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In JoVE (1)
Other Publications (20)
- Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
- Anti-cancer Drugs
- Chemical Communications (Cambridge, England)
- Organic & Biomolecular Chemistry
- Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
- Chemical Communications (Cambridge, England)
- Organic & Biomolecular Chemistry
- Molecular Therapy : the Journal of the American Society of Gene Therapy
- Chemistry & Biology
- Nucleic Acids Research
- Current Topics in Medicinal Chemistry
- Silence
- Biology of Blood and Marrow Transplantation : Journal of the American Society for Blood and Marrow Transplantation
- Oligonucleotides
- Science Translational Medicine
- Methods (San Diego, Calif.)
- Methods in Molecular Biology (Clifton, N.J.)
- Methods in Molecular Biology (Clifton, N.J.)
- Molecular Therapy : the Journal of the American Society of Gene Therapy
- Bioconjugate Chemistry
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Articles by Jiehua Zhou in JoVE
JoVE Immunology and Infection
Развитие сотового типа конкретных анти-ВИЧ gp120 аптамеры для доставки миРНК
1Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, 2Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, 3Shared Resource-DNA/RNA Peptide, Beckman Research Institute of City of Hope
Несколько 2'-фтор РНК аптамеры против ВИЧ-1BA-L gp120 с наномоля близость изолированы от библиотеки РНК
Other articles by Jiehua Zhou on PubMed
Bombesin/gastrin-releasing Peptide Receptor: a Potential Target for Antibody-mediated Therapy of Small Cell Lung Cancer
Bombesin/gastrin releasing peptide (BN/GRP) is a growth factor for small cell lung cancer (SCLC). The receptor (R) for BN/GRP is overexpressed on SCLC cells and other solid tumors. BN/GRP and its receptor form an autocrine loop to promote tumor growth. We developed a novel immunotherapeutic approach targeting cell surface BN/GRP-R on SCLC cells and an immune trigger molecule on host immune effector cells to direct immune effector cells to SCLC cells and mediate targeted cancer cell destruction. Targeted immunotherapy combined with chemotherapy enhanced cell killing.
Targeting Gastrin-releasing Peptide Receptors for Cancer Treatment
Growth factor receptors play critical roles in cancer cell proliferation and progression. A number of such receptors have been targeted for cancer treatment by either a monoclonal antibody or a specifically designed small molecule to inhibit the receptor function. Bombesin/gastrin-releasing peptide receptors (BN/GRP-Rs) are expressed in a variety of cancer cells and have limited distribution in normal human tissue. Inhibition of BN/GRP-Rs has been shown to block small cell lung cancer growth in vitro. Early phase clinical trials targeting human GRP-R showed anti-cancer activity. This review will focus on the study of the distribution of BN/GRP-Rs in normal and malignant tissues, and various approaches to targeting BN-GRP-Rs for cancer diagnosis and treatment.
Polycationic Dendrimers Interact with RNA Molecules: Polyamine Dendrimers Inhibit the Catalytic Activity of Candida Ribozymes
Various polyamine dendrimers with a triethanolamine core inhibit the activity of the Candida ribozyme by forming RNA-dendrimer complexes via electrostatic interactions.
Cooperative Binding and Self-assembling Behavior of Cationic Low Molecular-weight Dendrons with RNA Molecules
Tri(ethylene glycol) derived, low molecular-weight dendrons with various amine end groups were synthesized and characterized for their properties of binding and self-assembling with RNA using the Candida ribozyme as a model RNA molecule. These dendritic compounds form stable complexes and well-defined nanoscale particles with RNA molecules via electrostatic interactions and self-assembly process, while leaving the other terminal of the tri(ethylene glycol) chain accessible for targeting. This suggests that dendrimers of this type hold great promise for specific RNA targeting and RNA delivery.
Targeting Gastrin-releasing Peptide Receptors on Small Cell Lung Cancer Cells with a Bispecific Molecule That Activates Polyclonal T Lymphocytes
Gastrin-releasing peptide (GRP) is a growth factor for small cell lung cancer (SCLC). GRP belongs to the bombesin peptide family and has significant homology to bombesin. We constructed a bispecific molecule, OKT3xAntag2, by conjugating a monoclonal antibody OKT3 (anti-CD3) with a bombesin/GRP antagonist (Antag2) and evaluated cytotoxicity against SCLC cells.
