Inhibition of HIV-1 multiplication by antisense U7 snRNAs and siRNAs targeting cyclophilin A.

Human immunodeficiency virus 1 (HIV-1) multiplication depends on a cellular protein, cyclophilin A (CyPA), that gets integrated into viral particles. Because CyPA is not required for cell viability, we attempted to block its synthesis in order to inhibit HIV-1 replication. For this purpose, we used antisense U7 small nuclear RNAs (snRNAs) that disturb CyPA pre-mRNA splicing and short interfering RNAs (siRNAs) that target CyPA mRNA for degradation. With dual-specificity U7 snRNAs targeting the 3' and 5' splice sites of CyPA exons 3 or 4, we obtained an efficient skipping of these exons and a strong reduction of CyPA protein. Furthermore, short interfering RNAs targeting two segments of the CyPA coding region strongly reduced CyPA mRNA and protein levels. Upon lentiviral vector-mediated transduction, prolonged antisense effects were obtained for both types of antisense RNAs in the human T-cell line CEM-SS. These transduced CEM-SS cells showed a delayed, and for the siRNAs also reduced, HIV-1 multiplication. Since the two types of antisense RNAs function by different mechanisms, combining the two approaches may result in a synergistic effect.

Antisense properties of tricyclo-DNA.

Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides up to 17 nt in length, and evaluated their binding efficiency and selectivity towards complementary RNA, their biological stability in serum, their RNase H inducing potential and their antisense activity in a cellular assay. Relative to RNA or 2'-O-Me-phosphorothioate (PS)-RNA, fully modified tc-oligodeoxynucleotides, 10-17 nt in length, show enhanced selectivity and enhanced thermal stability by approximately 1 degrees C/modification in binding to RNA targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37 degree C. Moreover, tc-DNA-RNA duplexes are not substrates for RNase H. To test for antisense effects in vivo, we used HeLa cell lines stably expressing the human beta-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in beta-globin mRNA. Lipofectamine-mediated delivery of a 17mer tc-oligodeoxynucleotide complementary to the 3'-cryptic splice site results in correction of aberrant splicing already at nanomolar concentrations with up to 100-fold enhanced efficiency relative to a 2'-O-Me-PS-RNA oligonucleotide of the same length and sequence. In contrast to 2'-O-Me-PS-RNA, tc-DNA shows antisense activity even in the absence of lipofectamine, albeit only at much higher oligonucleotide concentrations.

Antisense derivatives of U7 and other small nuclear RNAs as tools to modify pre-mRNA splicing patterns

The importance of alternative splicing for the diversity of the proteome and the large number of genetic diseases that are due to splicing defects call for methods to modulate alternative splicing decisions. Although splicing can be modulated by antisense oligonucleotides, this approach is confronted with problems of efficient delivery and the need for repeated administrations of large amounts of the oligonucleotides. Therefore we have developed methods allowing us to modulate splicing with the help of modified derivatives of the U7 small nuclear RNA involved in histone RNA 3' end processing. Its nuclear accumulation as a stable ribonucleoprotein particle makes U7 snRNA especially useful for this purpose. In particular, U7 derivatives containing two tandem antisense sequences directed against targets upstream and downstream of an exon can induce the efficient and specific skipping of that exon. U7 expression cassettes have been successfully introduced into a great number of cell lines, primary cells or tissues with the help of lentiviral and adeno-associated viral vectors. Examples of these therapeutic strategies in the fields of β-thalassemia, Duchenne muscular dytrophy and HIV/AIDS are discussed.

