Utilization of Collagen Remodeling Pathways to Achieve Efficient, Controlled Gene Delivery in Chronic Wound Repair

摘要

Chronic, non-healing wounds represent a growing burden to patients, medical professionals, and the healthcare system. Over 7 million Americans suffer from these wounds and the total patient care costs $25 billion per year. Even after treatment, a large percent (~50%) of chronic wounds never completely heal, leaving patients susceptible to elevated rates of infection, amputation, and even death, demonstrating a grave need for new approaches to restore wound bed health and address causative deficiencies in growth factor(GF) stability accessibility, and activity. The application of GF gene activated matrices offer many compelling advantages over GF protein delivery within the aberrant, protease-rich wound bed. GF gene-approaches better mimic endogenous repair by utilizing host cells to orchestrate sustained GF expression, activity, and microlocalization which are crucial in management of chronic wounds due to extended healing over months, spatiotemporal heterogeneity, and elevated protease activation. However, clinical translation of gene-based therapies has been largely hindered by off-target responses, and inefficient gene transfer.;In this dissertation, a novel peptide-based approach for achieving efficient, controlled gene delivery through leveraging naturally elevated wound bed protease activity and subsequent collagen remodeling is presented. Specifically, collagen-mimetic peptides (CMPs)-collagen affinity was used to engineer DNA collagen matrices with tailored release profiles and improved activity. Variation in CMP-display on non-viral DNA carriers known as polyplex were demonstrated to increase retention/release time from collagen from 20 days to over a month, and in cell studies, bound polyplex exhibited enhanced stability in the presence of serum-containing media over a 2-week period as well as altered intracellular trafficking resulting in improved gene transfer efficiency. Moreover, transgene expression in CMP/polyplex/collagens was determined to be directly dependent on matrix metalloprotease(MMP)-stimulation, and fluorescent microscopy studies established the co-endocytosis and co-internalization of CMP-modified polyplex with collagen fragments, strongly suggesting CMP-modification can be used to harness collagen remodeling pathways to mediate controlled release and efficient cellular uptake, two major obstacles in gene delivery.;Furthermore, CMP/polyplex/collagens have been used to successfully tailor the expression of vital GFs platelet derived growth factor-BB (PDGF-BB) and keratinocyte growth factor (KGF), and decrease wound closure times through PDGF-BB expression when applied to an in vitro "wound" model. Experiments with an in vivo ECM depot model also demonstrated the capacity of CMP-modification to tailor the duration and extent of transgene expression in a more complex system, mediating expression for 10-fold longer time periods with average expression levels up to 2-orders of magnitude higher relative to samples with unmodified polyplexes. While the focus of this dissertation is application in chronic wound repair, this approach has enormous potential in improving delivery in other locations characterized by elevated collagen turnover, including tumor and joints. Its targeting of released ECM fragments to facilitate delivery also marks a significant deviation from typical gene delivery approaches which has already shown promise.