WO2001021837A1

WO2001021837A1 - Methods for potentiating antisense rna techniques

Info

Publication number: WO2001021837A1
Application number: PCT/US2000/025762
Authority: WO
Inventors: Brian Seed; Felipe X. Pimentel-Muinos
Original assignee: The General Hospital Corporation
Priority date: 1999-09-23
Filing date: 2000-09-20
Publication date: 2001-03-29
Also published as: WO2001021837A9; AU7595000A

Abstract

Disclosed herein is a method for potentiating antisense RNA activity, involving joining the antisense RNA to a protein-coding nucleic acid sequence. Also disclosed are nucleic acid sequences that include an antisense RNA joined to a protein-coding sequence, as well as nucleic acids which, when transcribed, generate such antisense products. Shown schematically in the accompanying figure is an engineered version of the yeast Flp site specific recombinase gene which is useful in such gene therapy vectors, whereby Flpe-mediated recombination results in expression of operably linked reporter protein upon expression of the Flpe protein in a host cell containing a Flpe substrate plasmid.

Description

METHODS FOR POTRNTTATTNCT ANTTSRNSK RNA TECHNIQUES

Background of the Invention In general, the invention involves improved antisense RNA techniques and related nucleic acid sequences.

Antisense RNA provides a means for the specific regulation of genes in eukaryotic organisms. In this approach, a single-stranded nucleic acid sequence is generated which is complementary to the coding strand, or mRNA, of a targeted gene. This antisense sequence interacts, by hybridization, with the targeted gene's mRNA and selectively inhibits the production of its protein product, while leaving the expression of other mRNAs in a population essentially unaffected.

Because of the specificity of the hybridization step, the antisense technique provides an extremely selective method for the regulation of gene expression. In addition, this technique may be used to inhibit the production of any protein for which the sequence of the coding strand has been determined. As a result of its universal applicability, the antisense approach has been used to regulate the genes of a significant number of different organisms, including mammalian, viral, and plant species (see, for example, Gene Regulation: Biology of Antisense RNA and DNA, ed., Erickson and Izant, Raven Press Ltd., New York, 1992; Antisense RNA and DNA, Wiley-Liss, Inc. 1992).

Summary of the Invention In general, applicants' invention features a novel means for enhancing the efficiency of any antisense technique. This improvement stems from applicants' surprising discovery that the inclusion of a protein-coding sequence in an antisense RNA transcript results in significantly greater inhibition of targeted genes. As described in more detail below, the exemplary protein-coding sequences utilized herein derive from green or blue fluorescent protein genes, but any open reading frame may be used for this purpose. Preferably, the open reading frame encodes a stable protein (i.e., a protein which is substantially non-thermal labile) and/or one in which at least 50% of the nascent protein chain is properly folded into a normal three-dimensional configuration. Any number of useful protein-coding sequences may be identified, for example, by cloning candidate cDNAs upstream of an antisense sequence known to reduce target gene expression, and then selecting out cells harboring cDNA inserts that increase the effectiveness of the antisense construct.

In addition, the present antisense RNA constructs preferably do not include RNA transcription or processing signals located between the antisense RNA and the protein-coding sequences. Specifically, polyA signals and transcriptional terminators (for example, a run of Ts followed by AT) should be avoided at internal positions within the transcript and particularly between the antisense and protein-encoding sequences. Moreover, mRNA instability signals (for example, ATTTA) preferably should be excluded entirely from the transcript. The orientation of the antisense RNA with respect to the protein- coding sequence within the transcript is not essential to the present method. The antisense RNA may be positioned either upstream or downstream of the protein-coding sequence, although constructs containing the antisense RNA positioned downstream of the protein-coding sequence are preferred. If desired, to further enhance the effectiveness of the antisense constructs described herein, mRNA stability sequences (for example, the 3' mRNA stability sequence from human growth hormone) may be included in applicants' constructs, typically at the 3' end. The 3' end of the transcripts (that is, the portion of the transcript downstream from both the antisense and the protein-encoding sequences) may also preferably include 3 '-untranslated regions and polyA signals. Intron sequences from any gene may also be included in the present antisense constructs to encourage export of the transcript from the nucleus. Typically, intron sequences are positioned upstream of, and relatively close to, the open reading frame sequence, although these sequences are functional anywhere in the antisense construct.

