Combinatorial ASO-mediated therapy with low dose SMN and the protective modifier Chp1 is not sufficient to ameliorate SMA pathology hallmarks.

Spinal muscular atrophy (SMA) is a devastating genetically inherited neuromuscular disorder characterized by the progressive loss of motor neurons in the spinal cord, leading to muscle atrophy and weakness. Although SMA is caused by homozygous mutations in SMN1, the disease severity is mainly determined by the copy number of SMN2, an almost identical gene that produces ~10% correctly spliced SMN transcripts. Recently, three FDA- and EMA-approved therapies that either increase correctly spliced SMN2 transcripts (nusinersen and risdiplam) or replace SMN1 (onasemnogen abeparvovec-xioi) have revolutionized the clinical outcome in SMA patients. However, for severely affected SMA individuals carrying only two SMN2 copies even a presymptomatic therapy might be insufficient to fully counteract disease development. Therefore, SMN-independent compounds supporting SMN-dependent therapies represent a promising therapeutic approach. Recently, we have shown a significant amelioration of SMA disease hallmarks in a severely affected SMA mouse carrying a mutant Chp1 allele when combined with low-dose of SMN antisense oligonucleotide (ASO) treatment. CHP1 is a direct interacting partner of PLS3, a strong protective modifier of SMA. Both proteins ameliorate impaired endocytosis in SMA and significantly restore pathological hallmarks in mice. Here, we aimed to pharmacologically reduce CHP1 levels in an ASO-based combinatorial therapy targeting SMN and Chp1. Chp1 modulation is a major challenge since its genetic reduction to ~50% has shown to ameliorate SMA pathology, while the downregulation below that level causes cerebellar ataxia. Efficacy and tolerability studies determined that a single injection of 30 µg Chp1-ASO4 in the CNS is a safe dosage that significantly reduced CHP1 levels to ~50% at postnatal day (PND)14. Unfortunately, neither electrophysiological predictors such as compound muscle action potential (CMAP) or motor unit number estimation (MUNE) nor histological hallmarks of SMA in neuromuscular junction (NMJ), spinal cord or muscle were ameliorated in SMA mice treated with Chp1-ASO4 compared to CTRL-ASO at PND21. Surprisingly, CHP1 levels were almost at control level 4-weeks post injection, indicating a rather short-term effect of the ASO. Therefore, we re-administrated Chp1-ASO4 by i.c.v. bolus injection at PND28. However, no significant improvement of SMA hallmarks were seen at 2 month-of-age either. In conclusion, in contrast to the protective effect of genetically-induced Chp1 reduction on SMA, combinatorial therapy with Chp1- and SMN-ASOs failed to significantly ameliorate the SMA pathology. Chp1-ASOs compared to SMN-ASO proved to have rather short-term effect and even reinjection had no significant impact on SMA progression, suggesting that further optimization of the ASO may be required to fully explore the combination.

Inhibition of survivin expression suppresses the growth of aggressive non-Hodgkin's lymphoma.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family and functions both as an apoptosis inhibitor and a regulator of cell division. Survivin overexpression is common in many human tumors and correlates with survival in large cell non-Hodgkin's lymphoma. To evaluate this molecule as a potential therapeutic target in large-cell lymphoma, we evaluated the effect of survivin inhibition both in vitro and in vivo. Using an antisense oligonucleotide (ASO) approach, cell growth was significantly inhibited in the DoHH2, RL and HT lymphoma cell lines. In a lymphoma xenograft model, the development of tumors as well as the growth of established tumors was inhibited in the survivin ASO-treated mice compared to controls. To assess the efficacy of the survivin ASO in combination with other biological agents, we combined the survivin ASO with an anti-CD20 monoclonal antibody, rituximab. The effect of survivin ASO and rituximab in combination was additive in vitro. In vivo, however, suppression of tumor growth with the combination was not significantly superior to controls. We conclude that inhibition of survivin expression is an attractive therapeutic strategy in aggressive non-Hodgkin's lymphomas, and that combining survivin ASO with rituximab may enhance the efficacy of this approach.