PAMAM Dendrimers for Efficient SiRNA Delivery and Potent Gene Silencing
Genuine, nondegraded PAMAM dendrimers self-assemble with siRNA into nanoscale particles that are efficient for siRNA delivery and induce potent endogenous gene silencing.
Importance of Size-to-charge Ratio in Construction of Stable and Uniform Nanoscale RNA/dendrimer Complexes
Formation of RNA/dendrimer complexes between various RNA molecules and PAMAM dendrimers was studied using atomic force microscopy. Our results demonstrate that effective construction of stable nanoscale and uniform RNA/dendrimer complexes depends critically on the size of the RNA molecule, the dendrimer generation and the charge ratio between the dendrimer and the RNA. Larger RNA molecules, higher generations of dendrimers and larger dendrimer-to-RNA charge ratios lead to the formation of stable, uniform nanoscale RNA/dendrimer complexes. These findings provide new insights in developing dendrimer systems for RNA delivery.
Novel Dual Inhibitory Function Aptamer-siRNA Delivery System for HIV-1 Therapy
The successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. In this study, we demonstrate cell type-specific delivery of anti-human immunodeficiency virus (anti-HIV) siRNAs through fusion to an anti-gp120 aptamer. The envelope glycoprotein is expressed on the surface of HIV-1-infected cells, allowing binding and internalization of the aptamer-siRNA chimeric molecules. We demonstrate that the anti-gp120 aptamer-siRNA chimera is specifically taken up by cells expressing HIV-1 gp120, and that the appended siRNA is processed by Dicer; this releases an anti-tat/rev siRNA which, in turn, inhibits HIV replication. We show for the first time a dual functioning aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. We also show that gp120 expressed on the surface of HIV-infected cells can be used for aptamer-mediated delivery of anti-HIV siRNAs.
Bivalent Aptamers Deliver the Punch
Aptamers, sometimes termed "chemical antibodies," have been engineered into multimerized versions for therapeutic application. The groups of Gilboa and Sullenger now report the development of a bivalent aptamer-molecular device as a receptor agonist that has the same functional properties, but stronger avidity than a corresponding antibody.
Selection, Characterization and Application of New RNA HIV Gp 120 Aptamers for Facile Delivery of Dicer Substrate SiRNAs into HIV Infected Cells
The envelope glycoprotein of human immunodeficiency virus (HIV) consists of an exterior glycoprotein (gp120) and a trans-membrane domain (gp41) and has an important role in viral entry into cells. HIV-1 entry has been validated as a clinically relevant anti-viral strategy for drug discovery. In the present work, several 2'-F substituted RNA aptamers that bind to the HIV-1(BaL) gp120 protein with nanomole affinity were isolated from a RNA library by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. From two of these aptamers we created a series of new dual inhibitory function anti-gp120 aptamer-siRNA chimeras. The aptamers and aptamer-siRNA chimeras specifically bind to and are internalized into cells expressing HIV gp160. The Dicer-substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells (PBMCs). Moreover, we have introduced a 'sticky' sequence onto a chemically synthesized aptamer which facilitates attachment of the Dicer substrate siRNAs for potential multiplexing. Our results provide a set of novel inhibitory agents for blocking HIV replication and further validate the use of aptamers for delivery of Dicer substrate siRNAs.
