Targeted cytotoxic effect of anti-JL1 immunotoxin against a human leukemic cell line and its clinical implications

We have previously reported the identification of a unique thymocyte-specific surface molecule, JL1, which was detected using the monoclonal antibody (mAb), anti-JL1. Interestingly, JL1 was shown to be expressed in most leukemias, irrespective of their immunophenotype, and subpopulations of normal bone marrow (BM) mononuclear cells (MNCs). Here we investigated the potential usefulness of the anti-JL1 mAb as a therapeutic tool for leukemia. We demonstrated that the proliferation of cultured human leukemia cells was dramatically inhibited in vitro by anti-JL1 mAb conjugated with the polypeptide toxin, gelonin, but not by gelonin alone. We then systematically investigated the reactivity of the anti-JL1 mAb against normal human tissues to evaluate possible side effects along with various hematopoietic and nonhematopoietic tumor cell lines. All of 33 types of normal tissues except thymus and subpopulation of BM MNCs were clearly devoid of JL1 expression. Among tumor cell lines, all the nonhematopoietic cell lines tested were negative for JL1 expression, while some hematopoietic cell lines contained JL1 antigen. Collectively, the results showed the cytotoxic effects of anti-JL1-based immunotoxin against JL1-positive leukemic cells, sparing most normal tissues other than thymocytes and some BM MNCs. Therefore, we strongly suggest that gelonin-conjugated anti-JL1 mAb immunotoxin could be developed as a potential immunotherapeutic agent in the treatment of various types of JL1-positive acute leukemias.     

Targeting of a developmentally regulated epitope of CD43 for the treatment of acute leukemia

Previously, we developed a JL1 mouse monoclonal antibody that specifically recognizes the leukemic cells of T, B, and myeloid lineages, but not the peripheral blood cells and pluripotent hematopoietic stem cells. Here, we identified that JL1 mAb recognized a specific epitope of human CD43 and validated its potential as an anti-leukemic targeting agent. After the comprehensive screening of JL1 Ag in the human thymocyte cDNA library, multiple fusion gene constructs encoding human CD43 were generated to identify its specific epitope to JL1 antibody. JL1 antibody interacted with a developmentally regulated and non-glycosylated epitope of the human CD43 extracellular domain (AA 73-81, EGSPLWTSI). In an in vivo leukemia model using NOD/SCID mice injected with CCRF-CEM7 cells, JL1 antibody induced effective cytotoxicity in tumor cells and prolonged survival (p < 0.05). Saporin conjugation to JL1 antibody effectively depleted tumor cells in in vitro cytotoxic assays and also prolonged survival in a leukemic mouse model (p < 0.001). These preclinical results further support the therapeutic potential of the JL1 antibody in the management of acute leukemia.     

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.     

Transcriptional targeting of small interfering RNAs into cancer cells

Small interfering RNAs (siRNAs) are widely used for analyzing gene function and have the potential to be developed into human therapeutics. However, persistent siRNA expression in normal cells may cause toxic side effects. Therefore, the therapeutic applications of RNAi in cancer require either the specific delivery of synthetic siRNAs into cancer cells or the control of siRNA expression. Accordingly, we have developed a cancer-specific vector that expresses siRNAs from the human survivin promoter. A plasmid vector expressing siRNAs under this promoter enabled efficient gene silencing of gene expression in different cancer cell lines. The levels of inhibition were comparable to that obtained with the constitutively active U6 promoter. By contrast to U6 promoter, no significant gene silencing was obtained with the Survivin promoter in normal mammary epithelial cells. Collectively, these data indicate that the survivin promoter is suitable for directing siRNA expression in cancer cells, but not normal cells.     

