Intratumor RNA interference of cell cycle genes slows down tumor progression.

Small interfering RNAs (siRNAs) are emerging as promising therapeutic tools. However, the widespread clinical application of such molecules as modulators of gene expression is still dependent on several aspects that limit their bioavailability. One of the most promising strategies to overcome the barriers faced by gene silencing molecules involves the use of lipid-based nanoparticles (LNPs) and viral vectors, such as adenoviruses (Ads). The primary obstacle for translating gene silencing technology from an effective research tool into a feasible therapeutic strategy remains its efficient delivery to the targeted cell type in vivo. In this study, we tested the capability of LNPs and Ad to transduce and treat locally tumors in vivo. Efficient knockdown of a surrogate reporter (luciferase) and therapeutic target genes such as the kinesin spindle protein (KIF11) and polo-like kinase 1 were observed. Most importantly, this activity led to a cell cycle block as a consequence and slowed down tumor progression in tumor-bearing animals. Our data indicate that it is possible to achieve tumor transduction with si/short hairpin RNAs and further improve the delivery strategy that likely in the future will lead to the ideal non-viral particle for targeted cancer gene silencing.

Gene therapy progress and prospects: transcription regulatory systems.

The clinical efficacy and safety as well as the application range of gene therapy will be broadened by developing systems capable of finely modulating the expression of therapeutic genes. Transgene regulation will be crucial for maintaining appropriate levels of a gene product within the therapeutic range, thus preventing toxicity. Moreover, the possibility to modulate, stop or resume transgene expression in response to disease evolution would facilitate the combination of gene therapy with more conventional therapeutic modalities. The development of ligand-dependent transcription regulatory systems is thus of great importance. Here, we summarize the most recent progress in the field.

Tight control of gene expression by a helper-dependent adenovirus vector carrying the rtTA2(s)-M2 tetracycline transactivator and repressor system.

Control of gene expression for gene therapy application requires the design of a sophisticated system embodying multiple properties. The ideal system should present the following features: (1) low or undetectable gene expression in the absence of inducer; (2) strong expression upon induction; and (3) fast kinetics of induction in the presence of inducers and rapid reversal of induction after its withdrawal. To evaluate these parameters, the features of the latest generation tetracycline-sensitive reverse-transactivator (rtTA2(s)-M2) alone or in combination with Tet-repressor (tTS-Kid) were explored in the context of helper-dependent adenovirus vector. Various genetic elements were assembled in a series of vectors and the ability to control secreted alkaline phosphatase expression evaluated in vitro in HeLa cells and in vivo by intramuscular injection in both C57/B6 and Balb/C nude mice. The results allow us to draw some general conclusions about the combination of transcription regulators and their relative orientation to the transgene to achieve maximal induction, while minimizing leakiness of expression.

betaMinor-globin messenger RNA accumulation in reticulocytes governs improved erythropoiesis in beta thalassemic mice after erythropoietin complementary DNA electrotransfer in muscles.

Mechanisms governing the induction of effective erythropoiesis in response to erythropoietin (Epo) oversecretion have been investigated in beta thalassemic C57Bl/6(Hbbth) mice. Naked DNA encoding an expression vector for mouse Epo was introduced into skeletal muscles by electrotransfer. A transient increase of serum Epo concentrations with a proportional augmentation of hematocrit values was observed. Various parameters relevant to beta thalassemia were surveyed in blood samples taken before treatment, at the peak of Epo secretion, and when the phenotype reverted to anemia. We measured globin messenger RNA (mRNA) levels in reticulocytes by real-time quantitative polymerase chain reaction, globin chain synthesis levels, and several indicators of erythrocyte membrane quality, including bound alpha chains, bound immunoglobulins, main protein components, and iron compartmentalization. Data indicated that high serum Epo levels primarily affect betaminor-globin mRNA accumulation in reticulocytes. Other changes subsequent to intense Epo stimulation, like increased betaminor/alpha-globin chain synthesis ratio, reduced levels of alpha chains and immunoglobulins bound to membranes, improved spectrin/band 3 ratio, increased red blood cell survival, and improved erythropoiesis appeared as consequences of increased betaminor-globin mRNA levels. This conclusion is consistent with models postulating that intense Epo stimulation induces the expansion and differentiation of erythroid progenitors committed to fetal erythropoiesis. Although phenotypic correction was partial in mice, and comparable achievements will probably be more difficult to obtain in humans, naked DNA electrotransfer may provide a safe and low-cost method for reassessing the potentials of Epo as an inducer of fetal erythropoiesis reactivation in patients with beta thalassemia.