The effectiveness of the antisense methods described herein may be further optimized by additional techniques for increasing the amount or stability of antisense RNAs, including the use of highly efficient promoters and polyA signals for antisense production and processing.

The technique described herein may be used to potentiate any antisense technique and may be utilized for the inhibition of any gene, including any eukaryotic (for example, mammalian), viral, or plant or lower protist gene. In addition, the constructs described herein may be co-delivered with other constructs for the purpose of monitoring the effectiveness of antisense-mediated inhibition of target gene expression. In one particular example, the constructs described herein may be co-delivered with reporter constructs for identifying cells in which expression of a target gene has been largely or completely abrogated. Such reporter constructs preferably include, in tandem, the coding sequence for the target gene positioned downstream from a gene encoding a detectable marker (for example, red fluorescent protein (RFP) or a cell surface protein for which a monoclonal antibody is available or can be generated by standard techniques). The cells in which the antisense is effective may be visualized or selected as a result of the decreased expression of the reporter construct. Accordingly, as claimed below, the present invention features a method for potentiating antisense RNA activity, involving joining the antisense RNA to a protein-coding nucleic acid sequence.

In preferred embodiments, the protein-coding sequence encodes a stable protein, and the antisense RNA is positioned downstream of the protein- coding sequence.

In a related aspect, the invention features a method for generating an antisense RNA, involving expressing a transcript which includes (i) the antisense RNA and (ii) a protein- coding sequence, whereby the transcript exhibits enhanced antisense activity relative to the antisense RNA in the absence of the protein-coding sequence.

In preferred embodiments, the protein-coding sequence encodes a stable protein, and the antisense RNA is positioned downstream of the protein- coding sequence. In another related aspect, the invention features a nucleic acid including an antisense RNA joined to a protein- coding nucleic acid sequence.

In preferred embodiments, the protein-coding nucleic acid sequence encodes a stable protein; the antisense RNA is positioned downstream of the protein-coding sequence; the nucleic acid further includes an mRNA stability sequence; the nucleic acid further includes a polyA signal positioned 3' to both the antisense RNA and the protein-coding nucleic acid sequence; and the nucleic acid further includes an intron sequence.

In a final related aspect, the invention features a nucleic acid which, when transcribed, produces an antisense RNA joined to a protein-coding sequence.

In preferred embodiments, the protein-coding sequence encodes a stable protein; the antisense RNA is positioned downstream of the protein- coding sequence; the nucleic acid further includes an mRNA stability sequence; the nucleic acid further includes a polyA signal positioned 3' to both the antisense RNA and the protein-coding nucleic acid sequence; the nucleic acid further includes an intron sequence; and the nucleic acid further includes a promoter sequence. As used herein, by "antisense activity" is meant antisense-mediated inhibition of target gene expression.

By "potentiating" is meant increasing (in this case, increasing antisense activity) by any mechanism including, but not limited to, by increasing RNA stability. By "joining" is meant to covalently bond, either directly (i.e., one end of the antisense RNA is immediately adjacent to one end of the protein- coding sequence) or indirectly (i.e., the antisense RNA sequence and protein- coding sequence are each included on a single nucleic acid, for example, a single RNA transcript, but are separated from each other by intervening nucleic acid sequence(s)).

By "protein-coding sequence" is meant a nucleic acid which contains an ATG (or AUG) start codon and which encodes a product of two or more amino acid residues. Preferably, this sequence encodes a "stable protein," or one which is not substantially thermally labile. Typically, stable proteins are characterized by sequences in which a large proportion (at least 50%, preferably, at least 75%, and, more preferably, at least 85% or even 95%>) of the nascent amino acid chain folds into a proper final configuration.

Other features and advantages of the present invention will be apparent from the following Detailed Description, and from the claims. Detailed Description Described below are experiments demonstrating that addition of protein- coding sequences to antisense RNA transcripts results in potentiation of antisense activity. As previously discussed, this approach is generally applicable and may be used to enhance the effectiveness of any antisense sequence or method. Accordingly, these examples are provided for the purpose of illustrating the invention, and should not be construed as limiting. The drawings are first briefly described.