Cytosolic delivery of antisense oligonucleotides by listeriolysin O-containing liposomes.

Antisense oligodeoxynucleotides (ODNs) possess great potential as sequence-specific therapeutic agents. Sufficient concentrations of intact ODN must bypass membrane barriers and access the cytosol and nucleus, for ODNs to be therapeutically effective. A cytosolic delivery strategy was designed to improve the efficiency of ODN delivery in bone-marrow-derived macrophages. This liposome-based formulation utilizes listeriolysin O (LLO), the endosomolytic hemolysin from Listeria monocytogenes, to mediate the escape of ODN from endocytic compartments into the cytosol. To monitor the cytosolic delivery of ODN, subcellular trafficking of fluorescently labeled ODNs was visualized using epifluorescence microscopy. The expression of target protein and mRNA after delivery was measured using flow cytometry and Northern blot analysis, respectively. ODN specific for murine intercellular adhesion molecule-1 (ICAM-1) encapsulated in LLO-liposomes was released to the cytosol and trafficked to the nucleus, efficiently and specifically suppressing activation-induced expression of ICAM-1 at both protein and mRNA levels. Delivery without LLO resulted in sequestration of ODN in vesicular compartments leading to little inhibition of ICAM-1 expression, which supports the requirement of LLO for efficient cytosolic delivery using this system. The data clearly demonstrate that LLO-mediated escape of ODN from intracellular vesicles is an effective approach to achieve full therapeutic antisense activity in cultured macrophages.

The CATH database: an extended protein family resource for structural and functional genomics.

The CATH database of protein domain structures (http://www.biochem.ucl.ac.uk/bsm/cath_new) currently contains 34 287 domain structures classified into 1383 superfamilies and 3285 sequence families. Each structural family is expanded with domain sequence relatives recruited from GenBank using a variety of efficient sequence search protocols and reliable thresholds. This extended resource, known as the CATH-protein family database (CATH-PFDB) contains a total of 310 000 domain sequences classified into 26 812 sequence families. New sequence search protocols have been designed, based on these intermediate sequence libraries, to allow more regular updating of the classification. Further developments include the adaptation of a recently developed method for rapid structure comparison, based on secondary structure matching, for domain boundary assignment. The philosophy behind CATHEDRAL is the recognition of recurrent folds already classified in CATH. Benchmarking of CATHEDRAL, using manually validated domain assignments, demonstrated that 43% of domains boundaries could be completely automatically assigned. This is an improvement on a previous consensus approach for which only 10-20% of domains could be reliably processed in a completely automated fashion. Since domain boundary assignment is a significant bottleneck in the classification of new structures, CATHEDRAL will also help to increase the frequency of CATH updates.

ICAM-1 and VCAM-1 antisense oligonucleotides attenuate in vivo leucocyte adherence and inflammation in rat inflammatory bowel disease.

BACKGROUND: Recruitment of circulating cells to the inflamed intestine is modulated by adhesion molecules expressed on the surface of both leucocytes and endothelial cells. AIMS: The objective of this study was to test whether 2'-O-methoxyethyl chimeric antisense oligonucleotides directed against endothelial intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) can downregulate leucocyte-endothelial interactions and thereby attenuate inflammation in rat experimental ileitis. METHODS: Indomethacin (7.5 mg/kg ) was injected subcutaneously into Sprague-Dawley rats 48 and 24 hours prior to intravital microscopy. Animals were treated with either ICAM-1 (ISIS 17470), VCAM-1 (ISIS 18155), or scrambled control antisense oligonucleotides administered subcutaneously or intravenously in parallel with indomethacin. Leucocyte trafficking was observed in ileal submucosal collecting venules. Macroscopic and histological grades of inflammation were measured 48 hours after the first indomethacin application. ICAM-1 and VCAM-1 expression in ileal submucosal venules was detected by immunohistochemistry. RESULTS: Intravenous administration of ICAM-1 oligonucleotides 2 mg/kg (rolling leucocytes 5.7 (2.4)/0.01 mm(2) endothelial surface, adherent leucocytes 0.8 (1.1)) and VCAM-1 oligonucleotides 8 mg/kg (9.2 (4.4), 0.6 (0.8)) significantly reduced leucocyte adhesion compared with diseased controls (27.8 (5.3), 14 (4.4)) in a dose dependent manner whereas subcutaneous treatment did not. Correspondingly, macroscopic and histological inflammation was significantly decreased. ICAM-1 oligonucleotides markedly reduced endothelial ICAM-1 expression while VCAM-1 oligonucleotides clearly diminished endothelial VCAM-1 expression. CONCLUSIONS: Both ICAM-1 and VCAM-1 2'-O-methoxyethyl chimeric antisense oligonucleotides attenuate rat ileitis by downregulation of leucocyte adherence and thus are potential candidates for anti-inflammatory treatment in inflammatory bowel disease.