Gel-based application of siRNA to human epithelial cancer cells induces RNAi-dependent apoptosis

Gene silencing by RNA interference (RNAi) operates at the level of mRNA that is targeted for destruction with exquisite sequence specificity. In principle, any disease-related mRNA sequence is a putative target for RNAi-based therapeutics. To develop this therapeutic potential, it is necessary to develop ways of inducing RNAi by clinically acceptable delivery procedures. Here, we ask if inducers of RNAi can be delivered to human cells via a gel-based medium. RNAi was induced using synthetic small interfering RNAs (siRNAs), which bypass the need for expression vectors and carry the added bonus of high potency and immediate efficacy. Established cultures of human cells of normal and tumor origin were overlaid with an agarose/liposome/siRNA gel formulation without adverse effects on cell viability or proliferation. Epithelial cancer cells (but not normal human fibroblasts) proved vulnerable to specific siRNAs delivered via the agarose/liposome/siRNA formulation. Moreover, proapoptotic siRNAs induced apoptosis of cervical carcinoma cells (treated with human papillomavirus [HPV] E7 siRNA) and of colorectal carcinoma cells (treated with Bcl-2 siRNA). Thus, we demonstrate successful topical gel-based delivery of inducers of RNAi to human epithelial cancer cells. Topical induction of RNAi opens an important new therapeutic approach for treatment of human diseases, including cervical cancer and other accessible disorders.     

Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras

Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type-specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics.     

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.     

Specific subcellular localization of siRNAs delivered by lipoplex in MCF-7 breast cancer cells

In order to better understand the mechanism of delivery of siRNAs by lipid-based vectors, we investigated the subcellular distribution of siRNAs directed against cyclin D1 delivered by the DLS system in the breast cancer cell line MCF-7. Cells were treated with cyclopentenone or 17beta-estradiol to modulate the level of expression of cyclin D1 mRNA. We qualitatively observed that siRNA localized to specific cytoplasmic compartments in the periphery of the nucleus in granular-like structures that do not correspond to early endosomal vesicles. In cells treated with either cyclopentenone or 17beta-estradiol cellular distribution of siRNAs was not affected but variations in the amount of siRNAs present in cells were found. We suggest these variations might be associated with the effects of cyclopentenone and 17beta-estradiol in cyclin D1 gene expression. Low cytotoxicity and highly cellular uptake of lipoplexes was observed in the presence of serum indicating that the DLS system could be a useful tool for siRNA vectorization in vitro and in vivo.     

Silencing early viral replication in macrophages and dendritic cells effectively suppresses flavivirus encephalitis

West Nile (WN) and St. Louis encephalitis (SLE) viruses can cause fatal neurological infection and currently there is neither a specific treatment nor an approved vaccine for these infections. In our earlier studies, we have reported that siRNAs can be developed as broad-spectrum antivirals for the treatment of infection caused by related viruses and that a small peptide called RVG-9R can deliver siRNA to neuronal cells as well as macrophages. To increase the repertoire of broad-spectrum antiflaviviral siRNAs, we screened 25 siRNAs targeting conserved regions in the viral genome. Five siRNAs were found to inhibit both WNV and SLE replication in vitro reflecting broad-spectrum antiviral activity and one of these was also validated in vivo. In addition, we also show that RVG-9R delivers siRNA to macrophages and dendritic cells, resulting in effective suppression of virus replication. Mice were challenged intraperitoneally (i.p.) with West Nile virus (WNV) and treated i.v. with siRNA/peptide complex. The peritoneal macrophages isolated on day 3 post infection were isolated and transferred to new hosts. Mice receiving macrophages from the anti-viral siRNA treated mice failed to develop any disease while the control mice transferred with irrelevant siRNA treated mice all died of encephalitis. These studies suggest that early suppression of viral replication in macrophages and dendritic cells by RVG-9R-mediated siRNA delivery is key to preventing the development of a fatal neurological disease.     

