No abstract

Current treatments for chronic pain rely largely on opioids despite their substantial side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing. In particular, a hereditary loss-of-function mutation in NaV1.7, a sodium channel protein associated with signaling in nociceptive sensory afferents, leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence and structural similarity between NaV subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 in primary afferents via epigenome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)–dCas9 and zinc finger proteins at the spinal level as a potential treatment for chronic pain. Toward this end, we first optimized the efficiency of NaV1.7 repression in vitro in Neuro2A cells and then, by the lumbar intrathecal route, delivered both epigenome engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain, and BzATP-induced pain. Our results show effective repression of NaV1.7 in lumbar dorsal root ganglia, reduced thermal hyperalgesia in the inflammatory state, decreased tactile allodynia in the neuropathic state, and no changes in normal motor function in mice. We anticipate that this long-lasting analgesia via targeted in vivo epigenetic repression of NaV1.7 methodology we dub pain LATER, might have therapeutic potential in management of persistent pain states.

Recent advances in tissue engineering and 3D bioprinting have enabled construction of cell-laden scaffolds containing perfusable vascular networks. Although these methods partially address the nutrient-diffusion limitations present in engineered tissues, they are still restricted in both their viable vascular geometries and matrix material compatibility. To address this, tissue constructs are engineered via encapsulation of 3D printed, evacuable, free standing scaffolds of poly(vinyl alcohol) (PVA) in biologically derived matrices. The ease of printability and water-soluble nature of PVA grant compatibility with biologically relevant matrix materials and allow for easily repeatable generation of complex vascular patterns. This study confirms the ability of this approach to produce perfusable vascularized matrices capable of sustaining both cocultures of multiple cell types and excised tumor fragments ex vivo over multiple weeks. The study further demonstrates the ability of the approach to produce hybrid patterns allowing for coculture of vasculature and epithelial cell-lined lumens in close proximity, thereby enabling ex vivo recapitulation of gut-like systems. Taken together, the methodology is versatile, broadly applicable, and importantly, simple to use, enabling ready applicability in many research settings. It is believed that this technique has the potential to significantly accelerate progress in engineering and study of ex vivo organotypic tissue constructs.

Pluripotent stem cell-derived organoids have transformed our ability to recreate complex three-dimensional models of human tissue. However, the directed differentiation methods used to create them do not afford the ability to introduce cross-germ-layer cell types. Here, we present a bottom-up engineering approach to building vascularized human tissue by combining genetic reprogramming with chemically directed organoid differentiation. As a proof of concept, we created neuro-vascular and myo-vascular organoids via transcription factor overexpression in vascular organoids. We comprehensively characterized neuro-vascular organoids in terms of marker gene expression and composition, and demonstrated that the organoids maintain neural and vascular function for at least 45 days in culture. Finally, we demonstrated chronic electrical stimulation of myo-vascular organoid aggregates as a potential path toward engineering mature and large-scale vascularized skeletal muscle tissue from organoids. Our approach offers a roadmap to build diverse vascularized tissues of any type derived entirely from pluripotent stem cells.