Selective cleavage of AAVS1 substrates by the adeno-associated virus type 2 rep68 protein is dependent on topological and sequence constraints.

The adeno-associated virus type 2 (AAV-2) Rep78 and Rep68 proteins are required for replication of the virus as well as its site-specific integration into a unique site, called AAVS1, of human chromosome 19. Rep78 and Rep68 initiate replication by binding to a Rep binding site (RBS) contained in the AAV-2 inverted terminal repeats (ITRs) and then specifically nicking at a nearby site called the terminal resolution site (trs). Similarly, Rep78 and Rep68 are postulated to trigger the integration process by binding and nicking RBS and trs homologues present in AAVS1. However, Rep78 and Rep68 cleave in vitro AAVS1 duplex-linear substrates much less efficiently than hairpinned ITRs. In this study, we show that the AAV-2 Rep68 endonuclease activity is affected by the topology of the substrates in that it efficiently cleaves in vitro in a site- and strand-specific manner the AAVS1 trs only if this sequence is in a supercoiled (SC) conformation. DNA sequence mutagenesis in the context of SC templates allowed us to elucidate for the first time the AAVS1 trs sequence and position requirements for Rep68-mediated cleavage. Interestingly, Rep68 did not cleave SC templates containing RBS from other sites of the human genome. These findings have intriguing implications for AAV-2 site-specific integration in vivo.

Characteristics of the adeno-associated virus preintegration site in human chromosome 19: open chromatin conformation and transcription-competent environment.

Adeno-associated virus (AAV) establishes latency in infected cells by integrating into the cellular genome, with a high preference for a unique region, called AAVS1, of the human chromosome 19. The AAV proteins Rep78 and -68 are postulated to initiate the site-specific integration process by binding to a Rep binding site (RBS) in AAVS1. We provide further evidence to corroborate this model by demonstrating that the AAVS1 RBS in human cell lines is located near a DNase I hypersensitive "open" chromatin region and therefore is potentially easily accessible to Rep proteins. This open conformation is maintained in transgenic rats which carry an AAVS1 3. 5-kb DNA fragment and are proficient for Rep-mediated site-specific integration. Interestingly, the core of the DNAse I hypersensitive site in AAVS1 corresponds to a sequence displaying transcriptional enhancer-like properties, suggesting that AAVS1 constitutes a transcription-competent environment. The implications of our findings for AAV physiology and gene therapy are discussed.

Conditional site-specific integration into human chromosome 19 by using a ligand-dependent chimeric adeno-associated virus/Rep protein.

It is of great interest for gene therapy to develop vectors that drive the insertion of a therapeutic gene into a chosen specific site on the cellular genome. Adeno-associated virus (AAV) is unique among mammalian viruses in that it integrates into a distinct region of human chromosome 19 (integration site AAVS1). The inverted terminal repeats (ITRs) flanking the AAV genome and the AAV-encoded nonstructural proteins Rep78 and/or Rep68 are the only viral elements necessary and sufficient for site-specific integration. However, it is also known that unrestrained Rep activity may cause nonspecific genomic rearrangements at AAVS1 and/or have detrimental effects on cell physiology. In this paper we describe the generation of a ligand-dependent form of Rep, obtained by fusing a C-terminally deleted Rep68 with a truncated form of the hormone binding domain of the human progesterone receptor, which does not bind progesterone but binds only its synthetic antagonist RU486. The activity of this chimeric protein, named Rep1-491/P, is highly dependent on RU486 in various assays: in particular, it triggers site-specific integration at AAVS1 of an ITR-flanked cassette in a ligand-dependent manner, as efficiently as wild-type Rep68 but without generating unwanted genomic rearrangement at AAVS1.

Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector.

Adenovirus (Ad) and adeno-associated virus (AAV) have attractive and complementary properties that can be exploited for gene transfer purposes. Ad vectors are probably the most efficient vehicles to deliver foreign genes both in vitro and in vivo. AAV exhibits the unique ability to establish latency by efficiently integrating at a specific locus of human chromosome 19 (AAVS1). Two viral elements are necessary for the integration at AAVS1: Rep68/78 and the inverted terminal repeats (AAV-ITRs). In this study, we report the development of two helper-dependent adenoviral (HD) vectors, one carrying the Rep78 gene, the other an AAV-ITR-flanked transgene. Although Rep proteins have been demonstrated to interfere with Ad replication, HD Rep78 vector was successfully amplified on serial passages in 293CRE4 cells with a yield of 50-100 transducing units per cell. DNA integration at the AAVS1 site also was demonstrated in hepatoma cells coinfected with the HD-expressing Rep78 and with the second HD vector carrying a transgene flanked by AAV-ITRs. The high transduction efficiency, large cloning capacity, and high titer of the HD, combined with the site-specific integration machinery provided by AAV-derived components, make the Ad/AAV hybrid viruses a promising vehicle for gene therapy.

Activation of gp130 signaling in vivo by the IL-6 super-agonist K-7/D-6 accelerates repopulation of lymphoid organs after irradiation.

Stimulation of the gp130 signaling pathway by IL-6 is known to contribute significantly to hematopoietic expansion in vitro, mostly in combination with other cytokines. In the present study we have investigated whether a similar effect can be observed also in vivo using short-term assays in which irradiated mice were analyzed for repopulation of lymphoid organs. Mice were injected with a combination of soluble IL-6Ralpha either with wild-type (wt) human IL-6 or with an IL-6 variant, called K-7/D-6, that shows a 70-fold higher IL-6Ralpha affinity. We observed that while wt IL-6 was able to induce a partial effect only in combination with IL-3, K-7/D-6 bypassed the need for IL-3 and yielded complete recovery. In lethally irradiated mice reconstituted with syngeneic bone marrow cells K-7/D-6 strongly accelerated the repopulation of thymus and spleen and hastened blood neutrophil recovery. These results underscore the potential of the gp130 signaling pathway in hematopoietic reconstitution after myeloablative regimens and open the possibility to fully exploit it with a super-active IL-6 variant.

Blocking signaling through the Gp130 receptor chain by interleukin-6 and oncostatin M inhibits PC-3 cell growth and sensitizes the tumor cells to etoposide and cisplatin-mediated cytotoxicity.

BACKGROUND: The mechanisms of drug resistance associated with advanced, hormone-independent prostate carcinoma are poorly understood. The human prostate carcinoma PC-3 cell line, derived from a metastatic tumor and lacking androgen receptors, represents a useful model to investigate drug resistance. METHODS: The effects of oncostatin M (OM), antiinterleukin-6 (IL-6) treatment, or interference with the gp130-mediated signaling on etoposide- or cisplatin-mediated cytotoxicity were investigated. RESULTS: Both endogenous and exogenous IL-6 and exogenous OM up-regulated cell growth and enhanced resistance of PC-3 tumor cells to both etoposide and cisplatin. The influence of IL-6 is controlled by treating PC-3 tumor cells with anti-IL-6 neutralizing antibody and, more efficiently, by a mutated IL-6, Sant7. Sant7 has a high affinity binding to the IL-6 receptor-alpha (IL-6Ralpha) subunit, but does not bind to the signaling subunit gp130; therefore, it behaves as a receptor antagonist. Both IL-6- and OM-mediated effects are inhibited by the treatment of PC-3 with an antisense oligodeoxynucleotide against gp130, the protein kinase inhibitor genistein (GNS), or the monoterpene perillic acid (PA), a posttranslational inhibitor of p21ras isoprenylation. CONCLUSIONS: These results demonstrate the protective roles in drug sensitivity of IL-6 and OM through signaling of the common chain gp130 and, most likely, a downstream ras-dependent pathway in PC-3 tumor cells. These findings suggest the potential clinical application of anticytokine therapy or interference with gp130 signaling in the treatment of drug resistant prostate carcinoma.

