ABSTRACT
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.
Subject(s)
HIV Infections/genetics , HIV Infections/therapy , RNA Interference , RNA, Small Interfering/metabolism , T-Lymphocytes/metabolism , Animals , Antigens, CD7/metabolism , Disease Models, Animal , Gene Expression , HIV-1/genetics , HIV-1/metabolism , Humans , Immunoglobulin Fragments/metabolism , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/metabolism , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/virology , Mice , Mice, Inbred NOD , Mice, SCID , RNA, Viral/metabolismABSTRACT
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.
Subject(s)
Antibodies, Immobilized/chemistry , Chitosan/chemistry , Nanoparticles/chemistry , RNA, Small Interfering/administration & dosage , T-Lymphocytes/metabolism , Antibodies, Immobilized/immunology , Antigens, CD7/immunology , Drug Delivery Systems , Humans , Jurkat Cells , Models, Molecular , RNA Interference , RNA, Small Interfering/genetics , T-Lymphocytes/immunologyABSTRACT
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.
Subject(s)
Arginine/chemistry , Cell-Penetrating Peptides/metabolism , RNA, Small Interfering/metabolism , CD4 Antigens/chemistry , CD4 Antigens/genetics , CD4 Antigens/metabolism , Cell Line , Cell Membrane/metabolism , Cell-Penetrating Peptides/chemistry , Cytoplasm/metabolism , Endocytosis , Endosomes/metabolism , Humans , Ligands , Microscopy, Confocal , RNA Interference , RNA, Messenger/metabolism , Superoxide Dismutase/antagonists & inhibitors , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Time-Lapse Imaging , TransfectionABSTRACT
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.
Subject(s)
HIV Infections/therapy , RNA Interference , RNA, Small Interfering/administration & dosage , Acquired Immunodeficiency Syndrome/genetics , Acquired Immunodeficiency Syndrome/therapy , Animals , Gene Targeting , Genetic Vectors , HIV Infections/genetics , Humans , RNA, Small Interfering/adverse effects , T-Lymphocytes/metabolismABSTRACT
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.