Project Summary/Abstract Chronic low back pain is a debilitating disorder of significant socio-economic importance and a major gateway to opioid use. Therefore, minimally invasive non-addictive treatments that aim to address pain by targeting the underlying disease pathology are critical to improve human health and limit the growing opioid crisis. Intervertebral disc (IVD) degeneration is strongly associated with the pathophysiology of chronic low back pain; specifically extracellular matrix (ECM) breakdown, inflammation, and aberrant nerve/vascular ingrowth, all of which are significantly correlated with “Discogenic back pain” (DBP). Therefore, the overall objective of this proposal is to develop novel non-viral reprogramming-based therapies to convert degenerate nucleus pulposus (NP) and annulus fibrosus (AF) IVD cells associated with DBP, into a healthy pro-anabolic, anti-nerve/vascular phenotype, using engineered extracellular vesicles (eEVs). Our central hypothesis is that non-viral reprogramming will restore IVD structure/mechanical function, and limit nerve/vascular ingrowth associated with pain, by converting the patient’s own degenerate IVD cells into a pro-anabolic phenotype in situ. To date, clinicians do not have access to the necessary biological tools to treat IVD degeneration in patients with DBP. A critical barrier to the success of current biologic strategies involves significant logistical and regulatory challenges such as a lack of sustained drug delivery systems, poor long-term cell viability or viral reprogramming that permanently integrates with the host DNA. Our non-addictive strategy focuses on addressing these limitations. Through our recent R61 award and published work, we have shown that human NP cells can be reprogrammed towards a healthier phenotype using developmental transcription factors Brachyury or FOXF1, showing increased ECM accumulation and decreased catabolic/ inflammatory/ neurotrophic factors - all key features of a healthy IVD. Furthermore, in our mouse DBP in vivo model we have also demonstrated significant improvement in NP tissue hydration and dampened pain behaviors in animals treated with eEVs loaded with FOXF1 for up-to 12 weeks, highlighting the potential of our proposed strategy to restore structure/function of the IVD while reducing pain. However, critical gaps remain such as i) understanding the synergistic reprogramming potential of multiple developmental transcription factors in NP and AF cells, ii) how sex and age influence our strategy, and iii) evaluating therapeutic efficiency using more clinically relevant in vivo animal models that simulate the human condition and functionalized eEVs to deliver transcription factors to specific cell types within the IVD (for example, NP or AF cells). Our first aim investigates the synergistic effects of eEVs loaded with multiple transcription factors using in vitro and in vivo clinically relevant models of DBP, and quantifying efficacy of our strategy v...