Lipofection of purified adeno-associated virus Rep68 protein: toward a chromosome-targeting nonviral particle.

Adeno-associated virus (AAV) integrates very efficiently into a specific site (AAVS1) of human chromosome 19. Two elements of the AAV genome are sufficient: the inverted terminal repeats (ITRs) and the Rep78 or Rep68 protein. The incorporation of the AAV integration machinery in nonviral delivery systems is of great interest for gene therapy. We demonstrate that purified recombinant Rep68 protein is functionally active when directly delivered into human cells by using the polycationic liposome Lipofectamine, promoting the rescue-replication of a codelivered ITR-flanked cassette in adenovirus-infected cells and its site-specific integration in noninfected cells. The sequencing of cloned virus-host DNA junctions confirmed that lipofected Rep68 protein triggers site-specific integration at the same sites in chromosome 19 already characterized in cells latently infected with AAV.

Differential effects of IL-6 on systemic and central production of TNF: a study with IL-6-deficient mice.

Interleukin 6 (IL-6) is known to inhibit the synthesis of tumour necrosis factor (TNF) in vitro and in vivo. In this study we investigated the possible role of IL-6 as an endogenous inhibitor of TNF production in the brain or in the periphery using IL-6-deficient mice or administering recombinant human IL-6 (rhIL-6). When IL-6-deficient mice were injected intracerebroventricularly (i.c.v.) with lipopolysaccaride (LPS), no differences were observed in the production of TNF in the brain, while in the periphery (serum or spleen) TNF levels were markedly increased (about four-fold). When normal mice were injected i.c.v. with a combination of LPS and rhIL-6, inhibition of TNF production was only slight (about 20%), while IL-6 had a stronger effect (> 80% inhibition) in the periphery. Co-administration of soluble IL-6 receptor (sIL-6R) did not enhance the effect of IL-6 on brain TNF, so this refractoriness cannot be attributed to a lack of IL-6 receptors. Interestingly, IL-6 potently inhibited LPS-induced TNF production by macrophagic cells but not by a microglial cell clone, suggesting that the defective response to IL-6 of the brain lies within the responsiveness TNF producing cells to IL-6. It thus appears that the TNF-inhibitory role of IL-6 is confined to the periphery.

Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment.

IL-6-/- mice showed impaired leukocyte accumulation in subcutaneous air pouches. Defective leukocyte accumulation was not due to a reduced migratory capacity of IL-6-/- leukocytes and was associated with a reduced in situ production of chemokines. These observations led to a reexamination of the interaction of IL-6 with endothelial cells (EC). EC express only the gp130 signal transducing chain and not the subunit-specific IL-6R and are therefore unresponsive to IL-6. However, EC are responsive to a combination of IL-6 and soluble IL-6R as measured by the activation of STAT3, chemokine expression, and augmentation of ICAM-1. Activation by IL-6-IL-6R complexes was inhibited by an IL-6 receptor antagonist and potentiated by a superagonist. Hence, in vivo and in vitro evidence supports the concept that the IL-6 system plays an unexpected positive role in local inflammatory reactions by amplifying leukocyte recruitment.

The receptor super-antagonist Sant7.