Targeting of gene expression by siRNA in CML primary cells

Development of array methods contributes to elucidation of many genes expressed during oncogenesis. Our array-based analyses of gene expression in patients with chronic myeloid leukemia (CML) revealed several genes (MMP8, MMP9, PCNA, JNK2, MAPK p38) with significant increased expression. We suppose that the genes may be implicated in the disease development and a siRNA-suppression can elucidate their functions in leukemogenesis. One of the crucial requirements for this purpose is a high efficiency of siRNA delivery into CML primary cells. Using fluorescein-labeled siRNAs we systematically tested a variety of physical and chemical non-vector based transfection methods in order to evaluate which of them gave the most suitable transfer. Chemically synthesized siRNAs against mentioned genes were transfected into the cells and level of knockdown was determined by real time RT-PCR. Chemical transfection reagents (Oligofectamine, Metafectene, siPORT Amine) commonly used to transfect siRNAs in CML cell lines showed very low siRNA delivery in CML primary cells—mRNA levels decreased at the most to 76%. Electroporation achieved better results (suppression to 63%) but it was associated with high degree of cell death (more than 60%). In the study we obtained the best transfection efficiency using nucleofector technology. Gene expressions ranged 22–37% that remained from original levels. According to our results, nucleofection appears to be the only suitable non-viral method for siRNA delivery into the hard-to-transfect CML primary cells.     

Cationic liposome-mediated delivery of siRNAs in adult mice

RNA interference mediated by small interfering RNAs (siRNAs) is a powerful tool for dissecting gene function and drug target validation. siRNAs can be synthesized in large quantities and thus can be used to analyze a large number of sequences emerging from genome projects in a cost-effective manner. However, the major obstacle to the use of siRNAs as therapeutics is the difficulty involved in effective in vivo delivery. We used a fluorescein-labeled siRNA to investigate cationic liposome-mediated intravenous and intraperitoneal delivery in adult mice. We show that this simple approach can deliver siRNAs into various cell types. In addition, we show that in contrast to mouse cells, siRNAs can activate the non-specific pathway in human freshly isolated monocytes, resulting in TNF-alpha and IL-6 production. Taken together, the data provide a basis for lipid-mediated systemic delivery of siRNAs and indicate that certain siRNA sequences can activate the innate immunity response genes that can be beneficial for the treatment of cancer.     

Hyaluronic acid complexed to biodegradable poly L‐arginine for targeted delivery of siRNAs

Background

Small interfering RNA (siRNA) has been recognized as a new therapeutic drug to treat various diseases by inhibition of oncogene or viral gene expression. Because hyaluronic acid (HA) has been described as a biocompatible biomaterial, we tested the nanoparticles formed by electrostatic complexation of negatively‐charged HA and cationic poly L ‐arginine (PLR) for siRNA delivery systems.

Methods

Different electrostatic complexes of HA and PLR (HPs) were formulated: HP101 with 50% (w/w) HA and HP110 with 9% (w/w) HA.

Results

Gel retardation assays showed that HP101 and HP110 could form complexes with siRNAs. The diameters of these complexes were less than 200 nm. Cellular delivery efficiency of siRNAs by HPs depended on cell surface CD44 density. The HP‐mediated delivery of siRNAs was highest in WM266.4 cells followed by B16F10 cells and COS‐7 cells, in parallel with CD44 surface densities of these cell lines. TC₅₀ values (i.e. the HP concentrations at which 50% of cells were viable after treatment) were used as indicators of cytotoxicity. HP101 showed TC₅₀ values that were 2‐fold and 23‐fold higher than those of HP110 and PLR, respectively. After delivery into cells, siRNA exerted target‐specific RNA interference effects on mRNA and protein levels. Three days after treatment of red fluorescent protein (RFP)‐expressing B16F10 cells with RFP‐specific siRNA complexed to HP101, cellular fluorescence signals were reduced. Intratumoral administration of RFP‐specific siRNA via HP101 delivery significantly reduced the expression of RFP in tumor tissues.

