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Background: The addition of phosphorothioate (PS) groups to natural phosphodiester (PD) antisense oligodeoxynucleotides (oligo) prevents their in vivo hydrolysis by nucleases allowing an RNase-dependent elimination of targeted mRNA. To further improve oligo function 2'-methoxyethyl (ME) groups were attached to selected nucleotides at the 3'-end because ME groups block RNase activity., Methods/results: ME modification of PS- or PD/PS-oligo targeting human intracellular adhesion molecule (ICAM)-1 mRNA significantly increased the degree and duration of the in vitro inhibitory effects without compromising selectivity and specificity. A 7-day intravenous or oral therapy with rat ME/PS-modified ICAM-1 antisense oligo extended the survivals of kidney allografts. In addition, ME/PS-modified ICAM-1 antisense oligo reduced ischemic-reperfusion injury in kidneys, as measured by glomerular filtration rate, creatinine levels, and infiltration with leukocytes. Finally, a 14-day treatment with cyclosporine (CsA)-induced nephrotoxicity in syngeneic kidney transplants correlated with both increased ICAM-1 protein expression and infiltration with leukocytes. Graft perfusion and treatment of recipients with ICAM-1 antisense ME/PS-oligo alleviated the nephrotoxic effect and decreased ICAM-1 expression and leukocyte infiltration., Conclusions: ME/PS-modified ICAM-1 antisense oligo is very effective in inhibiting the ICAM-1-dependent mechanism of graft infiltration and tissue damage involved in allograft rejection, ischemic-reperfusion injury, and CsA-induced nephrotoxicity.

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.

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.

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.

Background: We have previously shown that phosphorothioate intercellular adhesion molecule (ICAM)-1 antisense oligodeoxynucleotide (oligo) IP-9125 blocks the expression of rat ICAM-1 mRNA in rat L2 cells. A single ex situ perfusion of grafts with unformulated IP-9125, suspended in Euro-Collins solution, prolonged the survival of kidney allografts in rats. The present experiments examined whether perfusion of kidneys with unformulated IP-9125 prevents ischemic/reperfusion injury., Methods: Kidneys were perfused ex situ with 2 ml of Euro-Collins solution without or with IP-9125 and exposed to 30-min cold (4 degrees C storage time) and 30-min warm (anastomosis time) ischemia. Kidneys were then transplanted to syngeneic nephrectomized recipients., Results: Within 24 hr after transplantation, the glomerular filtration rate values were reduced by almost 60% to 0.49+/-0.14 ml/min from 1.20+/-0.27 ml/min in normal kidneys (P<0.001). Kidney perfusion with 10 mg of either IP-12140 (0.41+/-0.07 ml/min) or IP-13944 (0.47+/-0.07 ml/min) control oligo was ineffective. In contrast, perfusion with 10 mg of IP-9125 significantly improved kidney function (0.8+/-0.18 ml/min; P<0.005), whereas the lower doses of 2 mg (0.47+/-0.13 ml/min; NS) or 4 mg (0.54+/-0.04 ml/min; NS) had no significant effect. The glomerular filtration rate results were confirmed by measurements of blood creatinine (CR) levels at 24 hr after grafting: untreated recipients had a twofold higher CR value (0.70+/-0.14 mg/dl) compared with normal controls (0.65+/-0.07 mg/dl; P<0.001). Although perfusion with 10 mg of control IP-12140 (0.80+/-0.14 mg/dl) or IP-13944 (0.65+/-0.07 mg/dl) did not affect CR levels, perfusion with 10 mg of IP-9125 (0.45+/-0.07 mg/dl) lowered CR levels. The Western blots or reverse transcription-polymerase chain reaction experiments performed in kidney transplants within 24 hr after grafting showed that 10 mg of IP-9125 (but not control IP-12140) reduced the expression of ICAM-1 protein and ICAM-1 mRNA, respectively., Conclusions: Perfusion of grafts with unformulated ICAM-1 antisense oligo specifically reduces intragraft ICAM-1 protein expression and prevents ischemic/reperfusion injury.

Background: We designed an antisense phosphorothioate oligodeoxynucleotide (oligo) to specifically inhibit the expression of rat intercellular adhesion molecule-1 (ICAM-1) mRNA (IP-9125)., Methods: IP-9125 oligo was delivered intravenously by osmotic pump alone or in combination with cyclosporine (CsA) to recipients in order to prevent the rejection of kidney or heart allografts. In additional experiments, kidney allografts were perfused with IP-9125 before grafting., Results: IP-9125 inhibited ICAM-1 mRNA and ICAM-1 protein expression in rat aortic endothelial cells; scrambled controls IP-12140 and IP-13944 were ineffective. Untreated ACI (RT1a) recipients rejected Lewis (RT1l) kidney allografts at a mean survival time of 8.5+/-1.1 days. A 14-day intravenous administration of 2.5 mg/kg/day IP-9125 prolonged the survival of kidney allografts to 39.2+/-16.4 days; 5.0 mg/kg/day, to 43.0+/-17.5 days; and 10.0 mg/kg/day, to 50.4+/-21.6 days. In contrast, a scrambled control IP-12140 was not effective. A combination of 10 mg/kg/day IP-9125 and 1.0 mg/kg/day CsA delivered for 14 days synergistically extended kidney allograft survival times 88.5+/-7.5 days. In contrast, the combination of 10.0 mg/kg/day control IP-12140 with CsA was ineffective (20.7+/-3.2 days) when compared with CsA alone (20.2+/-4.0 days). Similar results were obtained for heart transplants in recipients treated with IP-9125 alone or in combination with CsA. Furthermore, in situ immunostaining showed that IP-9125 significantly reduced the expression of ICAM-1 protein in kidney allografts. Finally, perfusion of kidney grafts alone with 20.0 mg per 2 ml of IP-9125 protected kidney allografts from rejection (37.5+/-7.5 days; P < 0.001), whereas perfusion with 20 mg per 2 ml of control IP-12140 was ineffective (12.6+/-5.0 days)., Conclusions: Rat ICAM-1 IP-9125 oligo inhibits ICAM-1 protein expression in vitro and in vivo as well as blocks allograft rejection when used for pretreatment of donors, graft perfusion, or postoperative treatment of recipients.