Unlocking Intracellular Therapeutic Targets through Novel Nanostructured Biomaterials

Nucleic acid cargoes offer unmatched diversity in gene regulatory potential and therapeutics, and understanding of nucleic acid functionality continues to expand rapidly and dramatically through seminal discoveries including RNA interference approaches and gene editing technologies. In nature, the basis for gene regulation is ultimately encoded by the exquisite specificity with which cells are able to control both the location and accessibility of nucleic acid constructs to govern their activation states. My research program seeks to understand and control gene activation using synthetic constructs through nature-inspired approaches to control and quantify cell binding interactions and stability in polymer and peptide nanocarriers. The basis of our approaches is the design of stimuli-responsive polymers and peptides whose interactions with nucleic acids and cells can be controlled dynamically by specific intracellular or external triggers. We exploit our ability to control nucleic acid binding/release and cellular processing to gain new mechanistic insights over nucleic acid delivery, leading to design advances including histone-inspired DNA targeting, light-responsive gene silencing, and collagen turnover-stimulated gene expression. This talk will highlight two ways we have used nature-inspired peptides to control gene transfer in regenerative medicine. Our approaches are exemplified by our work in histone-targeted nanocarrier design. Histones have received great interest as potential gene carriers for several decades due to their seminal role in chromatin packaging and gene transfer, yet therapeutic efforts with histones have lacked both a well-controlled materials approach and a deeper knowledge of cellular processing mechanisms. Hence, histone-based carriers have failed to reach clinical efficacy. We have capitalized on newly recognized and highly pivotal roles for histone tails in native gene regulatory control to develop a gene transfer method that utilizes native, histone-based processing pathways via incorporation of post-translationally modified (PTM) histone tails within controllably-assembled DNA vehicles (polyplexes). Our efforts proved that polyplexes displaying PTM-modified histone tails promote nuclear accumulation, DNA release, transcription, and enhanced transfection. Moreover, our group has combined detailed nanostructure engineering with sophisticated cellular imaging to identify novel aspects in the cell biology framework regulating polyplex transport to the nucleus. We have also focused on novel mechanisms to exploit nature’s ability to harness extracellular matrix (ECM) proteins such as collagens to sequester and control delivery of bioactive nanostructures. Our specific approaches have capitalized on a class of peptides known as collagen-mimetic peptides, or CMPs, that have been recognized for their unique affinity for native collagen, which can be tailored through alterations in CMP amino acid sequence and molecular weight. CMPs incorporate themselves into the natural collagen triple helical structure via strand invasion, in a reversible process previously that has been used to modify extracted collagen in vitro and exclusively target remodeling collagen in vivo. In our studies, we employed a proline-rich CMP designed to act not only as an adjustable tether to regulate collagen-polyplex affinity, but also as an adhesive/endocytic ligand for polyplexes. The use of a collagen scaffold afforded our system structural support and innate bioactivity to encourage cellular ingrowth and proliferation, whereas altering the extent of the modification of our vector provided additional tunability to allow tailorable release for prolonged time periods. This CMP-based approach also consistently and fully maintained polyplex activity in the presence of serum for at least a week, whereas most bolus and substrate-mediated gene delivery approaches report rapid reductions within hours or a few days, and the level of transgene expression directly correlated with MMP-concentrations and the extent of collagen remodeling, demonstrating “on demand” release. The ability to tailor release over extended periods via physical attachments, combined with the ability to provide cell-trigger release and collagen-mediated uptake, make this approach very attractive for many applications in regenerative medicine

Development of a collagen-based scaffold for sequential delivery of antimicrobial agents and pdgf genes to chronic wounds

Chronic wounds are a global health burden affecting more than 5 million people in the United States alone. The complex wound microenvironment causes variable therapeutic outcomes following treatment with commercially available products. Wound infection is one of the major barriers in healing of wounds and localized delivery of antimicrobials is necessary for treatment. Furthermore, growth factors play a vital role in orchestrating the wound healing process through enhancement of cell proliferation, migration, and extracellular matrix remodeling. Accordingly, we have developed a collagen-based scaffold modified with combination of vancomycin-loaded liposomes and platelet derived growth factor (PDGF)-loaded DNA polyplexes. Both the liposomes and polyplexes were anchored to collagen using collagen mimetic peptides (CMPs). Our aim was to use CMP tethering to control the sequential release of vancomycin and PDGF polyplexes to immediately suppress infection and subsequently transfect wound bed fibroblasts with PDGF to assist the wound healing process. Vancomycin-loaded liposomes were prepared using dipalmitoylphosphatidylcholine (DPPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG). The liposomes were 160.7±2.1 nm in diameter based on dynamic light scattering (DLS) analyses, and the loading capacity of vancomycin was 51.5±0.7% in the liposomes. PDGF polyplexes (115.2±1.2 nm in diameter) were prepared by self-assembly of polyethyleneimine and PDGF plasmid DNA (N/P = 8) in 20 mM HEPES buffer (pH = 6.0), and successful PDGF gene loading was confirmed by agarose gel electrophoresis. Co-gels were prepared with collagen (4 mg/mL), fibrinogen (1.25 mg/mL), and thrombin (0.156 IU/mL) combinations that could successfully encapsulate both the vancomycin-loaded liposomes and PDGF polyplexes. Drug release studies confirmed that ~80% of the vancomycin was released during the 48 h study period, whereas PDGF polyplexes were retained longer (\u3e 5 days) in the gel because their release requires collagen degradation mediated by matrix metalloproteinases present in the wound bed. The ability of the PDGF polyplexes to transfect fibroblasts was confirmed by in vitro cell transfection studies using green fluorescent protein (GFP) as a model gene. Furthermore, polyplex-mediated PDGF transfection was evaluated in fibroblasts cultured in an in vitro culture wound model, which showed that PDGF transfection enhanced migration rates of fibroblasts by ~2.4 fold as compared to controls in which culture wounds were allowed to heal in the absence of polyplexes. These results showcase the capacity for sequential delivery of vancomycin and PDGF gene in vitro, using collagen-based scaffolds, for potential applications in in vivo chronic wound treatments

Importin‑4 Regulates Gene Delivery by Enhancing Nuclear Retention and Chromatin Deposition by Polyplexes

For successful gene delivery, plasmid DNA must be able to access the nucleus in order to be transcribed. Numerous studies have shown that gene delivery occurs more readily in dividing cells, which is attributed to increased nuclear access when the nuclear envelope disassembles during mitosis; however, nonviral carriers continue to have low transfection efficiencies and require large quantities of DNA per cell to achieve reasonable gene transfer, even in dividing cells. Therefore, we hypothesized that using histone-derived nuclear localization sequences (NLS)­s to target polyplexes might enhance nuclear delivery by facilitating interactions with histone effectors that mediate nuclear partitioning and retention during mitosis. We discovered a novel interaction between polyplexes linked to histone 3 (H3) N-terminal tail peptides and the histone nuclear import protein importin-4, as evidenced by strong spatial colocalization as well as significantly decreased transfection when importin-4 expression was reduced. A fraction of the histone-targeted polyplexes was also found to colocalize with the retrotranslocon of the endoplasmic reticulum, Sec61. Super resolution microscopy demonstrated a high level of polyplex binding to chromatin postmitosis, and there also was a significant decrease in the amount of chromatin binding following importin-4 knockdown. These results provide evidence that natural histone effectors mediate both nuclear entry and deposition on chromatin by histone-targeted polyplexes, and a translocation event from the endoplasmic reticulum into the cytosol may occur before mitosis to enable the polyplexes to interact with these essential cytoplasmic proteins

Histone-targeted Polyplexes Avoid Endosomal Escape and Enter the Nucleus During Postmitotic Redistribution of ER Membranes

Nonviral gene delivery is a promising therapeutic approach because of its safety and controllability, yet limited gene transfer efficacy is a common issue. Most nonviral strategies rely upon endosomal escape designs; however, endosomal escape is often uncorrelated with improved gene transfer and membranolytic structures are typically cytotoxic. Previously, we showed that histone-targeted polyplexes trafficked to the nucleus through an alternative route involving caveolae and the Golgi and endoplasmic reticulum (ER), using pathways similar to several pathogens. We hypothesized that the efficacy of these polyplexes was due to an increased utilization of native vesicular trafficking as well as regulation by histone effectors. Accordingly, using confocal microscopy and cellular fractionation, we determined that a key effect of histone-targeting was to route polyplexes away from clathrin-mediated recycling pathways by harnessing endomembrane transfer routes regulated by histone methyltransferases. An unprecedented finding was that polyplexes accumulated in Rab6-labeled Golgi/ER vesicles and ultimately shuttled directly into the nucleus during ER-mediated nuclear envelope reassembly. Specifically, super resolution microscopy and fluorescence correlation spectroscopy unequivocally indicated that the polyplexes remained associated with ER vesicles/membranes until mitosis, when they were redistributed into the nucleus. These novel findings highlight alternative mechanisms to subvert endolysosomal trafficking and harness the ER to enhance gene transfer

Histone H3 Tail Peptides and Poly(ethylenimine) Have Synergistic Effects for Gene Delivery

This goal of this work was to explore histone H3 tail peptides containing transcriptionally activating modifications for their potential as gene delivery materials. We have found that these H3 tail peptides, in combination with the cationic polymer poly­(ethylenimine) (PEI), can effectively bind and protect plasmid DNA. The H3/PEI hybrid polyplexes were found to transfect a substantially larger number of CHO-K1 cells <i>in vitro</i> compared to both polyplexes that were formed with only the H3 peptides and those that were formed with only PEI at the same total charge ratio; however, transfection was similarly high for polyplexes both with and without transcriptionally activating modifications. Transfections with the endolysosomal inhibitors chloroquine and bafilomycin A1 indicated that the H3/PEI hybrid polyplexes exhibited slower uptake and a reduced dependence on endocytic pathways that trafficked to the lysosome, indicating a potentially enhanced reliance on caveolar uptake for efficient gene transfer. In addition, whereas PEI polyplexes typically exhibit a cytotoxic effect, the H3/PEI hybrid polyplexes did not compromise cell viability. In total, the current studies provide new evidence for the potential role for histone-based materials as effective gene transfer agents, and support for the importance of subcellular trafficking for nonviral gene delivery

Using the Epigenetic Code To Promote the Unpackaging and Transcriptional Activation of DNA Polyplexes for Gene Delivery

Nonviral gene delivery has seen limited clinical application due in part to the inefficiency with which most nonviral vehicles navigate the intracellular gene delivery pathway. One key problem is the inability of most DNA-packaging materials to release DNA and enable its efficient transcription. Thus, our aim was to develop gene delivery polyplexes capable of initiating their own transcription upon arrival in the nucleus. We created nuclease-resistant polyplexes with plasmid DNA (pDNA) and post-translationally modified histone 3 (H3K4Me3) tail peptides known to signal transcriptional activation on chromosomal DNA. When the H3K4Me3–pDNA polyplexes were directly microinjected into the nuclei of NIH/3T3 mouse fibroblasts, protein expression occurred earlier and in a greater fraction of cells than when polyethylenimine–pDNA polyplexes were microinjected. The rate of protein expression initiated by the H3K4Me3–pDNA polyplexes was also significantly accelerated in comparison with the rate initiated by non-trimethylated H3–pDNA polyplexes. These differences in protein expression rates were quantified by the development of a noncompartmentalized cellular kinetics model. These results highlight the importance of polyplex unpackaging as a gene delivery barrier, and demonstrate for the first time that the epigenetic code can be utilized in nonviral gene delivery

Histone H3 Tail Peptides and Poly(ethylenimine) Have Synergistic Effects for Gene Delivery

This goal of this work was to explore histone H3 tail peptides containing transcriptionally activating modifications for their potential as gene delivery materials. We have found that these H3 tail peptides, in combination with the cationic polymer poly­(ethylenimine) (PEI), can effectively bind and protect plasmid DNA. The H3/PEI hybrid polyplexes were found to transfect a substantially larger number of CHO-K1 cells <i>in vitro</i> compared to both polyplexes that were formed with only the H3 peptides and those that were formed with only PEI at the same total charge ratio; however, transfection was similarly high for polyplexes both with and without transcriptionally activating modifications. Transfections with the endolysosomal inhibitors chloroquine and bafilomycin A1 indicated that the H3/PEI hybrid polyplexes exhibited slower uptake and a reduced dependence on endocytic pathways that trafficked to the lysosome, indicating a potentially enhanced reliance on caveolar uptake for efficient gene transfer. In addition, whereas PEI polyplexes typically exhibit a cytotoxic effect, the H3/PEI hybrid polyplexes did not compromise cell viability. In total, the current studies provide new evidence for the potential role for histone-based materials as effective gene transfer agents, and support for the importance of subcellular trafficking for nonviral gene delivery