ABSTRACT
The application of induced pluripotent stem cells (iPSCs) in advanced therapies is increasing at pace, but concerns remain over their clinical safety profile. We report the first-ever application of doggybone DNA (dbDNA) vectors to generate human iPSCs. dbDNA vectors are closed-capped linear double-stranded DNA gene expression cassettes that contain no bacterial DNA and are amplified by a chemically defined, current good manufacturing practice (cGMP)-compliant methodology. We achieved comparable iPSC reprogramming efficiencies using transiently expressing dbDNA vectors with the same iPSC reprogramming coding sequences as the state-of-the-art OriP/EBNA1 episomal vectors but, crucially, in the absence of p53 shRNA repression. Moreover, persistent expression of EBNA1 from bacterially derived episomes resulted in stimulation of the interferon response, elevated DNA damage, and increased spontaneous differentiation. These cellular activities were diminished or absent in dbDNA-iPSCs, resulting in lines with a greater stability and safety potential for cell therapy.
ABSTRACT
The Neuronal Ceroid Lipofuscinoses (NCL), otherwise known as Batten disease, are a group of neurodegenerative diseases caused by mutations in 13 known genes. All except one NCL is autosomal recessive in inheritance, with similar aetiology and characterised by the accumulation of autofluorescent storage material in the lysosomes of cells. Age of onset and the rate of progression vary between the NCLs. They are collectively one of the most common lysosomal storage diseases, but the enigma remains of how genetically distinct diseases result in such remarkably similar pathogenesis. Much has been learnt from cellular studies about the function of the proteins encoded by the affected genes. Such research has utilised primitive unicellular models such as yeast and amoeba containing gene orthologues, cells derived from naturally occurring (sheep) and genetically engineered (mouse) animal models or patient-derived cells. Most recently, patient-derived induced pluripotent stem cell (iPSC) lines have been differentiated into neural cell-types to study molecular pathogenesis in the cells most profoundly affected by disease. Here, we review how cell models have informed much of the biochemical understanding of the NCLs and how more complex models are being used to further this understanding and potentially act as platforms for therapeutic efficacy studies in the future.