Self-assembly into nanoparticles is essential for receptor mediated uptake of therapeutic antisense oligonucleotides

Antisense oligonucleotides (ASOs) have the potential of revolutionizing medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated with nanoparticles to enhance their stability and cellular uptake; however, one of the biggest challenges is the poor understanding of their uptake mechanism, which is needed for designing better ASOs with high activity and low toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (P-PMO), 2?Omethyl phosphorothioate (2?OMe) and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Deuchenne muscular dystrophy (DMD). We show that P-PMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. P-PMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations P-PMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in-vitro. In-vivo, the activity of P-PMO was significantly decreased in SCARA1 knock-out mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2?OMe as shown by competitive inhibition and co-localization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that P-PMO and tcDNA have higher binding profiles to the receptor compared to 2?OMe. These results demonstrate receptor-mediated uptake for a range of ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles.

Suprasellar chordoid neoplasm with expression of thyroid transcription factor 1: evidence that chordoid glioma of the third ventricle and pituicytoma may form part of a spectrum of lineage-related tumors of the basal forebrain.

Chordoid glioma of the third ventricle is a rare neuroepithelial tumor characterized by a unique histomorphology and exclusive association with the suprasellar/third ventricular compartment. Variously interpreted as either astrocytic- or ependymal-like, and speculatively ascribed to the lamina terminalis/subcommissural organ, its histogenesis remains, nevertheless, unsettled. Here, we report on a suprasellar chordoid glioma occurring in a 52-year-old man. Although displaying otherwise typical morphological features, the tumor was notable for expression of thyroid transcription factor 1, a marker of tumors of pituicytic origin in the context of the sellar region. We furthermore found overlapping immunoprofiles of this example of chordoid glioma and pituicytic tumors (pituicytoma and spindle cell oncocytoma), respectively. Specifically, phosphorylated ribosomal protein S6, a marker of mTOR pathway activation, was expressed in both groups. Based on these findings, we suggest that chordoid glioma and pituicytic tumors may form part of a spectrum of lineage-related neoplasms of the basal forebrain.

Consistent nuclear expression of thyroid transcription factor 1 in subependymal giant cell astrocytomas suggests lineage-restricted histogenesis.

Thyroid transcription factor 1 (TTF-1) is encoded by the NKX2-1 homeobox gene. Besides specifying thyroid and pulmonary organogenesis, it is also temporarily expressed during embryonic development of the ventral forebrain. We recently observed widespread immunoreactivity for TTF-1 in a case of subependymal giant cell astrocytoma (SEGA, WHO grade I) – a defining lesion of the tuberous sclerosis complex (TSC). This prompted us to investigate additional SEGAs in this regard. We found tumor cells in all 7 specimens analyzed to be TTF-1 positive. In contrast, we did not find TTF-1 immunoreactivity in a cortical tuber or two renal angiomyolipomas resected from TSC patients. We propose our finding of consistent TTF-1 expression in SEGAs to indicate lineage-committed derivation of these tumors from a regionally specified cell of origin. The medial ganglionic eminence, ventral septal region, and preoptic area of the developing brain may represent candidates for the origin of SEGAs. Such lineagerestricted histogenesis may also explain the stereotypic distribution of SEGAs along the caudate nucleus in the lateral ventricles.

FUS/TLS contributes to replication-dependent histone gene expression by interaction with U7 snRNPs and histone-specific transcription factors.

Replication-dependent histone genes are up-regulated during the G1/S phase transition to meet the requirement for histones to package the newly synthesized DNA. In mammalian cells, this increment is achieved by enhanced transcription and 3' end processing. The non-polyadenylated histone mRNA 3' ends are generated by a unique mechanism involving the U7 small ribonucleoprotein (U7 snRNP). By using affinity purification methods to enrich U7 snRNA, we identified FUS/TLS as a novel U7 snRNP interacting protein. Both U7 snRNA and histone transcripts can be precipitated by FUS antibodies predominantly in the S phase of the cell cycle. Moreover, FUS depletion leads to decreased levels of correctly processed histone mRNAs and increased levels of extended transcripts. Interestingly, FUS antibodies also co-immunoprecipitate histone transcriptional activator NPAT and transcriptional repressor hnRNP UL1 in different phases of the cell cycle. We further show that FUS binds to histone genes in S phase, promotes the recruitment of RNA polymerase II and is important for the activity of histone gene promoters. Thus, FUS may serve as a linking factor that positively regulates histone gene transcription and 3' end processing by interacting with the U7 snRNP and other factors involved in replication-dependent histone gene expression.

Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations.

We have used three beta-thalassemic mutations, IVS2-654, -705 and -745, that create aberrant 5' splice sites (5' ss) and activate a common cryptic 3' ss further upstream in intron 2 of the human beta-globin gene to optimize a generally applicable exon-skipping strategy using antisense derivatives of U7 small nuclear RNA (snRNA). Introducing a modified U7 snRNA gene carrying an antisense sequence against the cryptic 3' ss into cultured cells expressing the mutant beta-globin genes, restored correct beta-globin mRNA splicing for all three mutations, but the efficiency was much weaker for IVS2-654 than for the other mutations. The length of antisense sequence influenced the efficiency with an optimum of approximately 24 nucleotides. Combining two antisense sequences directed against different target sites in intron 2, either on separate antisense RNAs or, even better, on a single U7 snRNA, significantly enhanced the efficiency of splicing correction. One double-target U7 RNA was expressed on stable transformation resulting in permanent and efficient suppression of the IVS2-654 mutation and production of beta-globin. These results suggest that forcing the aberrant exon into a looped secondary structure may strongly promote its exclusion from the mRNA and that this approach may be used generally to induce exon skipping.

Sensitivity of splice sites to antisense oligonucleotides in vivo.

A series of HeLa cell lines which stably express beta-globin pre-mRNAs carrying point mutations at nt 654, 705, or 745 of intron 2 has been developed. The mutations generate aberrant 5' splice sites and activate a common 3' cryptic splice site upstream leading to aberrantly spliced beta-globin mRNA. Antisense oligonucleotides, which in vivo blocked aberrant splice sites and restored correct splicing of the pre-mRNA, revealed major differences in the sensitivity of these sites to antisense probes. Although the targeted pre-mRNAs differed only by single point mutations, the effective concentrations of the oligonucleotides required for correction of splicing varied up to 750-fold. The differences among the aberrant 5' splice sites affected sensitivity of both the 5' and 3' splice sites; in particular, sensitivity of both splice sites was severely reduced by modification of the aberrant 5' splice sites to the consensus sequence. These results suggest large differences in splicing of very similar pre-mRNAs in vivo. They also indicate that antisense oligonucleotides may provide useful tools for studying the interactions of splicing machinery with pre-mRNA.

Gas-phase fragmentation and conformation of the sugar-modified antisense oligonucleotide tcDNA

Tricyclo-DNA (tcDNA) is a sugar- and backbone-modified analogue of DNA that is currently tested as antisense oligonucleotide for the treatment of Duchenne muscular dystrophy. The name tricyclo-DNA is derived from the modified sugar-moiety: the deoxyribose is extended to a three-membered ring system. This modification is designed to limit the flexibility of the structure, thus giving rise to entropically stabilized hybrid duplexes formed between tcDNA and complementary DNA or RNA oligonucleotides. While the structural modifications increase the biostability of the therapeutic agent, they also render the oligonucleotide inaccessible to enzyme-based sequencing methods. Tandem mass spectrometry constitutes an alternative sequencing technique for partially and fully modified oligonucleotides. For reliable sequencing, the fragmentation mechanism of the structure in question must be understood. Therefore, the presented work evaluates the effect of the modified sugar-moiety on the gas-phase dissociation of single stranded tcDNA. Moreover, our experiments reflect the exceptional gas-phase stability of hybrid duplexes that is most noticeable in the formation of truncated duplex ions upon collision-induced dissociation. The stability of the duplex arises from the modified sugar-moiety, as the rigid structure of the tcDNA single strand minimizes the change of the entropy for the annealing. Moreover, the tc-modification gives rise to extended conformations of the nucleic acids in the gas-phase, which was studied by ion mobility spectrometry-mass spectrometry.