The Therapeutic Potential of Cell-internalizing Aptamers
Aptamers that are evolved by the SELEX procedure (Systematic Evolution of Ligands by Exponential enrichment) can specifically recognize and tightly bind their cognate targets by means of well-defined secondary and three-dimensional structures. In comparison to antibodies, nucleic acid-based aptamers offer some exciting advantages, including the potential for chemical synthesis, convenient modification, chemical versatility, stability and lack of immunogenicity. During the past 20 years, aptamers have been developed for various applications such as diagnostics, drug development, target validation and therapeutics. Aptamers targeting cell surface proteins are being explored as promising delivery vehicles to target a distinct disease or tissue in a cell-type-specific manner. In this review, we summarize the recent developments in creatively using cell-internalizing aptamers as drug delivery escorts to deliver, enhance and modulate the activity of other therapeutic agents, including chemical drugs, toxins, small interfering RNAs and nanoparticle-encapsulated drugs. Specifically, several attractive aptamer-mediated cell-type specific siRNA delivery systems are highlighted, and their promise in clinical development is also discussed.
Aptamer-targeted Cell-specific RNA Interference
This potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases. However, the efficient and safe delivery of siRNAs into specific cell populations is still the principal challenge in the clinical development of RNAi therapeutics. With the increasing enthusiasm for developing targeted delivery vehicles, nucleic acid-based aptamers targeting cell surface proteins are being explored as promising delivery vehicles to target a distinct disease or tissue in a cell-type-specific manner. The aptamer-based delivery of siRNAs can often enhance the therapeutic efficacy and reduce the unwanted off-target effects of siRNAs. In particular, for RNA interference-based therapeutics, aptamers represent an efficient agent for cell type-specific, systemic delivery of these oligonucleotides. In this review, we summarize recent attractive developments in creatively using cell-internalizing aptamers to deliver siRNAs to target cells. The optimization and improvement of aptamer-targeted siRNAs for clinical translation are further highlighted.
CTLA-4 Blockade Following Relapse of Malignancy After Allogeneic Stem Cell Transplantation is Associated with T Cell Activation but Not with Increased Levels of T Regulatory Cells
Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a key negative regulator of T cell activation and proliferation. Ipilimumab is a human monoclonal antibody that specifically blocks the binding of CTLA-4 to its ligand. To test the hypothesis that blockade of CTLA-4 by ipilimumab could augment graft-versus-malignancy (GVM) effects without a significant impact on graft-versus-host disease (GVHD), we conducted a phase I clinical trial of ipilimumab infusion in patients with relapsed malignancy following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Here, we report the analysis of peripheral blood T lymphocyte reconstitution, T regulatory cell (Treg) expression, and T cell activation markers after a single dose of ipilimumab in 29 patients. Peripheral blood samples were collected from all patients before and after ipilimumab infusion. Lymphocyte immunophenotyes, including levels of CD4(+)CD25(high) cells and T cell activation markers, were analyzed in all cases. Levels of CD4(+)CD25(high)Foxp3(+) cells and intracellular CTLA-4 in CD4(+) T cells also were evaluated in the last 11 cases. We found lower baseline levels of CD4(+) and CD45RO(+) T cells in patients compared with normal controls. More than 50% of the patients had abnormally low lymphocyte counts (CD4 or/and CD8 T cells), and some had no circulating B lymphocytes. The percentages of both CD4(+)CD25(high) and CD4(+)CD25(high)Foxp3(+) T cells were significantly higher in patients before ipilimumab infusion than in healthy donors. Twenty of 29 patients exhibited an elevated level of CD4(+)CD25(low) activated T cells at baseline, compared with only 3 of 26 healthy donors. Both CD4(+) and CD8(+) T lymphocyte counts were significantly increased after ipilimumab infusion. There was no consistent change in absolute lymphocyte count or in the number of T cells expressing the activation marker CD69. However, increases in CD4(+)CD25(low) T cells were seen in 20 of 29 patients and increases in CD4(+)HLA-DR(+) T cells were seen in the last 10 patients in the first 60 days after ipilimumab infusion. Although the percentages of both CD4(+)CD25(high) and CD4(+)CD25(high)Foxp3(+) T cells decreased significantly during the observation period, the absolute cell counts did not change. Intracellular CTLA-4 expression in CD4(+)CD25(lo/-) T cells increased significantly after ipilimumab infusion. We conclude that CTLA-4 blockade by a single infusion of ipilimumab increased CD4(+) and CD4(+)HLA-DR(+) T lymphocyte counts and intracellular CTLA-4 expression at the highest dose level. There was no significant change in Treg cell numbers after ipilimumab infusion. These data demonstrate that significant changes in T cell populations occur on exposure to a single dose of ipilimumab. Further studies with multiple doses are needed to explore this phenomenon further and to correlate changes in lymphocyte subpopulations with clinical events.
