ECM turnover-stimulated gene delivery through collagen-mimetic peptide-plasmid integration in collagen

Abstract

Gene therapies have great potential in regenerative medicine; however, clinical translation has been inhibited by low stability and limited transfection efficiencies. Herein, we incorporate collagen-mimetic peptide (CMP)-linked polyplexes in collagen scaffolds to increase DNA stability by up to 400% and enable tailorable in vivo transgene expression at 100-fold higher levels and 10-fold longer time periods. These improvements were directly linked to a sustained interaction between collagen and polyplexes that persisted during cellular remodeling, polyplex uptake, and intracellular trafficking. Specifically, incorporation of CMPs into polyethylenimine (PEI) polyplexes preserved serum-exposed polyplex-collagen activity over a period of 14 days, with 4 orders-of-magnitude more intact DNA present in CMP-modified polyplex-collagen relative to unmodified polyplex-collagen after a 10 day incubation under cell culture conditions. CMP-modification also altered endocytic uptake, as indicated by gene silencing studies showing a nearly 50% decrease in transgene expression in response to caveolin-1 silencing in modified samples versus only 30% in unmodified samples. Furthermore, cellular internalization studies demonstrated that polyplex-collagen association persisted within cells in CMP polyplexes, but not in unmodified polyplexes, suggesting that CMP linkage to collagen regulates intracellular transport. Moreover, experiments in an in vivo repair model showed that CMP modification enabled tailoring of transgene expression from 4 to 25 days over a range of concentrations. Overall, these findings demonstrate that CMP decoration provides substantial improvements in gene retention, altered release kinetics, improved serum-stability, and improved gene activity in vivo. This versatile technique has great potential for multiple applications in regenerative medicine.

Integration of growth factor gene delivery with collagen‐triggered wound repair cascades using collagen‐mimetic peptides

“…In particular, we demonstrated previously that GPP‐modification of PEI polyplexes greatly enhanced control over both the extent and duration of transgene expression through utilization of the reversible, serum‐stable affinity between CMPs and natural collagen. Enhanced control and activity were demonstrated both in vitro and in vivo, using a murine subdermal repair model . Additional studies concluded that these improvements were the result of superior serum‐stability and endocytic trafficking driven by increases in polyplex affinity for collagen and the natural process of collagen remodeling.…”

“…Through varying CMP display, DNA release/retention was tailored for over a month, two times longer than the retention/release periods of unmodified polyplexes. CMP‐modification also maintained polyplex activity in serum‐supplemented media for up to 2 weeks, in contrast with most gene delivery approaches which report losses to nuclease degradation within hours . Additionally, we demonstrated the novel ability to “hijack” collagen remodeling, a process that occurs in excess in the protease‐rich chronic wound environment .…”

“…CMPmodification also maintained polyplex activity in serum-supplemented media for up to 2 weeks, in contrast with most gene delivery approaches which report losses to nuclease degradation within hours. 34,56 Additionally, we demonstrated the novel ability to "hijack" collagen remodeling, 56 a process that occurs in excess in the proteaserich chronic wound environment. 3,8,9 Whereas previous studies have utilized proteolytically-sensitive materials to synchronize cell invasion with therapeutic release, the reversible, serum-stable nature of the CMP-collagen interaction allowed for continued association with collagen fragments, confirmed through colocalization microscopy studies.…”

Gene delivery by peptide-assisted transport

“…In vitro wound closure with PDGF polyplexes was improved by 2-fold and 2.5-fold with 20% CMP-PEI polyplexes and mixed polyplexes (20% CMP-PEI and 50% CMP-PEI mixtures), respectively, as compared to non-CMP-modified polyplexes [47]. On-demand polyplex release and gene expression delivery was also were demonstrated in vivo , wherein gene expression could be tailored to peak at 6-20 days depending on the levels of CMP modification [48]. …”

Rapid prototyping of proteins: Mail order gene fragments to assayable proteins within 24 hours

“…Custom proteins are finding increased utility in advanced material fields such as gene delivery (Urello, Kiick, & Sullivan, ), biomaterials (Hu, Cebe, Weiss, Omenetto, & Kaplan, ; Yang, Holmberg, & Olsen, ), enzyme design (Siegel et al, ), active peptide design (Ikonomova, Moghaddam‐Taaheri, Jabra‐Rizk, Wang, & Karlsson, ), and biosensors (Kallmyer, Musielewicz, Sutter, & Reuel, ; Pardee et al, ). In each case, the ability to quickly screen the phenotypic behavior of each candidate in a large library of proteins is critical to down‐select to a smaller pool for expression optimization and purification.…”