Brief Description of the Drawings FIGURES 1A-1C demonstrate TNF-mediated apoptosis of Jurkat transfectants expressing TNFR2 or IkB.DN. FIGURE 1 A is a photograph of an immunoblot analysis of transfectant cell lines. Wild-type (WT) and RIP(-) Jurkat cells were stably transfected with vectors either without insert (-), or expressing TNFR2 or a dominant negative form (IkB.DN) of IkB-α (IkB-α S32A, S36A) (+). The presence of two TNFR2 bands is likely due to glycosylation. Both endogenous and transfected IkB-α were detected by the antibody used, and the DN form has a peptide tag at the amino terminus that gives it a slightly higher molecular mass. IkB.DN expression blocked NF-kB activation induced by a wide variety of stimuli (not shown). The blots were reprobed with an anti-RIP antibody to show that levels of RIP were unaffected by transfection. FIGURE IB is a graph and a photograph demonstrating that cycloheximide (CHX) unmasks TNFR1 -mediated cell death in Jurkat cells. When indicated, cells were preincubated for 1 hour in 10 μg/ml of cycloheximide and, after that, TNF (20 ng/ml) or anti-fas/CD95 (100 ng/ml) were added for 6 hours. Upper panel, cells were stained with

Annexin-V-FITC, and the percentage of Annexin-V-positive cells was measured by flow cytometry. Lower panel, cells were lysed and low molecular weight DNA was fractionated on a 1% agarose gel. FIGURE 1C is a series of photographs showing that cells expressing TNFR2 or IkB.DN undergo RIP-dependent apoptosis. Cells were cultured for the indicated times in the absence or presence of 20 ng/ml of TNF, or for 5 hour in the presence of 100 ng/ml of anti-CD95 antibody. Low molecular weight DNA was isolated and analyzed as in IB. Annexin-V stainings confirmed these results, and showed that cell death was always greater than 50% in those experimental points showing DNA ladder formation (not shown). Cell death always reached more than 85% when treatments were done overnight (not shown). Similar results were obtained when CHX was used instead of IkB.DN as a sensitizing agent (not shown).

FIGURES 2A-2D illustrate the TNFR2 apoptosis pathway in T cells. FIGURE 2 A is a series of graphs demonstrating that agonistic and antagonistic anti-TNFRl antibodies define a predominant role for TNFR2 in TNF-induced apoptosis in Jurkat cells expressing TNFR2. Jurkat cells expressing either TNFR2 or IkB.DN were incubated overnight in the presence or absence of 10 μg/ml of an agonistic anti-TNFRl antibody (upper panel). Jurkat cells expressing either TNFR2 or IkB.DN were incubated with TNF (20 ng/ml) for 24 hours, in the absence or presence of 50 μg/ml of a neutralizing anti-TNFRl antibody (lower panel). The percent death was measured using flow cytometry. FIGURE 2B is a graph showing that the TRAF2 binding region of TNFR2 is required for TNF-mediated cell death. Jurkat cells were electroporated with a mixture of 2 μg of a plasmid expressing GFP and 10 μg of a plasmid encoding the indicated C-terminal deletions of TNFR2. After 24 hours, cells were split and incubated overnight in the absence or presence of 20 ng/ml of TNF. Cells were subjected to flow cytometry, and the percentage of live transfected cells was evaluated. (+), > 45% death; (-), < 3% death. FIGURE 2C is a series of photographs showing that TNF-induced death in cells expressing TNFR2 or IkB.DN is blocked by dominant negative FADD/MORT1 (FADD.DN), Bcl-X_L and CrmA. Cells expressing TNFR2 or IkB.DN were electroporated with a mixture of 2 μg of CD 14 expression plasmid and 10 μg of either vector alone or vector bearing FADD.DN (amino acids 80-205), Bcl-X_L or CrmA. After 36 hours, the percentage of CD 14 positive cells was evaluated by flow cytometry, and equal numbers of cells expressing CD 14 were selected with anti-CD 14 magnetic beads. The purified cells were either treated for 6 hours with 20 ng/ml of TNF or left untreated, then harvested and low molecular weight DNA was fractionated on a 1% agarose gel. FIGURE 2D is a graph illustrating that FADD accumulates in a TNFR2 signaling complex in the presence of TRAF2 and RIP. 293 EBNA/T cells were transfected as indicated with DNA mixtures containing equal amounts of plasmids expressing TRAF2, RIP, FADD.DN, and a chimeric membrane protein having an extracellular domain comprising the human IgGl constant region, a CD7 transmembrane domain, and either the complete intracellular domain of TNFR2 or a deleted version lacking residues 415 to 465 (spanning the TRAF2 binding site). An anti-apoptotic plasmid expressing CrmA and Bcl-X_L was cotransfected in each case. 36 hours after transfection, cells were lysed and the Ig.TNFR2 chimera were immunoprecipitated using Protein A-Sepharose. The presence of FADD.DN in the precipitates was detected by blot analysis with an antibody that recognizes the death domain of FADD.

