Targeted Delivery of Recombinant Heat Shock Protein 27 to Cardiomyocytes Promotes Recovery from Myocardial Infarction.

Ischemic heart disease, especially myocardial infarction (MI), is the leading cause of death worldwide. Apoptotic mechanisms are thought to play a significant role in cardiomyocyte death after MI. Increased production of heat shock proteins (Hsps) in cardiomyocytes is a normal response to promote tolerance and to reduce cell damage. Hsp27 is considered to be a therapeutic option for the treatment of ischemic heart disease due to its protective effects on hypoxia-induced apoptosis. Despite its antiapoptotic effects, the lack of strategies to deliver Hsp27 to the heart tissue in vivo limits its clinical applicability. In this study, we utilized an antibody against the angiotensin II type 1 (AT1) receptor, which is expressed immediately after ischemia/reperfusion in the heart of MI rats. To achieve cardiomyocyte-targeted Hsp27 delivery after ischemia/reperfusion, we employed the immunoglobulin-binding dimer ZZ, a modified domain of protein A, in conjunction with the AT1 receptor antibody. Using the AT1 receptor antibody, we achieved systemic delivery of ZZ-TAT-GFP fusion protein into the heart of MI rats. This approach enabled selective delivery of Hsp27 to cardiomyocytes, rescued cells from apoptosis, reduced the area of fibrosis, and improved cardiac function in the rat MI model, thus suggesting its applicability as a cardiomyocyte-targeted protein delivery system to inhibit apoptosis induced by ischemic injury.

Attachment of cell-binding ligands to arginine-rich cell-penetrating peptides enables cytosolic translocation of complexed siRNA.

Cell-penetrating peptides (CPPs), such as nona-arginine (9R), poorly translocate siRNA into cells. Our studies demonstrate that attaching 9R to ligands that bind cell surface receptors quantitatively increases siRNA uptake and importantly, allows functional delivery of complexed siRNA. The mechanism involved accumulation of ligand-9R:siRNA microparticles on the cell membrane, which induced transient membrane inversion at the site of ligand-9R binding and rapid siRNA translocation into the cytoplasm. siRNA release also occurred late after endocytosis when the ligand was attached to the L isoform of 9R, but not the protease-resistant 9DR, prolonging mRNA knockdown. This critically depended on endosomal proteolytic activity, implying that partial CPP degradation is required for endosome-to-cytosol translocation. The data demonstrate that ligand attachment renders simple polycationic CPPs effective for siRNA delivery by restoring their intrinsic property of translocation.

T cell-specific siRNA delivery using antibody-conjugated chitosan nanoparticles.

The intracellular delivery of small interfering RNA (siRNA) plays a key role in RNA interference (RNAi) and provides an emerging technique to treat various diseases, including infectious diseases. Chitosan has frequently been used in gene delivery applications, including siRNA delivery. However, studies regarding the modification of chitosan with antibodies specifically targeting T cells are lacking. We hypothesized that chitosan nanoparticles modified with T cell-specific antibodies would be useful for delivering siRNA to T cells. CD7-specific single-chain antibody (scFvCD7) was chemically conjugated to chitosan by carbodiimide chemistry, and nanoparticles were prepared by a complex coacervation method in the presence of siRNA. The mean diameter and zeta potential of the scFvCD7-chitosan/siRNA nanoparticles were approximately 320 nm and +17 mV, respectively, and were not significantly influenced by the coupling of antibody to chitosan. The cellular association of antibody-conjugated nanoparticles to CD4+ T cell lines as well as gene silencing efficiency in the cells was significantly improved compared to nonmodified chitosan nanoparticles. This approach to introducing T cell-specific antibody to chitosan nanoparticles may find useful applications for the treatment of various infectious diseases.

Delivering antiviral siRNA into human T-cells: New approaches in RNAi-based HIV therapy.

The ability to block the expression of any disease-causing gene or disease-related protein highlights the potential use of RNAi technology in the therapy of 'undruggable' human diseases. However, considering the risks associated with RNAi therapy, targeting and restricting the action of siRNA to specific cells could greatly minimize toxic side effects. However, this is a major challenge, as many primary cell types are highly recalcitrant to siRNA uptake. This review discusses advances in siRNA targeting methods for human T-cells, with an emphasis on the potential use of an RNAi-based therapy for the treatment of HIV/AIDS.

Preparation and characterization of chitosan/polyguluronate nanoparticles for siRNA delivery.

Small interfering RNA (siRNA) has been widely investigated as a potential therapeutic for treatment of various diseases. However, the use of siRNA is limited due to its rapid degradation and low intracellular association in vitro and in vivo. Chitosan nanoparticles encapsulating siRNA were prepared using a coacervation method in the presence of polyguluronate (PG), which is isolated from alginate and is strongly related to ionic interactions of negatively charged alginate. Various physicochemical properties of chitosan/PG nanoparticles, including size, surface charge, morphology, and interaction with siRNA, were characterized. The mean diameter of siRNA-loaded chitosan-based nanoparticles ranged from 110 to 430 nm, depending on the weight ratio between chitosan and siRNA. Nanoparticles showed low cytotoxicity and were useful in delivering siRNA to HEK 293FT and HeLa cells. Chitosan/PG nanoparticles were considered promising for siRNA delivery due to their low cytotoxicity and ability to transport siRNA into cells, which can effectively inhibit induction of targeting mRNA.

The heat shock protein 27 (Hsp27) operates predominantly by blocking the mitochondrial-independent/extrinsic pathway of cellular apoptosis.

Heat shock protein 27 (Hsp27) is a molecular chaperone protein which regulates cell apoptosis by interacting directly with the caspase activation components in the apoptotic pathways. With the assistance of the Tat protein transduction domain we directly delivered the Hsp27 into the myocardial cell line, H9c2 and demonstrate that this protein can reverse hypoxia-induced apoptosis of cells. In order to characterize the contribution of Hsp27 in blocking the two major apoptotic pathways operational within cells, we exposed H9c2 cells to staurosporine and cobalt chloride, agents that induce mitochondria-dependent (intrinsic) and -independent (extrinsic) pathways of apoptosis in cells respectively. The Tat-Hsp27 fusion protein showed a greater propensity to inhibit the effect induced by the cobalt chloride treatment. These data suggest that the Hsp27 predominantly exerts its protective effect by interfering with the components of the extrinsic pathway of apoptosis.

T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice.

Evaluation of the therapeutic potential of RNAi for HIV infection has been hampered by the challenges of siRNA delivery and lack of suitable animal models. Using a delivery method for T cells, we show that siRNA treatment can dramatically suppress HIV infection. A CD7-specific single-chain antibody was conjugated to oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2rgamma-/- mice reconstituted with human lymphocytes (Hu-PBL) or CD34+ hematopoietic stem cells (Hu-HSC). In HIV-infected Hu-PBL mice, treatment with anti-CCR5 (viral coreceptor) and antiviral siRNAs complexed to scFvCD7-9R controlled viral replication and prevented the disease-associated CD4 T cell loss. This treatment also suppressed endogenous virus and restored CD4 T cell counts in mice reconstituted with HIV+ peripheral blood mononuclear cells. Moreover, scFvCD7-9R could deliver antiviral siRNAs to naive T cells in Hu-HSC mice and effectively suppress viremia in infected mice. Thus, siRNA therapy for HIV infection appears to be feasible in a preclinical animal model.