon.

We provide an overview of the regulatory framework, pathways and underlying regulatory authority for cell, gene and tissue-engineered therapies in Canada. Canada's regulatory approach uses three sets of regulations, namely, the Cells, Tissues and Organs Regulations, the Food and Drug Regulations and the Medical Devices Regulations. We provide an overview of each these sets of regulations as they apply to clinical investigation to post-market product lifecycle stages. Information is provided on the current sources of relevant Health Canada guidance documents. We highlight several regional success stories including Prochymal, a cell therapy product that achieved Canadian regulatory approval using the conditional marketing approval system. We also examine the perceived gaps in the Canadian regulations and how those gaps are being addressed by interactions between the government, stakeholders and international bodies. We conclude that the risk-benefit approach used by Health Canada for regulatory approval processes is sufficiently flexible to enable to development of novel cell and gene therapy products in Canada, yet stringent enough to protect patient safety.

Bone Marrow Mesenchymal Stromal Cell Treatment in Patients with Osteoarthritis Results in Overall Improvement in Pain and Symptoms and Reduces Synovial Inflammation.

Patients with late-stage Kellgren-Lawrence knee osteoarthritis received a single intra-articular injection of 1, 10, or 50 million bone marrow mesenchymal stromal cells (BM-MSCs) in a phase I/IIa trial to assess safety and efficacy using a broad toolset of analytical methods. Besides safety, outcomes included patient-reported outcome measures (PROMs): Knee Injury and Osteoarthritis Outcome Score (KOOS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC); contrast-enhanced magnetic resonance imaging (MRI) for cartilage morphology (Whole Organ MRI Scores [WORMS]), collagen content (T2 scores), and synovitis; and inflammation and cartilage turnover biomarkers, all over 12 months. BM-MSCs were characterized by a panel of anti-inflammatory markers to predict clinical efficacy. There were no serious adverse events, although four patients had minor, transient adverse events. There were significant overall improvements in KOOS pain, symptoms, quality of life, and WOMAC stiffness relative to baseline; the 50 million dose achieved clinically relevant improvements across most PROMs. WORMS and T2 scores did not change relative to baseline. However, cartilage catabolic biomarkers and MRI synovitis were significantly lower at higher doses. Pro-inflammatory monocytes/macrophages and interleukin 12 levels decreased in the synovial fluid after MSC injection. The panel of BM-MSC anti-inflammatory markers was strongly predictive of PROMs over 12 months. Autologous BM-MSCs are safe and result in significant improvements in PROMs at 12 months. Our analytical tools provide important insights into BM-MSC dosing and BM-MSC reduction of synovial inflammation and cartilage degradation and provide a highly predictive donor selection criterion that will be critical in translating MSC therapy for osteoarthritis. Stem Cells Translational Medicine 2019;8:746&757.

Strategy for an abbreviated in-house qualification of a commercially available Rapid Microbiology Method (RMM) for canadian regulatory approval.

BACKGROUND AIMS: Cell therapy products (CTP) typically require full sterility, endotoxin and Mycoplasma testing before product release. Often this is not feasible with fresh cells, and sponsors may rely on rapid microbiological methods (RMM). RMM must be qualified in-house using the sponsor's facilities, equipment, consumables, cells and matrices to meet regulatory approval. Herein, we present a cost-effective strategy to conduct an in-house abbreviated qualification of a commercially available RMM kit to meet Health Canada regulatory requirements. METHODS: We performed an abbreviated qualification using a polymerase chain reaction (PCR)-based Mycoplasma testing method involving assay sensitivity and ruggedness, based on an experimental plan that was pre-approved by Health Canada. Briefly, investigational CTPs were tested in-house using a PCR-based Mycoplasma detection kit. Assay sensitivity was determined using a 10-fold dilution series of genomic DNA of only two Mycoplasma species, Mycoplasma arginini and Mycoplasma hominis in the absence of CTP-matrix as the kit had been previously validated against nine species. Matrix interference was measured by testing independent CTP samples. Testing by different operators on different days measured ruggedness. RESULTS: The RMM Mycoplasma qualification exceeded sensitivity (4 genome copies per reaction for M. arginini and 0.12 genome copies per reaction for M. hominis) and met ruggedness requirements without matrix interference, as required by the Pharmacopoeial guidelines (Ph. Eur. 2.6.7 and USP <1223>). DISCUSSION: Our approach represents a minimal qualification that can be performed by an academic institution while ensuring regulatory compliance for implementing RMM testing for in-process and product-release testing of CTPs.

Workshop to address gaps in regulation of minimally manipulated autologous cell therapies for homologous use in Canada.

In Canada, minimally manipulated autologous cell therapies for homologous use (MMAC-H) are either regulated under the practice of medicine, or as drugs or devices under the Food and Drugs Act, Food and Drug Regulations (F&DR) or Medical Device Regulations (MDR). Cells, Tissues and Organs (CTO) Regulations in Canada are restricted to minimally manipulated allogeneic products for homologous use. This leaves an important gap in the interpretation of existing regulations. The purposes of this workshop co-organized by the Stem Cell Network and the Centre for Commercialization of Regenerative Medicine (CCRM) were to discuss the current state of regulation of MMAC-H therapies in Canada and compare it with other regulatory jurisdictions, with the intent of providing specific policy recommendations to Health Canada. Participants came to a consensus on the need for well-defined common terminology between regulators and stakeholders, a common source of confusion and misinformation. A need for a harmonized national approach to oversight of facilities providing MMAC-H therapies based on existing standards, such as Canadian Standards Association (CSA), was also voiced. Facilities providing MMAC-H therapies should also participate in collection of long-term data to ensure patient safety and efficacy of therapies. Harmonization across provinces of the procedures and practices involving administration of MMAC-H would be preferred. Participants felt that devices used to process MMAC-H are adequately regulated under existing MDR. Overly prescriptive regulation will stifle innovation, whereas insufficient regulation might allow unsafe or ineffective therapies to be offered. Until a clear, balanced and explicit approach is articulated, regulatory uncertainty remains a barrier.