Interleukin-6 (IL-6) plays a central role in the pathogenesis of multiple myeloma, acting both as a growth and a survival factor for myeloma cells. IL-6 has been recently shown to possess three topologically distinct receptor binding sites: site 1 for binding to the subunit specific chain IL-6R alpha and sites 2 and 3 for the interaction with two separate subunits of the signalling chain gp130. We have generated a set of IL-6 receptor antagonists carrying substitutions that abolish interaction with gp130 at either site 2 alone (site 2 antagonist) or at both sites 2 and 3 (site 2+3 antagonist). In addition, substitutions were introduced at site 1 that increased affinity for IL-6R alpha. When tested as growth inhibitors on a representative set of IL-6-dependent human myeloma cell lines (XG-1, XG-2, XG-4 and XG-6), although site 2 antagonists were effective on 3 out of 4 of the cell lines, only the site 2+3 antagonist Sant7 showed full antagonism on the entire spectrum of cells tested. Moreover, IL-6 receptor antagonists were also pro-apoptotic factors for myeloma cells. Their capacity to induce cell death was directly related to the impairment of binding to gp130 and to their ability to fully block intracellular signalling. In fact, the most potent inducer of apoptosis was again Sant7, which also counteracted the protective autocrine effect excercised by the endogenously produced IL-6. On the basis of these results we propose the super-antagonist Sant7 as a possible candidate for the immunotherapy of multiple myeloma.

IL-6 is an in vitro and in vivo autocrine growth factor for middle T antigen-transformed endothelial cells.

Murine endothelial cells immortalized with the middle-size Ag of polyomavirus (PmT) cause vascular tumors in syngenic mice by recruitment of host normal endothelial cells. This pathogenic process is similar to that occurring in Kaposi's sarcoma, in which the core of the lesion is constituted by "spindle cells," which recruit normal vascular mesenchymal cells. In murine endothelial cells, PmT induces modification of the expression of genes, including that of IL-6. Since IL-6 is a pleiotrophic cytokine that also regulates endothelial cell functions related to angiogenesis, we studied the relevance of IL-6 in the tumorigenicity of PmT-endothelial cells. In vitro studies demonstrated that the spontaneous PmT-endothelial cell proliferation rate was slow during the first 6 days of culture and then increased rapidly and paralleled the IL-6 release. The addition of recombinant IL-6 during the first days of culture induced a marked proliferation in a dose-dependent manner. PmT-endothelial cells expressed on their surface a high-affinity binding site for IL-6 constituted by both IL-6Ralpha and gp130 transmembrane receptors. The growth-promoting effect of exogenous IL-6 or that released by PmT-endothelial cells was abrogated by mAbs anti-IL-6Ralpha, whereas a mAb recognizing the endothelial cell CD31 molecule was inactive. 15A7 mAb anti-murine IL-6Ralpha was also active in vivo, reducing the number of metastases forming after transplantation of PmT-endothelial cells in DBA/2 mice. 15A7 mAb also increased the survival of mice bearing vascular tumors. We conclude that IL-6 is involved in the progression of vascular tumors induced by PmT, and that the blockage of IL-6-mediated intercellular circuits could be useful in the management of human vascular tumors, including Kaposi's sarcoma.

Engineering human interleukin-6 to obtain variants with strongly enhanced bioactivity.

Interleukin-6 (IL-6) triggers the formation of a high affinity receptor complex with the ligand binding subunit IL-6Ralpha and the signal transducing chain gp130. Since the intracytoplasmic region of the IL-6Ralpha does not contribute to signaling, soluble forms of the extracytoplasmic domain (sIL-6Ralpha), potentiate IL-6 bioactivity and induce a cytokine-responsive status in cells expressing gp130 only. This observation, together with the detection of high levels of circulating soluble human IL-6Ralpha (shIL-6Ralpha) in sera, suggests that the hIL-6-shIL-6Ralpha complex is an alternative form of the cytokine. Here we describe the generation of human IL-6 (hIL-6) variants with strongly enhanced shIL-6Ralpha binding activity and bioactivity. Homology modeling and site-directed mutagenesis of hIL-6 suggested that the binding interface for hIL-6Ralpha is constituted by the C-terminal portion of the D-helix and residues contained in the AB loop. Four libraries of hIL-6 mutants were generated by each time fully randomizing four different amino acids in the predicted AB loop. These libraries were displayed monovalently on filamentous phage surface and sorted separately for binding to immobilized shIL-6Ralpha. Mutants were selected which, when expressed as soluble proteins, showed a 10- to 40-fold improvement in shIL-6Ralpha binding; a further increase (up to 70-fold) was achieved by combining variants isolated from different libraries. Interestingly, high affinity hIL-6 variants show strongly enhanced bioactivity on cells expressing gp13O in the presence of shIL-6Ralpha at concentrations similar to those normally found in human sera.