FIGURES 3A-3B show NF-kB activation induced by TNF in wild-type and RIP(-) cells expressing TNFR2. FIGURE 3 A is a graph illustrating that TNF strongly activates NF-kB in Jurkat cells expressing TNFR2. Wild- type cells bearing either vector or TNFR2 expression plasmid were electroporated with an NF-kB-luciferase reporter (4 μg) and a vector expressing both CrmA and Bcl-X_L (16 μg). After 36 hours, cells were split into three wells and treated for 6 hours with 20 ng/ml of TNF, 10 ng/ml of phorbol-12-myristate-13-acetate (PMA) plus 1 μM of ionomycin, or left untreated. They were then lysed and the luciferase activity was measured. Values shown are averages of triplicate measurements, normalized as the fraction of luciferase activity induced by treatment with PMA/ionomycin. FIGURE 3B is a graph showing that RIP is not required for TNFR2-mediated activation of NF-kB. RIP(-) cells transfected with either vector or TNFR2 expression plasmid were electroporated with 4 μg of an NF-kB-luciferase reporter plasmid. After 16 hours, cultures were exposed for 6 hours to either 20 ng/ml of TNF, 10 ng/ml of PMA plus 1 μM of ionomycin, or no agent. Luciferase assay and normalization were conducted as described in 3 A.

FIGURES 4A-4D show an analysis of TNF-induced cell death and NF-kB activation during human T cell activation. FIGURE 4A is a graph illustrating that activated T cells acquire sensitivity to TNF-induced cell death after IL2 exposure. Peripheral blood mononuclear cells (PBMCs) were purified and activated with 2 μg/ml of PHA for 2 days. CD4 and CD8 cells were purified with magnetic beads and subsequently incubated for 8 days in the presence of 30 U/ml of IL-2. At the indicated times, cells were harvested, washed, and incubated for 72 hours in the absence or presence of 50 ng/ml TNF. Where indicated, 30 U/ml of IL-2 were present during TNF treatment. Cell death was evaluated by Annexin-V-FITC staining. FIGURE 4B is a photograph demonstrating that TNF-induced NF-kB activation does not change during T cell activation and proliferation. Cells were activated, purified using a mixture of CD4 and CD8-specific magnetic beads, and cultured as above. Day 2 or day 10 cells were collected, washed, and incubated overnight to reduce spontaneous activation of NF-kB. T cells were then treated with TNF (50 ng/ml) for the indicated times, lysed and nuclear extracts isolated. Equal amounts of nuclear protein were subjected to SDS-PAGE and the p65 (RelA) subunit was detected by immunoblot. FIGURE 4C is a series of graphs showing that TNFR2 expression does not vary significantly during IL-2-dependent human T cell expansion. Cells were activated with PHA, purified and cultured as in 4A with the exception of TNF treatment, and subjected to flow cytometry using an anti-TNFR2 or an irrelevant control antibody. Shown is the mean fluorescence intensity (MF) obtained with anti-TNFR2 antibody (light line). FIGURE 4D is a photograph illustrating that RIP expression is induced during IL-2-dependent T cell expansion, whereas FADD expression remains constant. Cells were activated and purified as in 4A, washed and lysed in a buffer containing 1% NP-40. Extracts were normalized for protein content and equal amounts of protein were loaded in each well, resolved by SDS-PAGE and subjected to immunoblotting using a specific anti-RIP antibody. Western-blots were reprobed with an anti-FADD specific antibody.

