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Articles by Swiderski Piotr in JoVE

 JoVE Immunology and Infection

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

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

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Several 2’-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

Other articles by Swiderski Piotr on PubMed

Truncated Amplification: a Method for High-fidelity Template-driven Nucleic Acid Amplification

The error rate of conventional PCR is problematic when amplifying from single cells or amplifying segments for protein functional analysis by in vitro translation. We describe truncated amplification, a method for high-fidelity amplification in which DNA polymerase errors are not propagated efficiently and original DNA templates exert greater influence on the amplification process. Truncated amplification utilizes pairs of oligonucleotides and thermal cycling, but it differs from PCR. Truncated amplification amplifies non-exponentially with one or two chimeric oligonucleotides and produces truncated terminal products that are no more than three rounds of replication from the original template. Exon 6 of the p53 gene was utilized as a model system to demonstrate proof of principle. Chimeric oligonucleotides containing three 3'-->5' reversed-deoxynucleotides or 2'-OMe-ribonucleotides at 6-8 nucleotides from the 3 'terminus retained sequence specificity and primer extension activity. With PfuTurbo but not with Taq or Vent (exo-) DNA polymerases, the modified nucleotides completely truncated the DNA polymerase elongation. The resulting truncated terminal products are not templates for further amplification because of the short length of the 3' complementary region. Truncated amplific ation can amplify quadratically or geometrically depending on whether two or one chimeric oligonucleotides are used. Truncated amplification is a promising approach when template-driven amplification is desired to increase thefrequency of error-free products.

Mobility-shift Analysis with Microfluidics Chips

Electrophoretic mobility shift analysis (EMSA) is a well-characterized and widely used technique for the analysis of proten-DNA interaction and the analysis of transcription factor combinatorics. Currently implemented EMSA generally involves the time-consuming use of radiolabeled DNA and polyacrylamide gel electrophoresis. We are studying the bionanoscience of self-assembling supramolecular protein-nucleic nanostructures. We have undertaken these studies because they promise to enhance our understanding of assemblies formed during prebiotic evolution, provide tools for analysis of biological processes like DNA recombination, and may lead to the development of nanoscale biosensors designed for site-specific molecular targeting. During the course of that work, we noted that EMSA of these complex structures could be effectively implemented with microfluidics chips designed for the separation of DNA fragments. In this report we compare the two techniques and demonstrate that the microfluidics system is also capable of resolving complex mixtures produced by decorating DNA recombination intermediates with mixtures of DNA binding proteins. Moreover, the microfluidics chip system improves EMSA by permitting analysis with smaller samples, avoiding the use of radiolabeling, and reducing the time involved to a matter of minutes.

Construction of Ordered Protein Arrays

Artificially ordered protein arrays provide a facile approach to a variety of problems in biology and nanoscience. Current demonstration systems use either nucleic acid tethers or methyltransferase fusions in order to target proteins or peptides of interest to nucleic acid scaffolds. These demonstrations point to the large number of useful devices and assemblies that can be envisioned using this approach, including smart biological probes and drug delivery systems. In principle, these systems are now capable of imitating the earliest forms of prebiotic organisms and can be expected to reach the complexity of a small virus in the near future. Third-generation methyltransferase inhibitors provide an example of a smart chemotherapeutics that can be constructed with this approach. We describe the use of mechanistic enzymology, computer-aided design, and microfluidic chip-based capillary electrophoresis in assessing the final assembly and testing of designs of this type.

Proinflammatory Effects of Advanced Lipoxidation End Products in Monocytes

The reactions of carbohydrate- or lipid-derived intermediates with proteins lead to the formation of Maillard reaction products, which subsequently leads to the formation of advanced glycation/lipoxidation end products (AGE/ALEs). Levels of AGE/ALEs are increased in diseases like diabetes. Unlike AGEs, very little is known about ALE effects in vitro. We hypothesized that ALEs can have proinflammatory effects in monocytes.

Effects of Cholesterol-tagged Small Interfering RNAs Targeting 12/15-lipoxygenase on Parameters of Diabetic Nephropathy in a Mouse Model of Type 1 Diabetes