Amelioration of chronic and spontaneous intestinal inflammation with an antisense oligonucleotide (ISIS 9125) to intracellular adhesion molecule-1 in the HLA-B27/beta2 microglobulin transgenic rat model.

Adhesion molecules are known to be an important part of leukocyte migration and extravasation in both homeostatic and inflammatory conditions. Intracellular adhesion molecule-1 (ICAM-1 or CD54) is constitutively expressed on endothelial cells and is up-regulated during acute and chronic inflammation. We investigated the efficacy and consequences of interfering with CD54 after administration of an antisense oligonucleotide to ICAM-1 (CD54) in the transgenic HLA-B27/beta2 microglobulin rat model. One hundred percent of the HLA-B27 transgene + animals will spontaneously develop chronic inflammation (some more severely than others) in the gastric mucosa, cecum, and colon. We carried out two studies, i.p. injection and rectal administration of antisense. Following i.p. and rectal treatment, there were significant decreases in colonic mucosal wall thickness, histologic inflammation, CD54 expression in the colon and peripheral blood, and the percentage of colon weight per end body weight. Furthermore, decreased expression of CD49d, CD18, and tumor necrosis factor-alpha was observed in antisense treated rats. Therefore, the HLA-B27 transgenic model of spontaneous and chronic inflammatory bowel disease, which has increased expression of adhesion molecules, responds to both routes of administration of ICAM-1 antisense oligonucleotides. These studies support the regulatory role of adhesion molecules in chronic intestinal inflammation, the need for an understanding of how the route of drug delivery can alter the dose and area affected, and finally the role of antisense oligonucleotides as a therapeutic modality in chronic spontaneous inflammatory bowel diseases.

ETV6/AML1 fusion by FISH in adult acute lymphoblastic leukemia.

Dual-color interphase fluorescence in situ hybridization (FISH) with ETV6 and AML1 probes was used for the first time on a series of 159 adult patients with acute lymphoblastic leukemia (ALL), for detection of the t(12;21)(p13;q22) translocation. Seven patients (4.4%) were found, with 50-100% of positive cells, of whom one of two tested, proved negative for the fusion product by RT-PCR. Two of them, aged 43 and 50 years, are the oldest patients so far confirmed to have the translocation. Three who relapsed at 10, 11 and 24 months, suggest that adults may not enjoy the good short-term prognosis reported for t(12;21)-positive children. Thirty-one-negative cases had signal numbers differing from the two expected for each gene. In 15 cases these results were consistent with the karyotype. In nine cases with uninformative cytogenetics, the numbers were consistent with those for centromeres and indicated a hidden aneuploidy. Loss of ETV6 genes in two cases and AML1 amplification in three others were not suspected from the cytogenetics. In conclusion, FISH proved to be reliable in defining ETV6/AML1 positivity in this group of patients as well as providing valuable insights into negative cases.

Selective inhibition of IL-2 gene expression by IL-2 antisense oligonucleotides blocks heart allograft rejection.