Conclusions

HP101 may function as a biocompatible polymeric carrier of siRNAs and have possible application to localized siRNA delivery in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

The apparent uptake of fluorescently labeled siRNAs by electroporated cells depends on the fluorochrome

Transfection of mammalian cells with preformed small interfering RNAs (siRNAs) permits a transient and often specific reduction of gene expression. It is possible to rapidly examine the uptake of siRNAs by transfection with fluorescently labeled siRNAs. We examined the apparent uptake of such siRNAs by several leukemic cell lines after electroporation. We show that Cy3 and Cy5-labeled siRNAs cause a significant amount of cell fluorescence, as judged by flow cytometry. In contrast, several fluorescein-labeled siRNAs could not be detected. Nevertheless, such fluoresceinated siRNAs efficiently suppressed a leukemic target gene, demonstrating that siRNA uptake must have taken place. Therefore, for cell electroporation, fluorescein-labeled siRNAs may lead to false negative results and should not be used to examine electroporation-mediated siRNA uptake.     

Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors

Delivery of small interfering RNAs (siRNAs) into cells is a key obstacle to their therapeutic application. We designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells. The fusion protein (F105-P) was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope. Additionally, siRNAs targeted against the HIV-1 capsid gene gag, inhibited HIV replication in hard-to-transfect, HIV-infected primary T cells. Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelope-negative B16 cells; injection of F105-P with siRNAs targeting c-myc, MDM2 and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. This study demonstrates the potential for systemic, cell-type specific, antibody-mediated siRNA delivery.     

Targeted delivery of siRNA to hepatocytes and hepatic stellate cells by bioconjugation

Previously, we successfully conjugated galactosylated poly(ethylene glycol) (Gal-PEG) to oligonucleotides (ODNs) via an acid labile ester linker (Zhu et al., Bioconjugate Chem. 2008, 19, 290-8). In this study, sense strands of siRNA were conjugated to Gal-PEG and mannose 6-phosphate poly(ethylene glycol) (M6P-PEG) for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells (HSCs), respectively. These siRNA conjugates were purified by ion exchange chromatography and verified by gel retardation assay. To evaluate their RNAi functions, the validated siRNA duplexes targeting firefly luciferase and transforming growth factor beta 1 (TGF-β1) mRNA were conjugated to Gal-PEG and M6P-PEG, and their gene silencing efficiencies were determined after transfection into HepG2 and HSC-T6 cells. The disulfide bond between PEG and siRNA was cleaved by dithiothreitol, leading to the release of intact siRNA. Both Gal-PEG-siRNA and M6P-PEG-siRNA conjugates could silence luciferase gene expression by about 40% without any transfection reagents, while the gene silencing effects reached more than 98% with the help of cationic liposomes at the same dose. Conjugation of TGF-β1 siRNA with Gal-PEG and M6P-PEG could silence endogenous TGF-β1 gene expression as well. In conclusion, these siRNA conjugates have the potential for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells for efficient gene silencing in vivo.     

Mechanisms and optimization of in vivo delivery of lipophilic siRNAs

Cholesterol-conjugated siRNAs can silence gene expression in vivo. Here we synthesize a variety of lipophilic siRNAs and use them to elucidate the requirements for siRNA delivery in vivo. We show that conjugation to bile acids and long-chain fatty acids, in addition to cholesterol, mediates siRNA uptake into cells and gene silencing in vivo. Efficient and selective uptake of these siRNA conjugates depends on interactions with lipoprotein particles, lipoprotein receptors and transmembrane proteins. High-density lipoprotein (HDL) directs siRNA delivery into liver, gut, kidney and steroidogenic organs, whereas low-density lipoprotein (LDL) targets siRNA primarily to the liver. LDL-receptor expression is essential for siRNA delivery by LDL particles, and SR-BI receptor expression is required for uptake of HDL-bound siRNAs. Cellular uptake also requires the mammalian homolog of the Caenorhabditis elegans transmembrane protein Sid1. Our results demonstrate that conjugation to lipophilic molecules enables effective siRNA uptake through a common mechanism that can be exploited to optimize therapeutic siRNA delivery.     