Structural elucidation and antisense properties of a sugar-modified DNA analogue

Antisense oligonucleotides deserve great attention as potential drug candidates for the treatment of genetic disorders. For example, muscle dystrophy can be treated successfully in mice by antisense-induced exon skipping in the pre-mRNA coding for the structural protein dystrophin in muscle cells. For this purpose a sugar- and backbone-modified DNA analogue was designed, in which a tricyclic ring system substitutes the deoxyribose. These chemical modifications stabilize the dimers formed with the targeted RNA relative to native nucleic acid duplexes and increase the biostability of the antisense oligonucleotide. While evading enzymatic degradation constitutes an essential property of antisense oligonucleotides for therapeutic application, it renders the oligonucleotide inaccessible to biochemical sequencing techniques and requires the development of alternative methods based on mass spectrometry. The set of sequences studied includes tcDNA oligonucleotides ranging from 10 to 15 nucleotides in length as well as their hybrid duplexes with DNA and RNA complements. All samples were analyzed on a LTQ Orbitrap XL instrument equipped with a nano-electrospray source. For tandem mass spectrometric experiments collision-induced dissociation was performed, using helium as collision gas. Mass spectrometric sequencing of tcDNA oligomers manifests the applicability of the technique to substrates beyond the scope of enzyme-based methods. Sequencing requires the formation of characteristic backbone fragments, which take the form of a-B- and w-ions in the product ion spectra of tcDNA. These types of product ions are typically associated with unmodified DNA, which suggests a DNA-like fragmentation mechanism in tcDNA. The loss of nucleobases constitutes the second prevalent dissociation pathway observed in tcDNA. Comparison of partially and fully modified oligonucleotides indicates a pronounced impact of the sugar-moiety on the base loss. As this event initiates cleavage of the backbone, the presented results provide new mechanistic insights into the fragmentation of DNA in the gas-phase. The influence of the sugar-moiety on the dissociation extends to tcDNA:DNA and tcDNA:RNA hybrid duplexes, where base loss was found to be much more prominent from sugar-modified oligonucleotides than from their natural complements. Further prominent dissociation channels are strand separation and backbone cleavage of the single strands, as well as the ejection of backbone fragments from the intact duplex. The latter pathway depends noticeably on the base sequence. Moreover, it gives evidence of the high stability of the hybrid dimers, and thus directly reflects the affinity of tcDNA for its target in the cell. As the cellular target of tcDNA is a pre-mRNA, the structure was designed to discriminate RNA from DNA complements, which could be demonstrated by mass spectrometric experiments.

The Rice Transcription Factor WRKY53 Suppresses Herbivore-Induced Defenses by Acting as a Negative Feedback Modulator of Mitogen-Activated Protein Kinase Activity

The mechanisms by which herbivore-attacked plants activate their defenses are well studied. By contrast, little is known about the regulatory mechanisms that allow them to control their defensive investment and avoid a defensive overshoot. We characterized a rice (Oryza sativa) WRKY gene, OsWRKY53, whose expression is rapidly induced upon wounding and induced in a delayed fashion upon attack by the striped stem borer (SSB) Chilo suppressalis. The transcript levels of OsWRKY53 are independent of endogenous jasmonic acid but positively regulated by the mitogen-activated protein kinases OsMPK3/OsMPK6. OsWRKY53 physically interacts with OsMPK3/OsMPK6 and suppresses their activity in vitro. By consequence, it modulates the expression of defensive, MPK-regulated WRKYs and thereby reduces jasmonic acid, jasmonoyl-isoleucine, and ethylene induction. This phytohormonal reconfiguration is associated with a reduction in trypsin protease inhibitor activity and improved SSB performance. OsWRKY53 is also shown to be a negative regulator of plant growth. Taken together, these results show that OsWRKY53 functions as a negative feedback modulator of MPK3/MPK6 and thereby acts as an early suppressor of induced defenses. OsWRKY53 therefore enables rice plants to control the magnitude of their defensive investment during early signaling.