PURPOSE: We tested the effects of selective inhibition of interleukin (IL)-2 gene expression by IL-2 antisense oligonucleotide (oligo) with phosphorothioate (PS)/phosphodiester (PD)/2'-methoxyethyl (ME) modifications (17359) on T-cell function and the survival of heart allografts in mice. METHODS: The PS- (17328) or PS/PD/ME- (17359) IL-2 oligo was electroporated to mouse T cell lymphoma cells (TIB 155) stimulated with concanavalin A (Con A). Expression of IL-2 was analyzed by an ELISA spot assay and a reverse transcript polymerase chain reaction method. C3H (H-2k) mice transplanted with BALB/c (H-2d) heart grafts were treated i.v. with a 7-day osmotic pump with 20 mg/kg 17359 alone or in combination with sirolimus (SRL). RESULTS: In comparison with untreated controls, 500 to 2000 nM 17328 inhibited IL-2 protein production by 21.8% to 47.2%, whereas 500 to 2000 nM 17359 did so by 35.5% to 83.5% (both P<0.001). In vivo, 20 mg/kg 17359 prolonged survivals to a mean survival time (MST) of 18.3+/-2.6 days (P<0.001) in comparison with only 8.2+/-0.8 days in untreated controls. Although 0.2 mg/kg SRL alone produced a MST of 18.8+/-6.0 days (P<0.01), addition of 20 mg/kg 17539 synergistically extended survivals to 54.3+/-12.1 days (P<0.001). As expected, IL-2 mRNA, but not IL-7, IL-9, or IL-15 mRNA, was reduced in allografts from recipients treated with 17359 compared with untreated controls. Lymph node cells from the same recipients displayed reduction in proliferative response to donor alloantigen and in generation of alloantigen-specific cytotoxic T cells. CONCLUSION: Selective inhibition of IL-2 mRNA in vivo inhibits T-cell function and extends allograft survival.

Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting.

The protective genes that mediate endothelial cell (EC) survival during angiogenesis have not been completely characterized. Here, we show that an antisense oligonucleotide to the apoptosis inhibitor survivin suppressed de novo expression of survivin in ECs by vascular endothelial cell growth factor (VEGF). In contrast, the survivin antisense oligonucleotide did not affect anti-apoptotic bcl-2 levels in endothelium. When assessed in cell death assays, antisense targeting of survivin abolished the anti-apoptotic function of VEGF against tumor necrosis factor-alpha- or ceramide-induced cell death, enhanced caspase-3 activity, promoted the generation of a approximately 17-kd active caspase-3 subunit, and increased cleavage of the caspase substrate, polyADP ribose polymerase. In contrast, the survivin antisense oligonucleotide had no effect on EC viability in the absence of VEGF. Antisense oligonucleotides to platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), lymphocyte function-associated molecule-3 (LFA-3, CD58), or intercellular adhesion molecule-1 (ICAM-1, CD54) did not reduce the anti-apoptotic function of VEGF in endothelium. When tested on other angiogenic activities mediated by VEGF, survivin antisense treatment induced rapid regression of three-dimensional vascular capillary networks, but did not affect EC migration/chemotaxis. These data suggest that the anti-apoptotic properties of VEGF during angiogenesis are primarily mediated by the induced expression of survivin in ECS: Manipulation of this pathway may increase EC viability in compensatory angiogenesis or facilitate EC apoptosis and promote vascular regression during tumor angiogenesis.

ADAM17 but not ADAM10 mediates tumor necrosis factor-alpha and L-selectin shedding from leukocyte membranes.

The release of tumor necrosis factor-alpha (TNF-alpha) from cellular membranes has been shown by different laboratories to be controlled by a disintegrin and metalloprotease, ADAM10 or ADAM17. In contrast, only ADAM17 has shown to be involved in L-selectin shedding. To determine the specific roles of ADAM10 and ADAM17 in the processing of TNF-alpha and L-selectin shedding, antisense oligonucleotides (ASO) targeting both ADAM10 and ADAM17 were identified. We show that ISIS 16337 reduces ADAM17 mRNA and ISIS 100750 reduces ADAM10 mRNA in a sequence-specific and dose-dependent manner in both Jurkat and THP-1 cells. The ADAM17 ASO (ISIS 16337) inhibited both TNF-alpha secretion in THP-1 cells and L-selectin shedding in Jurkat cells, whereas the ADAM10 ASO (ISIS 100750) did not significantly inhibit release of either protein. These results suggest that ADAM17 is one of the major metalloproteases involved in L-selectin shedding as well as TNF-alpha processing. The biologic substrates for ADAM10 in Jurkat and THP-1 cells remain to be elucidated.

