Alternative approaches to LV are non viral vectors

Alternative approaches to LV are non-viral vectors such as RNA or DNA, which are considered safer due to their integration-free nature (Hardee et al., 2017). However, manufacturing RNA is expensive and difficult because RNA has a propensity to degrade (Yin et al., 2014). On the other hand, DNA plasmids are relatively economical and stable but are inefficient in terms of cellular uptake and intracellular transport into the host (Nehlsen et al., 2006). A miniaturized plasmid lacking bacterial DNA sequences, known as a minicircle (MC) shows several benefits over these vectors, including: i) much smaller size of DNA for higher diffusion rate and increased biological activity both in vitro and in vivo (Chabot et al., 2013; Chen et al., 2003); ii) lack of bacterial-related genes and CpG motifs (Ismail et al., 2012; Mayrhofer and Iro, 2012); and iii) reduced risk of possible transgene integration into host genome. It has been previously shown that a MC encoding the pluripotent transcription factors Oct4, Nanog, Lin28 and Sox2, enables the reprogramming of human adipose JNJ26481585 cost into iPS cells (Jia et al., 2010; Narsinh et al., 2010). However, the application of MC for lineage-specific differentiation from PS cells has not yet been reported. In the present study, we assess the feasibility of delivering PAX7 through MC to generate scalable and engraftable PAX7+ myogenic progenitors from PS cells, equivalent to those generated using LV.
Material and methods
Results
Discussion Although numerous protocols are available for the in vitro differentiation of hPS cells into myogenic cells, the method that generates progenitors with functional force-generating potential, suitable for translation into a potential cell replacement therapy, is the one involving conditional lentiviral expression of PAX7 or PAX3 (Darabi et al., 2012; Darabi et al., 2008; Darabi et al., 2011; Filareto et al., 2012; Filareto et al., 2013). The LV system allows for the generation of large quantities of PAX7+ myogenic progenitors that upon transplantation, contribute to significant muscle regeneration in vivo. Here we tested an alternative non-viral delivery approach to induce PAX7 expression, and generate PS cell-derived myogenic progenitors. The in vitro results were comparable between MC- (after 3 transductions) and LV-generated myogenic progenitors. However, because the transgene expression mechanism of MC is non-integrative and PAX7+ myogenic progenitors rapidly divide when they are induced with PAX7 expression, we identified several disadvantages for the use of this approach for the generation of PS cell-derived myogenic progenitors: i) requirement of multiple transfections to sustain high levels of PAX7 expression, necessary to enable expansion and maintenance of PAX7+ myogenic progenitor identity, which is labor intensive and costly; ii) multiple transfections introduce toxicity, reducing the rate of cell growth even when the cell death inhibitor Y-27632 was used; and iii) inability to expand and maintain a homogeneous population of PAX7+ myogenic progenitors after FACS purification. On the other hand, the LV system only requires a single transduction, which results in stable and efficient transgene expression (>90%). This enables the expansion of a homogeneous population of PAX7+ myogenic progenitors with minimal cell death upon Dox induction. However, efficient in vitro differentiation does not necessarily predict efficient in vivo function (Kim et al., 2017). The major caveat observed for the MC approach relates to the very limited in vivo regenerative potential of generated PAX7+ myogenic progenitors. This is likely due to the dilution of the MC, resulting in diminished PAX7 expression and subsequent rapid differentiation towards myoblast. It is shown here and previously that the transplantation of heterogeneous population of cells greatly reduces the efficacy of myogenic contribution in vivo (Kim et al., 2017). It might be possible to transplant a homogeneous population of MC-induced PAX7+ myogenic progenitors by directly sorting GFP+ (PAX7+) progenitors. However, FACS is highly stressful and transplanted cells are prone to apoptosis, limiting likelihood of successful engraftment. In the LV approach, in contrast, a homogeneous population of iPAX7 myogenic progenitors can be harvested with minimal stress in a significantly shorter time frame, and these have more efficient muscle regeneration contribution.