Human interleukin-6 receptor super-antagonists with high potency and wide spectrum on multiple myeloma cells.

Interleukin-6 (IL-6) is the major growth factor for myeloma cells and is believed to participate in the pathogenesis of chronic autoimmune diseases and postmenopausal osteoporosis. IL-6 has been recently shown to possess three topologically distinct receptor binding sites: site 1 for binding to the subunit specific chain IL-6R alpha and sites 2 and 3 for the interaction with two subunits of the signaling chain gp130. We have generated a set of IL-6 variants that behave as potent cytokine receptor super-antagonists carrying substitutions that abolish interaction with gp130 at either site 2 alone (site 2 antagonist) or at both sites 2 and 3 (site 2 + 3 antagonist). In addition, substitutions have been introduced in site 1 that lead to variable increases in binding for IL-6R alpha up to 70-fold. IL-6 super-antagonists inhibit wild-type cytokine activity with efficacy proportional to the increase in receptor binding on a variety of human call lines of different origin, and the most potent molecules display full antagonism at low molar excess to wild-type IL-6. When tested on a representative set of IL-6-dependent human myeloma cell lines, although site 2 super-antagonists were in general quite effective, only the site 2 + 3 antagonist Sant7 showed antagonism on the full spectrum of cells tested. In conclusion, IL-6 super-antagonists are a useful tool for the study of myeloma in vitro and might constitute, in particular Sant7, effective IL-6 blocking agents in vivo.

Impaired neutrophil response and CD4+ T helper cell 1 development in interleukin 6-deficient mice infected with Candida albicans.

To define the role of interleukin (IL)6 in Candida albicans infection, IL-6 deficient mice were assessed for susceptibility to systemic or gastrointestinal infection, as well as for parameters of elicited T helper cell (Th) immunity. IL-6-deficient mice were more susceptible than wild-type mice to either type of infection caused by virulent C. albicans. In response to systemic challenge with a live vaccine strain of yeast, IL-6-deficient mice failed to mount Th1-associated protective immunity, but the resulting Th2-biased response could be redirected to the Th1 phenotype by IL-10 neutralization. Severe impairment of the macrophage and neutrophil response to infection was observed in IL-6-deficient mice, but administration of IL-6 would increase both neutrophil response and resistance to infection. IL-6 seems to oppose the Th2-promoting role of IL-10 in candidiasis, its early regulatory activity involving effects on neutrophil function.

Interleukin (IL)-6 gene expression in the central nervous system is necessary for fever response to lipopolysaccharide or IL-1 beta: a study on IL-6-deficient mice.

Interleukin (IL)-6, IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) are considered to act as endogenous pyrogens. Because of the complex pattern of cross-inductions between these cytokines, the relative role of the central and peripheral production of these cytokines in eliciting the fever response has not yet been clarified. The purpose of this study was to determine the role of IL-6 in the fever response by making use of mice carrying a null mutation in the IL-6 gene. The intraperitoneal injections of lipopolysaccharide (LPS) (50 micrograms/kg) and recombinant murine (rm) IL-1 beta (10 micrograms/kg), respectively, failed to evoke fever response in IL-6-deficient mice, whereas the same doses of LPS and rmIL-1 beta caused fever response in wild-type mice. The fever response could be induced in the IL-6-deficient mice by intracerebroventricular injection of recombinant human (rh) IL-6 (500 ng/mouse), whereas intracerebroventricular injection of rmIL-1 beta (100 ng/mouse) failed to produce fever response in the IL-6-deficient mice. These results suggest that central IL-6 is a necessary component of the fever response to both endogenous (IL-1 beta) and exogenous (LPS) pyrogens in mice and that IL-6 acts downstream from both peripheral and central IL-1 beta.