A constitutive cytoprotective pathway protects endothelial cells from lipopolysaccharide-induced apoptosis.

Lipopolysaccharide (LPS) has been implicated as the bacterial component responsible for much of the endothelial cell injury/dysfunction associated with Gram-negative bacterial infections. Protein synthesis inhibition is required to sensitize the endothelium to lipopolysaccharide-induced apoptosis, suggesting that a constitutive or inducible cytoprotective protein(s) is required for endothelial survival. We have identified two known endothelial anti-apoptotic proteins, c-FLIP and Mcl-1, the expression of which is decreased markedly in the presence of cycloheximide. Decreased expression of both proteins preceded apoptosis evoked by lipopolysaccharide + cycloheximide. Caspase inhibition protected against apoptosis, but not the decreased expression of c-FLIP and Mcl-1, suggesting that they exert protection upstream of caspase activation. Inhibition of the degradation of these two proteins with the proteasome inhibitor, lactacystin, prevented lipopolysaccharide + cycloheximide-induced apoptosis. Similarly, lactacystin protected against endothelial apoptosis induced by either tumor necrosis factor-alpha or interleukin-1beta in the presence of cycloheximide. That apoptosis could be blocked in the absence of new protein synthesis by inhibition of the proteasome degradative pathway implicates the requisite involvement of a constitutively expressed protein(s) in the endothelial cytoprotective pathway. Finally, reduction of FLIP expression with antisense oligonucleotides sensitized endothelial cells to LPS killing, demonstrating a definitive role for FLIP in the protection of endothelial cells from LPS-induced apoptosis.

Fully modified 2' MOE oligonucleotides redirect polyadenylation.

Many genes have been described and characterized that have alternative polyadenylation signals at the 3'-end of their pre-mRNAs. Many of these same messages also contain destabilization motifs responsible for rapid degradation of the mRNA. Polyadenylation site selection can thus determine the stability of an mRNA. Fully modified 2'-O:-methoxy ethyl/phosphorothioate oligonucleotides that hybridize to the 3'-most polyadenylation site or signal of E-selectin were able to inhibit polyadenylation at this site and redirect it to one of two upstream cryptic sites. The shorter transcripts produced after antisense treatment have fewer destabilization sequences, increased mRNA stability and altered protein expression. This study demonstrates that antisense oligonucleotides can be successfully employed to redirect polyadenylation. This is the first demonstration of the use of oligonucleotides to increase, rather than decrease, abundance of a message.

Intercellular adhesion molecule-1 suppression in skin by topical delivery of anti-sense oligonucleotides.

We topically applied 20 nucleotide phosphorothioate intercellular adhesion molecule-1 anti-sense oligodeoxynucleotide in a cream formulation. It effectively inhibited tumor necrosis factor-alpha-induced expression of intercellular adhesion molecule-1 in human skin transplanted on severe compromised immunodeficient mice. The effects were concentration dependent, sequence specific, and resulted from reduction of intercellular adhesion molecule-1 mRNA levels in the skin. Intravenous administration of the drug did not show pharmacologic effects, probably due to insufficient drug concentrations in skin. Topical delivery, however, produced a rapid and a significantly higher accumulation of oligodeoxynucleotide in the epidermis and dermis. The results strongly suggest that topically applied anti-sense oligonucleotides can be delivered to target sites in the skin and may be of considerable value in the treatment of psoriasis and other inflammatory skin disorders.

Pharmacokinetic and toxicity profile of a phosphorothioate oligonucleotide following inhalation delivery to lung in mice.