Cell-specific Aptamer-mediated Targeted Drug Delivery
Nucleic acid aptamers are in vitro-selected small, single-stranded DNA or RNA oligonucleotides that can specifically recognize their target on the basis of their unique 3-dimensional structures. Recent advances in the development of escort aptamers to deliver and enhance the efficacy of other therapeutic agents have drawn enthusiasm in exploiting cell-type-specific aptamers as drug delivery vehicles. This review mainly focuses on the recent developments of aptamer-mediated targeted delivery systems. We also place particular emphasis on aptamers evolved against cell membrane receptors and possibilities for translation to clinical applications.
An Aptamer-siRNA Chimera Suppresses HIV-1 Viral Loads and Protects from Helper CD4(+) T Cell Decline in Humanized Mice
Therapeutic strategies designed to treat HIV infection with combinations of antiviral drugs have proven to be the best approach for slowing the progression to AIDS. Despite this progress, there are problems with viral drug resistance and toxicity, necessitating new approaches to combating HIV-1 infection. We have therefore developed a different combination approach for the treatment of HIV infection in which an RNA aptamer, with high binding affinity to the HIV-1 envelope (gp120) protein and virus neutralization properties, is attached to and delivers a small interfering RNA (siRNA) that triggers sequence-specific degradation of HIV RNAs. We have tested the antiviral activities of these chimeric RNAs in a humanized Rag2(-/-)γc(-/-) (RAG-hu) mouse model with multilineage human hematopoiesis. In this animal model, HIV-1 replication and CD4(+) T cell depletion mimic the situation seen in human HIV-infected patients. Our results show that treatment with either the anti-gp120 aptamer or the aptamer-siRNA chimera suppressed HIV-1 replication by several orders of magnitude and prevented the viral-induced helper CD4(+) T cell decline. In comparison to the aptamer alone, the aptamer-siRNA combination provided more extensive inhibition, resulting in a significantly longer antiviral effect that extended several weeks beyond the last injected dose. The aptamer thus acts as a broad-spectrum HIV-neutralizing agent and an siRNA delivery vehicle. The combined aptamer-siRNA agent provides an attractive, nontoxic therapeutic approach for treatment of HIV infection.
Dual Functional RNA Nanoparticles Containing Phi29 Motor PRNA and Anti-gp120 Aptamer for Cell-type Specific Delivery and HIV-1 Inhibition
The potent ability of small interfering RNA (siRNA) to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for diseases including HIV. However, efficient delivery of siRNAs remains a key obstacle to successful application. A targeted intracellular delivery approach for siRNAs to specific cell types is highly desirable. HIV-1 infection is initiated by the interactions between viral glycoprotein gp120 and cell surface receptor CD4, leading to fusion of the viral membrane with the target cell membrane. Once HIV infects a cell it produces gp120 which is displayed at the cell surface. We previously described a novel dual inhibitory anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. We also demonstrated that gp120 can be used for aptamer mediated delivery of anti-HIV siRNAs. Here we report the design, construction and evaluation of chimerical RNA nanoparticles containing a HIV gp120-binding aptamer escorted by the pRNA of bacteriophage phi29 DNA-packaging motor. We demonstrate that pRNA-aptamer chimeras specifically bind to and are internalized into cells expressing HIV gp120. Moreover, the pRNA-aptamer chimeras alone also provide HIV inhibitory function by blocking viral infectivity. The Ab' pRNA-siRNA chimera with 2'-F modified pyrimidines in the sense strand not only improved the RNA stability in serum, but also was functionally processed by Dicer, resulting in specific target gene silencing. Therefore, this dual functional pRNA-aptamer not only represents a potential HIV-1 inhibitor, but also provides a cell-type specific siRNA delivery vehicle, showing promise for systemic anti-HIV therapy.
