Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue

Abstract

Although cellular therapies represent a promising strategy for a number of conditions, current approaches face major translational hurdles, including limited cell sources and the need for cumbersome pre-processing steps (for example, isolation, induced pluripotency)1–6. In vivo cell reprogramming has the potential to enable more-effective cell-based therapies by using readily available cell sources (for example, fibroblasts) and circumventing the need for ex vivo pre-processing7,8. Existing reprogramming methodologies, however, are fraught with caveats, including a heavy reliance on viral transfection9,10. Moreover, capsid size constraints and/or the stochastic nature of status quo approaches (viral and non-viral) pose additional limitations, thus highlighting the need for safer and more deterministic in vivo reprogramming methods11,12. Here, we report a novel yet simple-to-implement non-viral approach to topically reprogram tissues through a nanochannelled device validated with well-established and newly developed reprogramming models of induced neurons and endothelium, respectively. We demonstrate the simplicity and utility of this approach by rescuing necrotizing tissues and whole limbs using two murine models of injury-induced ischaemia.

Nonviral Transfection With Brachyury Reprograms Human Intervertebral Disc Cells to a Pro‐Anabolic Anti‐Catabolic/Inflammatory Phenotype: A Proof of Concept Study

“…Live/Dead staining of human NP cells suggested that bulk electroporation of T or SHAM plasmids had no detrimental effects on NP cell viability up to 4 weeks in high density 3D agarose cultures for both Autopsy and Surgical specimens. The investigators have chosen this method of physical transfection due to (i) higher transfection efficiency and lower toxicity evidenced by studies comparing electroporation to chemical transfection such as lipofection in human embryonic stem cells (hESCs) and (ii) This proof‐of‐concept study serves as a basis for studying initial effects of T transfection for downstream applications using novel engineered vesicles or tissue nanotransfection . However, it is important to acknowledge that direct comparisons of chemical and physical methods for transfection of NP cells have not been previously compared and thus it is difficult to draw conclusions on which method is more efficient.…”

“…Similar to the effects of T on healthy phenotypic markers, these effects were more pronounced in AT cells versus ST, highlighting a need for further optimization of experimental conditions and delivery protocol while taking into account the pathological state of tissue. In addition previous studies by the authors have demonstrated that multiple transcription factors are often necessary to reprogram somatic cells therefore warranting the potential need for a cocktail of transcription factors for improved reprogramming …”

“…However, this does not exclude the possibility of other unwanted cellular effects and future cytotoxicity studies involving apoptotic and senescent markers would be required. Because of potential problems associated with bulk electroporation and the route toward clinical translation, we propose using novel nanotransfection or engineered vesicles as a means to deliver nonviral plasmids into tissue/animal models, which has shown great promise in cellular reprogramming . Second, but related to bulk transfection, we observed temporal effects in expression of phenotypic, inflammatory/neurotrophic and ECM related genes and thus plasmid quantity and transfection parameters require further refinement and would be likely improved by nano‐transfection.…”

“…In addition previous studies by the authors have demonstrated that multiple transcription factors are often necessary to reprogram somatic cells therefore warranting the potential need for a cocktail of transcription factors for improved reprogramming. 27…”

“…However, the use of viral vectors for gene delivery poses concerns of insertional mutagenesis and unwarranted immune responses . Nonviral plasmid‐based vector systems eliminate much of the risk associated with viral vectors and have been used successfully in conjunction with novel tissue‐nanotransfection for rescue of mice skin tissue under ischemic conditions . Therefore, transfection of a transcription factor that promotes a “healthy NP phenotype” via a nonviral vehicle is a promising therapeutic approach for treatment of IVD degeneration.…”