Antisense oligonucleotides are currently being investigated for the treatment of a variety of diseases. Antisense drugs are being administered primarily by parenteral injection. To explore more convenient patient delivery methods, we have characterized the tissue kinetics and tolerability of an inhaled aerosol formulation of a phosphorothioate oligonucleotide in mice. Concentrations of oligonucleotide in bronchioalveolar lavage fluid, plasma, and tissue and immunohistochemical localization were used to assess deposition and pharmacokinetic parameters. Significant concentrations of oligonucleotide in lung, as well as systemic tissues, were measured following a pulmonary dose of 12 mg/kg. Doses as low as 1-3 mg/kg also produced significant concentrations of oligonucleotide (>50 microg oligonucleotide per gram of tissue), and these were maintained in the lung with a halflife of 20 hours or greater. Oligonucleotide was localized to bronchiolar epithelium and alveolar epithelium and endothelium. Toxicity was mild at the 12 mg/kg level and minimal to absent at doses of 3 mg/kg or below. Based on a favorable pharmacokinetic profile and a relative lack of toxicity, inhalation delivery appears to be a therapeutic option for antisense oligonucleotides.

Inhibition of C-raf expression by antisense oligonucleotides extends heart allograft survival in rats.

BACKGROUND: C-raf is a well-characterized serine/ threonine (Ser/Thr) protein kinase that is involved in the transduction of multiple signals of T cells. We demonstrate that the inhibition of C-raf mRNA expression prolongs heart allograft survival. METHODS: Three 20-mer C-raf antisense oligonucleotides, each with identical sequences, were synthesized with different chemical modifications: one as a uniform phosphorothioate oligodeoxynucleotide (PS oligo), a second with a PS backbone and 2'-methoxyethyl (ME) substitutions at the 2'-sugar positions in the first and last five nucleotides, and a third with a mixed PS and phosphodiester (PD) backbone and ME modifications on the first and last five nucleotides. RESULTS: Both ME-modified C-raf antisense oligos were at least 5-fold more effective than the PS C-raf antisense oligo in blocking C-raf mRNA expression in two cell lines. Similarly, each of the ME C-raf antisense oligos produced better heart allograft survival rates than did PS C-raf oligo. Furthermore, although the combination of PS C-raf antisense oligo with sirolimus (SRL) acted synergistically to extend heart allograft survival, the effect was potentiated by either of the ME-modified oligos. CONCLUSIONS: C-raf inhibition extends heart allograft survival, and ME-modification potentiates antisense activity.

Attenuation of cytomegalovirus-induced endothelial intercellular adhesion molecule-1 mRNA/protein expression and T lymphocyte adhesion by a 2'-O-methoxyethyl antisense oligonucleotide.

BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) is strongly induced under inflammatory conditions associated with allograft rejection, thereby promoting leukocyte recruitment and activation at the site of inflammation. Enhancement of ICAM-1 expression can also be the result of viral infection, in particular human cytomegalovirus (CMV), a frequent source of complications in the transplant recipient. In vitro studies have shown that CMV infection of endothelial cells (EC) results in the direct enhancement of ICAM-1 expression and consequent leukocyte adhesion/activation suggesting mechanisms by which CMV exacerbates graft vascular disease. Although treatment of EC with ICAM-1-specific antisense oligonucleotides has been shown to attenuate ICAM-1 induction under simulated inflammatory conditions (i.e., TNF-alpha), no studies have addressed their effectiveness on virally-induced ICAM-1 expression. RESULTS: In the current investigation, we show that the progressive increase in endothelial ICAM-1 protein expression that follows inoculation with CMV correlates with a progressive accumulation of ICAM-1 mRNA. Furthermore, we demonstrate that treatment of EC with a partially 2'-O-methoxyethyl modified ICAM-1-specific antisense oligonucleotide before viral inoculation significantly reduces CMV-associated induction of ICAM-1 protein and mRNA expression. Finally, we show that antisense-mediated attenuation in ICAM-1 expression results in a significant reduction of T lymphocyte adhesion to CMV-infected EC monolayers, an interaction that has been implicated in allogeneic T lymphocyte activation, in viral transmission to transiently adherent leukocytes and subsequent hematogenous dissemination. CONCLUSIONS: These findings demonstrate for the first time that antisense oligonucleotides can effectively reverse virally-induced host cellular protein expression, specifically ICAM-1, as well as consequent T lymphocytes adhesion, thus broadening the potential clinical utility of antisense oligonucleotides.