Progress in RNAi-based Antiviral Therapeutics
RNA interference (RNAi) refers to the conserved sequence-specific degradation of message RNA mediated by small interfering (si)RNA duplexes 21-25 nucleotides in length. Given the ability to specifically silence any gene of interest, siRNAs offers several advantages over conventional drugs as potential therapeutic agents for the treatment of human maladies including cancers, genetic disorders, and infectious diseases. Antiviral RNAi strategies have received much attention and several compounds are currently being tested in clinical trials. In particular, the development of siRNA-based HIV (human immunodeficiency virus) therapeutics has progressed rapidly and many recent studies have shown that the use of RNAi could inhibit HIV-1 replication by targeting a number of viral or cellular genes. Therefore, the present chapter mainly focuses on the recent progress of RNAi-based anti-HIV gene therapeutics, with particular attention to molecular targets and delivery strategies of the siRNAs.
Aptamer-targeted RNAi for HIV-1 Therapy
The highly specific mechanism of RNA (RNAi) that inhibits the expression of disease genes is increasingly being harnessed to develop a new class of therapeutics for a wide variety of human maladies. The successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. Herein, we demonstrate novel cell type-specific dual inhibitory function anti-gp120 aptamer-siRNA delivery systems for HIV-1 therapy, in which both the aptamer and the siRNA portions have potent anti-HIV activities. The envelope glycoprotein is expressed on the surface of HIV-1 infected cells, allowing binding and internalization of the aptamer-siRNA chimeric molecules. The Dicer substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells. Our results provide a set of novel aptamer-targeted RNAi therapeutics to combat HIV and further validate the use of anti-gp120 aptamers for delivery of Dicer substrate siRNAs.
Systemic Administration of Combinatorial DsiRNAs Via Nanoparticles Efficiently Suppresses HIV-1 Infection in Humanized Mice
We evaluated the in vivo efficacy of structurally flexible, cationic PAMAM dendrimers as a small interfering RNA (siRNA) delivery system in a Rag2(-)/-γc-/- (RAG-hu) humanized mouse model for HIV-1 infection. HIV-infected humanized Rag2-/-γc-/- mice (RAG-hu) were injected intravenously (i.v.) with dendrimer-siRNA nanoparticles consisting of a cocktail of dicer substrate siRNAs (dsiRNAs) targeting both viral and cellular transcripts. We report in this study that the dendrimer-dsiRNA treatment suppressed HIV-1 infection by several orders of magnitude and protected against viral induced CD4(+) T-cell depletion. We also demonstrated that follow-up injections of the dendrimer-cocktailed dsiRNAs following viral rebound resulted in complete inhibition of HIV-1 titers. Biodistribution studies demonstrate that the dendrimer-dsiRNAs preferentially accumulate in peripheral blood mononuclear cells (PBMCs) and liver and do not exhibit any discernable toxicity. These data demonstrate for the first time efficacious combinatorial delivery of anti-host and -viral siRNAs for HIV-1 treatment in vivo. The dendrimer delivery approach therefore represents a promising method for systemic delivery of combinations of siRNAs for treatment of HIV-1 infection.
Structurally Flexible Triethanolamine Core PAMAM Dendrimers Are Effective Nanovectors for DNA Transfection in Vitro and in Vivo to the Mouse Thymus
With the aim of developing dendrimer nanovectors with a precisely controlled architecture and flexible structure for DNA transfection, we designed PAMAM dendrimers bearing a triethanolamine (TEA) core, with branching units pointing away from the center to create void spaces, reduce steric congestion, and increase water accessibility for the benefit of DNA delivery. These dendrimers are shown to form stable nanoparticles with DNA, promote cell uptake mainly via macropinocytosis, and act as effective nanovectors for DNA transfection in vitro on epithelial and fibroblast cells and, most importantly, in vivo in the mouse thymus, an exceedingly challenging organ for immune gene therapy. Collectively, these results validate our rational design approach of structurally flexible dendrimers with a chemically defined structure as effective nanovectors for gene delivery, and demonstrate the potential of these dendrimers in intrathymus gene delivery for future applications in immune gene therapy.