STRUCTURAL ANALYSIS OF THE TRANSCRIPTION PRODUCTS OF A TEMPERATURE-SENSITIVE PHENOTYPIC MUTANT OF MOLONEY SARCOMA VIRUS--MUSV TS110

Cells infected with a temperature sensitive phenotypic mutant of Moloney sarcoma virus (MuSVts110) exhibit a transformed phenotype at 33('(DEGREES)) and synthesize two virus specific proteins, p85('gag-mos), a gag-mos fusion protein and p58('gag), a truncated gag precursor protein (the gag gene codes for viral structural proteins and mos is the MuSV transforming gene). At 39('(DEGREES)) only p58('gag) is synthesized and the morphology of the cells is similar to uninfected NRK parental cells. Two MuSVts110 specific RNAs are made in MuSVts110-infected cells, one of 4.0 kb in length, the other of 3.5 kb. Previous work indicated that each of these RNAs arose by a single central deletion of parental MuSV genetic material, and that p58('gag) was made by the 4.0 kb RNA and p85('gag-mos) from the 3.5 kb RNA. The objective of my dissertation research was to map precisely the deletion boundaries of both of the MuSVts110 RNAs, and to determine the proper reading frame across both deletion borders. This work succeeded in arriving at the following conclusions: (a) Using S-1 nuclease analysis and primer extension sequencing, it was found that the 4.0 kb MuSVts110 RNA arose by a 1488 base deletion of 5.2 kb parental MuSV genomic RNA. This deletion resulted in an out of frame fusion of the gag and mos genes that resulted in the formation of a "stop" codon which causes termination of translation just beyond the c-terminus of the gag region. Thus, this RNA can only be translated into the truncated gag protein p58('gag). (b) S-1 analysis of RNA from cells cultivated at different temperatures demonstrated that the 4.0 kb RNA was synthesized at all temperatures but that synthesis of the 3.5 kb RNA was temperature sensitive. These observations supported the data derived from blot hybridization experiments the interpretation of which argued for the existence of a single provirus in MuSVts110 infected cells, and hence only a single primary transcript (the 4.0 kb RNA). (c) Analyses similar to those described in (a) above showed that the 3.5 kb RNA was derived from the 4.0 kb MuSVts110 RNA by a further deletion of 431 bases, fusing the gag and mos genes into a continuous reading frame capable of directing synthesis of the p85('gag-mos) protein. These sequence data and the presence of only one MuSVts110-specific provirus, indicate that a splice mechanism is employed to generate the 3.5 kb RNA since the gag and mos genes are observed to be fused in frame in this RNA. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Ovarian cancer angiogenesis, biology and therapy