Systemic siRNA delivery to leukocyte-implicated diseases

Short interfering RNA (siRNA), a small duplex of RNA fragment, has proved as an extremely useful research tool to interrogate gene functions in test tubes. However, the transformation of siRNAs from a functional genomic tool into a new therapeutic modality has been hindered by ineffective delivery methods for systemic administration. In this review, we will discuss the recent advances in formulating new delivery strategies that target siRNAs to specific cells following systemic administration. Special emphasis will be given to leukocytes, since siRNA delivery remains exceptionally challenging here due to the unavailability of effective delivery technologies. We will not only detail new platforms that utilize leukocyte integrins as receptor targets for siRNAs delivery, but also show how one of these strategies has been utilized for in vivo drug target validation of a novel anti-inflammatory target, cyclin D1, for inflammatory bowel diseases.     

Matrix metalloproteinase-2 and -9 secreted by leukemic cells increase the permeability of blood-brain barrier by disrupting tight junction proteins

Central nervous system (CNS) involvement remains an important cause of morbidity and mortality in acute leukemia, the mechanisms of leukemic cell infiltration into the CNS have not yet been elucidated. The blood-brain barrier (BBB) makes CNS become a refugee to leukemic cells and serves as a resource of cells that seed extraneural sites. How can the leukemic cells disrupt this barrier and invasive the CNS, even if many of the currently available chemotherapies can not cross the BBB? Tight junction in endothelial cells occupies a central role in the function of the BBB. Except the well known role of degrading extracellular matrix in metastasis of cancer cells, here we show matrix metalloproteinase (MMP)-2 and -9, secreted by leukemic cells, mediate the BBB opening by disrupting tight junction proteins in the CNS leukemia. We demonstrated that leukemic cells impaired tight junction proteins ZO-1, claudin-5 and occludin resulting in increased permeability of the BBB. However, these alterations reduced when MMP-2 and -9 activities were inhibited by RNA interference strategy or by MMP inhibitor GM6001 in an in vitro BBB model. We also found that the disruption of the BBB in company with the down-regulation of ZO-1, claudin-5 and occludin and the up-regulation of MMP-2 and -9 in mouse brain tissues with leukemic cell infiltration by confocal imaging and the assay of in situ gelatin zymography. Besides, GM6001 protected all mice against CNS leukemia. Our findings suggest that the degradation of tight junction proteins ZO-1, claudin-5 and occludin by MMP-2 and -9 secreted by leukemic cells constitutes an important mechanism in the BBB breakdown which contributes to the invasion of leukemic cells to the CNS in acute leukemia.     

bcr rearrangement and C-abl gene expression in Ph1-positive hybrid acute leukemia with simultaneous proliferation of lymphoid and myeloid blasts

bcr gene rearrangement and c-abl gene expression were analyzed in a patient with Philadelphia chromosome (Ph1)-positive hybrid acute leukemia with simultaneous proliferation of lymphoid and myeloid blasts. These data were compared with those from a patient with chronic myelogenous leukemia (CML) in mixed crisis. The leukemic cells of both patients showed immuno-phenotypic profiles such as non-T, non-B common ALL with some MPO-positive leukemic cells and rearranged JH genes. On analysis of molecular events associated with the Ph1 chromosome, the leukemic cells of a patient with CML in mixed crisis showed bcr rearrangement and an 8.5-kb bcr-abl chimeric mRNA, but those of a patient with Ph1-positive hybrid acute leukemia showed no 8.5-kb bcr-abl mRNA, as previously reported in a number of Ph1-positive acute lymphoblastic leukemia (ALL) cases. These results revealed that the molecular event found in Ph1-positive ALL is not only restricted to lymphoid lineage but may play an important role in the proliferation of the myeloid lineage.