We previously showed that the 12/15-lipoxygenase (12/15-LO) pathway of arachidonate acid metabolism is involved in multiple events related to diabetic nephropathy (DN), including glomerular hypertrophy and extracellular matrix deposition (Kang SW, Adler SG, Nast CC, LaPage J, Gu JL, Nadler JL, Natarajan R. Kidney Int 59: 1354-1362, 2001; Kang SW, Natarajan R, Shahed A, Nast CC, LaPage J, Mundel P, Kashtan C, Adler SG. J Am Soc Nephrol 14: 3178-3187, 2003; Kim YS, Lanting L, Adler SG, Natarajan R. Kindney Int 64: 1702-1714, 2003; Reddy MA, Adler SG, Kim YS, Lanting L, Rossi JJ, Kang SW, Nadler JL, Shahed A, Natarajan R. Am J Physiol Renal Physiol 283: F985-F994, 2002). In this study, we investigated whether in vivo delivery of small interfering RNAs (siRNAs) targeting 12/15-LO can ameliorate renal injury and DN in a streptozotocin-injected mouse model of type 1 diabetes. To achieve greater in vivo access and siRNA expression in the kidney, we used double-stranded 12/15-LO siRNA oligonucleotides conjugated with cholesterol. Diabetic DBA/2J mice were injected subcutaneously with either cholesterol-tagged 12/15-LO siRNA, mismatched control siRNA, or vehicle alone, twice weekly for 7 wk. Relative to controls, mice that received 12/15-LO siRNA showed significant reduction in albuminuria, kidney-to-body weight ratios, glomerular mesangial matrix expansion, renal structural damage, and monocyte/macrophage infiltration. These effects were associated with lower renal cortical or glomerular levels of profibrotic markers transforming growth factor-beta, connective tissue growth factor, type I and type IV collagens, plasminogen activator inhibitor 1, and fibronectin. The diabetes-induced increase in glomerular cyclin-dependent kinase inhibitors that are associated with hypertrophy was also prevented by siRNA administration. Our results show for the first time that systemic delivery of cholesterol-tagged siRNAs targeting 12/15-LO has renoprotective effects under diabetic conditions and therefore could be a novel therapeutic approach for DN.

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.

In Vivo Delivery of SiRNA to Immune Cells by Conjugation to a TLR9 Agonist Enhances Antitumor Immune Responses

Efficient delivery of small interfering (si)RNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We show that siRNA synthetically linked to a CpG oligonucleotide agonist of toll-like receptor (TLR)9 targets and silences genes in TLR9(+) myeloid cells and B cells, both of which are key components of the tumor microenvironment. When a CpG-conjugated siRNA that targets the immune suppressor gene Stat3 is injected in mice either locally at the tumor site or intravenously, it enters tumor-associated dendritic cells, macrophages and B cells. Silencing of Stat3 leads to activation of tumor-associated immune cells and ultimately to potent antitumor immune responses. Our findings demonstrate the potential of TLR agonist-siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment.

Stability of Thioester Intermediates in Ubiquitin-like Modifications

Ubiquitin-like modifications are important mechanisms in cellular regulation, and are carried out through several steps with reaction intermediates being thioester conjugates of ubiquitin-like proteins with E1, E2, and sometimes E3. Despite their importance, a thorough characterization of the intrinsic stability of these thioester intermediates has been lacking. In this study, we investigated the intrinsic stability by using a model compound and the Ubc9 approximately SUMO-1 thioester conjugate. The Ubc9 approximately SUMO-1 thioester intermediate has a half life of approximately 3.6 h (hydrolysis rate k = 5.33 +/- 2.8 x10(-5) s(-1)), and the stability decreased slightly under denaturing conditions (k = 12.5 +/- 1.8 x 10(-5) s(-1)), indicating a moderate effect of the three-dimensional structural context on the stability of these intermediates. Binding to active and inactive E3, (RanBP2) also has only a moderate effect on the hydrolysis rate (13.8 +/- 0.8 x 10(-5) s(-1) for active E3 versus 7.38 +/- 0.7 x 10(-5) s(-1) for inactive E3). The intrinsically high stability of these intermediates suggests that unwanted thioester intermediates may be eliminated enzymatically, such as by thioesterases, to regulate their intracellular abundance and trafficking in the control of ubiquitin-like modifications.

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.

Humanized Lewis-Y Specific Antibody Based Delivery of STAT3 SiRNA

The clinical application of siRNA is limited largely by the lack of efficient, cell-specific delivery systems. Antibodies are attractive delivery vehicles for targeted therapy due to their high specificity. In this study we describe the use of a humanized monoclonal antibody (mAb), hu3S193, against Lewis-Y (Le(y)), as a delivery vehicle for STAT3 siRNA. This mAb is rapidly internalized into Le(y)-expressing cancer cells via antigen recognition, and when coupled to STAT3 siRNA, a potentially powerful molecularly targeted delivery agent is created. Selective silencing of STAT3 is associated with tumor suppression. Two hu3S193 based siRNA delivery systems using STAT3 siRNA as a prototype were developed and tested in Le(y)-positive cancer cells: (a) a covalent construct based on a reductive disulfide linker that is expected to undergo cleavage within cells and (b) a noncovalent construct based on (d-arginine)(9) (9r) modified hu3S193. Le(y)-specific binding and internalization of both the covalent and noncovalent constructs were confirmed by flow cytometry and confocal microscopy. Both the covalent and the noncovalent system led to efficient STAT3 silencing in Le(y)-positive cancer cells (A431) but not in Le(y)-negative cancer cells (MDA-MB-435). The covalent construct, however, required co-treatment with reagents such as chloroquine or 9r that facilitate the escape of the siRNA from endosomes to achieve significant gene silencing. The 9r modified noncovalent construct induced ∼70% STAT3 knockdown at submicromolar siRNA concentrations when used at an optimal vehicle-to-siRNA ratio of 5:1. The STAT3 knockdown also led to ∼50% inhibition of cell proliferation of Le(y)-positive cells. Noncovalent linked STAT3 siRNA-hu3S193 has great promise for targeted knockdown of STAT3 in tumor cells.

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.

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