FIGURES 5A-5C show that retroviral transduction of RIP antisense inhibits the IL2-driven transition of CD8 T cells to a death-sensitive state. FIGURE 5 A is a graph demonstrating potentiation of antisense activity by an upstream open reading frame. 293T cells were transfected as indicated with 10 mg of a mixture of mammalian expression plasmids containing GFP, RIP and either antisense RIP or a construct containing the GFP open reading frame upstream of antisense RIP (GFP-RIP antisense), at a 1 : 1 : 3 molar ratio. The percentage of GFP-positive cells undergoing apoptotic cell death was scored 36 hours after transfection. FIGURE 5B is a graph showing that GFP-RIP antisense inhibits TNF-induced apoptosis in CD 8 T cell blasts. PBMCs from four independent donors were isolated and cultured in the presence of PHA as described in Figure 4. After 2 days, cells were transduced overnight with retrovirus expressing either GFP or GFP-RIP antisense. A day later, CD8-positive cells were isolated using magnetic beads, and cultured for 8 days in the presence of IL2 (30 U/ml) to induce competency for TNF-triggered cell death. After that, cells were washed and incubated for 72 hours in the absence or presence of TNF (50 ng/ml). Apoptosis assays were performed with Annexin-V-phycoerythrin. The death-protective activity was assessed for each culture by comparison of the percent death in the transduced (green) and nontransduced (non-green) cells. Data for the antisense constructs are presented as a percentage of the death protection (nil) provided by GFP transduction alone. FIGURE 5C is a graph demonstrating that antisense CD59 or CD44H do not significantly interfere with TNF-induced apoptosis of CD8 T cell blasts. Cells were cultured and processed as in 5B with the exception that GFP or GFP-CD59 antisense and GFP or GFP-CD44H antisense constructs were used to transduce activated PBMCs. Susceptibility to TNF-induced apoptosis was calculated as in 5B.

FIGURE 6 is a series of diagrams illustrating exemplary antisense constructs according to the invention. In this figure, "Flpe" denotes an engineered version of the yeast Flp site specific recombinase gene useful in gene therapy vectors, "Anti-Flpe" denotes an antisense Flpe sequence, "BFP" denotes a blue fluorescent protein-coding sequence, "IRES" denotes an internal ribosome entry site sequence, and "PA" denotes a polyA signal.

FIGURE 7 is a diagram of a Flpe substrate plasmid. As indicated, upon expression of Flpe protein in a host cell containing this plasmid, Flpe- mediated recombination results in the expression of detectable green fluorescent protein ("GFP").

FIGURE 8 is a series of photographs demonstrating that the activity of Flpe antisense RNA is potentiated by the inclusion of a blue fluorescent protein-coding sequence in the antisense transcript. 293 cells were transfected with the Flpe substrate plasmid, a Flpe-expressing plasmid, and either carrier DNA or the indicated Flpe antisense construct, and green fluorescent protein was measured. "Flpe" cells express the Flpe gene only; "+ anti-Flpe" cells express the Flpe gene in combination with a construct expressing a standard Flpe antisense sequence; "+ anti-Flpe IRES BFP" cells express the Flpe gene in combination with a construct which includes the Flpe antisense sequence positioned upstream from the blue fluorescent protein-coding sequence containing an internal ribosome binding site; and "+ BFP anti-Flpe" cells express the Flpe gene in combination with a construct which includes the Flpe antisense sequence positioned downstream from the blue fluorescent protein- coding sequence.

Tumor Necrosis Factor (TNFR) Signaling Cell populations in the immune system undergo expansion and contraction in the absence of the cell-cell and cell-matrix contacts that normally regulate cell number in solid organs. For these populations, regulatory mechanisms that function in the absence of specific organ context cues are required, and the regulated control of programmed cell death is an important function (Rothstein, Curr. Opin. Immunol. 8:362-71 (1996); Nagato, Cell 88:355-65 (1997); and Winoto, Curr. Opin. Immunol. 9:365-70 (1997)).

