Safety Assessment of Advanced Therapies for the Treatment of Human Diseases

In the 21st century, a new class of treatments called “Advanced Therapy Medicinal Products” (ATMPs) emerged as promising option for a wide range of diseases with an unmet medical need. According to the European Medicines Agency (EMA) and the legislative framework of the European Union, ATMPs include: 1) gene therapy medicinal products, 2) somatic-cell therapy medicinal products and 3) tissue-engineered products. Gene therapy products contain recombinant nucleic acids that are designed to either alter or introduce a gene into a patient. Cell therapy and tissue-engineered products both describe cell-based treatments where patient or donor-derived cells have either undergone substantial manipulation to alter intrinsic properties, e.g. by the activation with growth factors, or the biological function in the recipient differs from the donor. If the indication of such a cell-based product is the treatment, prophylaxis or diagnosis of a disease, it is categorized as cell therapy product. In contrast, the regeneration, repair or replacement of tissues are indications that lead to a classification as tissue-engineered product.
With ATMPs emerging as new class of treatments, questions about their safety for patients arise. This thesis focuses on the preclinical safety testing of ATMPs, in particular on tumorigenicity and carcinogenicity risk assessment. By definition, tumorigenicity is the ability of autologous or allogenic inoculated cells to give rise to tumors in the host, while carcinogenicity is the ability of a substance to cause cancer. This thesis evaluates three different ATMPs and is divided accordingly into three parts, each containing an independent manuscript. The first manuscript includes the establishment and validation of two in vitro transformation assays, SACF (soft agar colony forming assay) and GILA (growth in low attachment assay), to determine the tumorigenic potential of genome edited-cells, which belong to the first category of ATMPs. In particular, cells that were genetically modified with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) technology were tested. In the second manuscript, it is demonstrated how these assays could be adapted to other nucleic acid-based applications, namely, to the carcinogenicity testing of an adeno associated viruses (AAV)-based vaccine. Strictly speaking, vaccines against infectious diseases do not belong to the category of ATMPs. However, since the safety concerns regarding potential AAV carcinogenicity remain the same irrespective of their use, the AAV-based vaccine was analyzed as example for AAV-based products. Lastly, the third part of this thesis addresses the tumorigenicity evaluation of induced pluripotent stem cell (iPSC)-derived products, which could belong to the second and third group of ATMPs depending on the indication. Due to their pluripotency and self-renewable capacities, iPSCs could form teratomas when injected into patients, which could pose a safety risk. Here, marker genes for the detection of iPSC contaminations in iPSC-derived products were identified by meta-analysis of ribonucleic acid (RNA)-sequencing (RNA-seq) data and confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation.