Histone-mimetic delivery systems

The field of gene therapy has garnered significant interest over the past two decades as a method for revolutionizing the treatment of various diseases such as Alzheimer's, Parkinson's, and many types of cancer.  In recent years, non-viral methods of delivery have received particular attention due to safety concerns and production limitations associated with viral vectors.  However, inefficient DNA release is a common cause of ineffective non-viral DNA delivery.  A novel solution to this constraint is the development of biomimetic scaffolds capable of regulating DNA accessibility. 

The presented study involves the design of histone-mimetic gold nanoparticles (HMGNs) as gene therapy packaging materials.  Colloidal gold serves as a scaffold for the incorporation of histone H3 tail peptides trimethylated at lysine 4 (H3K4Me3).  H3K4Me3 has a high density of positively charged residues that provide a DNA condensation template and impart protection from nuclease degradation.  H3K4Me3 is known to be highly enhanced at the transcription start site for essentially all active genes.  In addition, recognition of this trimethylated K4-containing peptide sequence by nucleosome remodeling factors has been implicated in mechanisms for chromatin activation. 

The purpose of this work is to assemble and characterize these HMGNs as well as to investigate the influence of HMGN functionalization on DNA binding, protection, and release.  To this end, functionalized gold particles have been prepared.  Their size and morphology have been characterized by methods such as TEM and SANS.  In addition, the self-assembly of H3K4Me3 with plasmid DNA has also been investigated by dynamic light scattering, zeta-potential, nuclease assays, and cell transfection studies .  These selected studies are aimed at validating the HMGN approach to gene delivery and will provide the framework for further development of our system.

ECM fragment-targeted delivery systems

Collagen is a primary structural component of the human body and is found in all connective tissues. Due to its biocompatible and bioactive properties, collagen proves to be an attractive scaffold for mimicking the cellular microenvironment and has been used in both tissue regeneration and wound healing. Natural collagen is, however, difficult to physically modify with biologically relevant molecules without significantly altering its physical properties. In our work we have developed systems with collagen-mimetic peptides (CMPs) that are able to physically interact with natural collagen. These systems containing cell responsive domains along with our well-developed PEI/peptide polyplex system will allow for enhanced transfection within this three-dimensional microenvironment and provide a strategy for targeted and controlled gene delivery.

Surface-mediated delivery systems

Gene delivery enables mammalian cell manipulation necessary for disease treatment, tissue development, and comprehension of biochemical functions and cellular response. Currently, non-viral gene delivery is accomplished by bolus delivery of cationic polymer-complexed DNA, which is limited by mass transport and deactivation processes. For tissue engineering applications, incorporation of DNA onto biomaterial scaffolds may avoid these limitations and increase cell transfection by maintaining a high concentration of DNA in the cellular microenvironment. Surface immobilization of DNA also allows spatial targeting of cells. One method for DNA inclusion in a biomaterial consists of non-covalent surface immobilization. Presently, vector immobilization is dependent upon carrier-surface molecular interactions and requires careful design to support cellular uptake of the DNA. Covalent binding of the vector to a substrate via a labile peptide sequence would allow surface immobilization and greater control over cellular transfection. In this design, chemical functionalization of DNA is necessary for covalent binding. This is accomplished using a peptide nucleic acid (PNA) clamp, a DNA analog that sequence-specifically hybridizes with DNA to form a highly stable conjugate but does not interfere with the DNA transcriptional activity. The use of a maleimide-functionalized PNA clamp allows peptide coupling to the DNA-PNA hybrids to form chemically stable DNA-PNA-peptide conjugates. The use of coupling peptides that include cell adhesive and matrix metalloproteinase-1 (MMP-1) degradable sequences will allow for release and uptake of the complexes in a cell-responsive manner.  Conjugate attachment to model surfaces was accomplished using self-assembled monolayer (SAM) technology on gold substrates.  After further validation of the tethering system via surface analysis techniques as well as cellular transfection studies, this chemistry will be translated from 2D model scaffolds to 3D tissue engineering scaffolds, and will be employed to promote the correct cellular responses necessary for tissue engineering.

Nucleic acid nanoconjugates

The long-term goal of this project is to develop biomaterials for the efficient delivery of anti-tumor small interfering RNA (siRNA).  siRNAs are short double-stranded RNA molecules with highly potent gene silencing capacity.  Safe, synthetic siRNAs can now be designed against essentially any gene, and siRNA-based anti-cancer therapies have generated significant interest because of their exquisite specificity and potency.  The development of targeted, efficient, and safe methods for in vivo RNA delivery is widely considered the limiting factor towards its effective therapeutic implementation. Successful vehicles must function appropriately in both the extra- and intracellular environments to promote the selective delivery of functional RNA to the target cell’s cytoplasm.  These materials must be capable of protecting their RNA payloads from degradation and immune recognition; furthermore, they must direct these RNA molecules through the step-wise journey from the bloodstream, through the target tissue, and into the cell of interest.  A major challenge has been the development of materials that can accomplish these complex functional requirements while still remaining simple from the standpoint of formulation and clinical administration.  Thus, our specific goal is to create siRNA delivery conjugates containing protective and targeting features as well as components that enable their “bedside” formulation.  Ongoing work is focused on the synthesis and biological evaluation of these conjugates for efficient and safe siRNA delivery to the cytoplasm of tumor cells.

Peptide-functionalized polymer assemblies

The delivery of macromolecules and drugs to specific cellular and intracellular compartments is critically important for therapeutic efficacy.  Unfortunately, methods for drug delivery are often highly inefficient due to non-ideal serum-stability, transport across biological barriers, and release at the target site.  To address these problems, we are creating bio-responsive, polymeric nano-containers for the selective delivery of therapeutic compounds to pre-determined cellular locations.  We will combine the solution self-assembly of block copolymers with peptide linkages designed to permit vesicle evolution in response to critical environmental stimuli, eventually leading to the site-specific release of encapsulated payloads.  Ongoing work is focused on the synthesis and aqueous self-assembly of these novel copolymers, as well as the biological evaluation of the assembled vesicles for targeted drug delivery.