Studies in mice have shown that IL-2, an important cytokine for clonal expansion, also programs T cells for eventual death (Lenardo, Nature 353:858-61 (1991). The significance of this observation has been underscored by findings that mice deficient in either the IL-2 gene or the genes for the β or γ chains of the IL-2 receptor show lymphoproliferative disorders (Sadlack et al., Cell 75:253-61 (1993); Kneitz et al, Eur. J. Immunol. 25:2572-7 (1995); Suzuki et al., Science 268: 1472-6 (1995); and Nakajima and Leonard, J. Immunol. 159:4737-44 (1997)). These reports point to an important role for IL-2 signaling in population contraction in the immune system. On resting or submaximally stimulated naive T cells TNF promotes proliferation through a pathway involving TNFR2 (Scheurich et al., J. Immunol. 138: 1786-90 (1987); Yokota et al., J. Immunol. 140:531-6 (1988); Gehr et al., J. Immunol. 149:911-7 (1992); Tartaglia et al., J. Immunol. 151 :4637-41 (1993); and Grell et al., Eur. J. Immunol. 28:257-63 (1998)). This pathway has high disease relevance because TNF is an important inducer of HIV-1 transcription in resting or newly activated human T cells (Folks et al., Proc. Natl. Acad. Sci. USA 86:2365-8 (1989); and Vyakarnam et al., Aids 4:21-7 (1990)). However, late in activation, TNF induces apoptosis (Sarin et al., J. Immunol. 155:3716-8 (1995)), and a TNFR2-dependent cell death pathway has been described that affects activated CD8 positive cells (Zheng et al., Nature 377:348-51 (1995); Alexander-Miller et al., J. Exp. Med. 188: 1391- 9 (1998); and Herbein et al., Nature 395: 189-94 (1998)). Susceptibility to death is controlled by prior activation, so that T cells that have been exposed to IL-2 are prone to apoptosis, but resting cells are not (Lenardo, Nature 353:858- 61 (1991).

Jurkat (human T cell leukemia) cells express TNFR1 but not TNFR2, and are largely insensitive to TNF-induced cell death (Figure IB) (Ting et al., EMBO J. 15:6189-96 (1996)). To explore the apoptotic effects of TNF in these cells we created lines that express TNFR2 (Smith et al., Science 248: 1019-23 (1990)), or a mutated form of IkB-α (IkB.DN) (Brockman et al., Mol. Cell Biol. 15:2809-18 (1995)) that behaves as a dominant negative regulator of NF-kB activation (Brockman et al., Mol. Cell Biol. 15:2809-18 (1995); Brown et al, Science 267: 1485-8 (1995); and Traenckner et al., EMBO J. 14:2876-83 (1995)). Figure 1A documents the expression of these molecules in the transfectant cell lines. In accord with previous reports (Wallach, J. Immunol. 132:2464-9 (1984); Holtmann et al, Immunobiology 177:7-22 (1988); Beyaert and Fiers, FEBS Lett. 340:9-16 (1994); and Van Antwerp et al., Science 274:787-9 (1996)), preincubation of cells with cycloheximide (CHX) unmasks the apoptotic activity of TNFRl (Figure IB), and Jurkat cells expressing IkB.DN rapidly succumb to TNF-induced apoptosis (Figure IC). However, expression of TNFR2 in Jurkat cells renders them sensitive to TNF-dependent cell death in the absence of cycloheximide or IkB.DN (Figure IC), suggesting the existence of a death pathway able to overcome the protective effect of NF-kB activation.