The progressive growth of epithelial ovarian cancer tumor is regulated by proangiogenic molecules and growth factors released by tumor cells and the microenvironment. Previous studies showed that the expression of interleukin-8 (IL-8) directly correlates with the progression of human ovarian carcinomas implanted into the peritoneal cavity of nude mice. We examined the expression level of IL-8 in archival specimens of primary human ovarian carcinoma from patients undergoing curative surgery by in situ mRNA hybridization technique. The expression of IL-8 was significantly higher in patients with stage III disease than in patients with stage I disease. To investigate the role of IL-8 in the progressive growth of ovarian cancer, we isolated high- and low-IL-8 producing clones from parental Hey-A8 human ovarian cancer cells, and compared their proliferative activity and tumorigenicity in nude mice. The effect of exogenous IL-8 and IL-8 neutralizing antibody on ovarian cancer cell proliferation was investigated. Finally, we studied the modulation of IL-8 expression in ovarian cancer cells by sense and antisense IL-8 expression vector transfection and its effect on proliferation and tumorigenicity. We concluded that IL-8 has a direct growth potentiating activity in human ovarian cancer cells. ^ The expression level of IL-8 directly correlates with disease progression of human ovarian cancer, but the mechanism of induction is unknown. Since hypoxia and acidic pH are common features in solid tumors, we determined whether hypoxic and acidic conditions could regulate the expression of IL-8. Culturing the human ovarian cancer cells in hypoxic or acidic medium led to a significant increase in IL-8 mRNA and protein. Hypoxic- and acidosis-mediated transient increase in IL-8 expression involved both transcriptional activation of the IL-8 gene and enhanced stability of the IL-8 mRNA. Furthermore, we showed that IL-8 transcription activation by hypoxia or acidosis required the cooperation of NF-κB and AP-1 binding sites. ^ Finally, we studied novel therapies against human ovarian cancer. First, we determined whether inhibition of the catalytic tyrosine kinase activity of the receptors for vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) inhibits the formation of malignant ascites and the progressive growth of human ovarian carcinoma cells implanted into the peritoneal cavity of nude mice. Our results suggest that blockade of the VEGF/VPF receptor may be an efficient strategy to inhibit formation of malignant ascites and growth of VEGF/VPF-dependent human ovarian carcinomas. Secondly, we determined whether local sustained production of murine interferon-β could inhibit the growth of human ovarian cancer cells in the peritoneal cavity of nude mice. Our results showed that local production of IFN-β could inhibit the in vivo growth of human ovarian cancer cells by upregulating the expression of the inducible nitric oxide synthase (NOS) in host macrophages. ^

Alteration of CEACAM1 gene transcription in prostate cancer cells: The role of AR, Sp2, and HDAC

Cell to cell adhesion molecule (CEACAM1), a type II tumor suppressor, has been found to be down-regulated in prostate cancer cells. The mechanism that causes CEACAM1's down-regulation in tumorigenesis is unknown. Here we show that the transcriptional activity of CEACAM1 is decreased in prostate cancer cells. This decrease is not due to methylation of the CEACAM1's promoter, but rather to the alteration of transcription factors regulating CEACAM1 expression. ^ Since androgen/androgen receptors (AR) are potent regulators of prostate growth and differentiation, their role on CEACAM1 gene transcription was examined. The androgen receptor could directly increase CEACAM1 transcriptional activity in a ligand dependent manner by interacting with an AR consensus element that resides in the CEACAM1 promoter. However, AR binding to the CEACAM1 promoter is not related to the loss of CEACAM1 during prostate cancer progression. ^ Further analysis enabled us to determine the particular region in the CEACAM1 promoter that mediates a decrease in CEACAM1 transcriptional activity in prostate cancer cells. Upon further examination, we found that this CEACAM1 promoter region interacts with the Sp1, Sp2, and Sp3 transcription factors. However, only Sp2 expression was found to increase in prostate cancer cells. Inhibiting Sp2 from binding to the CEACAM1 promoter caused an increase in CEACAM1 transcriptional activity in prostate cancer cells. In addition, over-expressing Sp2 in normal prostate cells resulted in a decrease in CEACAM1 transcriptional activity and endogenous protein expression. These observations suggest that Sp2 is a transcription repressor of CEACAM1. Furthermore, prostate cancer cells treated with trichostatin A (TSA), a specific histone deacetylase (HDAC) inhibitor, activated CEACAM1 transcriptional activity. This implies that HDACs are involved in CEACAM1 transcriptional activity. Mutation of the Sp2 DNA binding region on the CEACAM1 promoter inhibited TSA activation of CEACAM1 transcriptional activity. This indicates that HDACs inhibit CEACAM1 transcriptional activity through Sp2. Base on these results, we propose that Sp2 is critical for down-regulating CEACAM1 expression, and one mechanism by which Sp2 represses CEACAM1 expression is by recruiting HDAC to the CEACAM1 promoter in prostate cancer cells. Collectively, these findings provide novel insights into mechanisms that cause the down-regulation of CEACAM1 expression in prostate cancer cells. ^