Specific stimulation of TNFRl with agonistic antibodies in Jurkat cells expressing TNFR2 failed to induce apoptosis (Figure 2A, upper panel), whereas those antibodies are able to efficiently induce cell death in cells expressing IkB.DN (Figure 2 A, upper panel). In addition, ligand-blocking anti-TNFRl antibodies substantially inhibited TNF-induced cell death in Jurkat cells expressing IkB.DN, but not in those expressing TNFR2 (Figure 2 A, lower panel). Although these findings point to a principal role for TNFR2 in transmitting the apoptotic signal, some requirement for TNFRl costimulation cannot be excluded. Transfection of C-terminally deleted versions of TNFR2 into Jurkat cells showed that the TRAF2 binding site (Rothe et al., Cell 78:681- 92 (1994)) is essential for efficient TNF-induced apoptosis (Figure 2B). This agrees with results in other cell types (Weiss et al., J. Immunol. 158:2398-404 (1997); Weiss et al, J. Immunol. 161 :3136-42 (1998); and Declercq et al., J. Immunol. 161:390-9 (1998)), and indicates that the TNFR2 intracellular domain is required for death.

RIP is required for TNFRl -mediated NF-kB induction, but not for CD95-mediated cell death (Ting et al., EMBO J. 15:6189-96 (1996); and Kelliher et al., Immunity 8:297-303 (1998)), and any proapoptotic role it might play in TNF-dependent death in Jurkat cells might be masked by the strong protective effect of NF-kB activation (Van Antwerp et al., Science 274:787-9 (1996); Liu et al., Cell 87:565-76 (1996); Beg and Baltimore, Science 274:782- 4 (1996); Wang et al, Science 274:784-7 (1996); and Wu et al., Science 281 :998-1001 (1998)). RIP deficient cells expressing IkB.DN are highly resistant to TNF-induced cell death (Figure IC), indicating that RIP is required for TNFRl -dependent apoptosis in this setting. RIP deficient Jurkat cells expressing TNFR2 did not die when exposed to TNF but retained the capacity to undergo apoptosis when triggered by CD95 (Figure IC). Thus, cell death induced by TNF in Jurkat cells seems to require RIP whether it proceeds through TNFRl or TNFR2.

Although RIP null cells did not undergo apoptosis, it remained possible that the mutant cell line harbors other mutations that are responsible for the death-resistance phenotype. To show that RIP itself was sufficient to correct the phenotypic defect, we reintroduced it into RIP deficient cells by transfection. Transient transfection of wild- type RIP into RIP(-) cells expressing TNFR2 or IkB.DN resulted in cell death after TNF treatment (data not shown). The dependence of cell death on the presence or absence of the different domains of RIP was similar in the two systems (Ting et al., EMBO J. 15:6189-96 (1996)) (data not shown), suggesting that a common function of RIP was exploited by the two death pathways.

To explore the mechanism by which TNF provokes apoptosis in Jurkat cells, plasmids expressing anti-apoptotic proteins, including dominant negative FADD/MORT1 (FADD.DN), Bcl-X_L and CrmA (Chinnaiyan et al., J. Biol. Chem. 271 :4961-5 (1996); Boise et al, Cell 74:597-608 (1993); Hu et al, Proc. Natl. Acad. Sci. USA 95:4386-91 (1998); Tewari and Dixit, J. Biol. Chem. 270:3255-60 (1995); and Miura et al., Proc. Natl. Acad. Sci. USA 92:8318-22 (1995)), were transfected into wild-type cells expressing either TNFR2 or IkB.DN. As shown in Figure 2C, all three proteins tested were able to inhibit cell death mediated by TNFR2 and TNFRl to a similar extent. This suggests that TNFR2, like TNFRl, mediates apoptosis through a FADD/MORT1 -dependent caspase pathway that can be inhibited by CrmA and

Bcl-X_L. Because the apoptotic signals mediated by TNFR2 required the

TRAF2 binding site we looked for a mechanism coupling TRAF2 to apoptosis.

TRAF2 has been previously shown to bind RIP (Hsu et al., Immunity 4:387-96 (1996)), and RIP and FADD/MORT1 are known to interact (Varfolomeev et al., J. Exp. Med. 183: 1271-5 (1996)), although the precise geometry of these associations is not understood.

Cotransfection and coimmunoprecipitation studies showed that RIP could bridge TNFR2 -bound TRAF2 to FADD by simultaneously interacting with both molecules (Figure 2D). Thus a probable logic for the pathway is that

TNFR2 engagement leads to the recruitment of TRAF2, association of RIP, and binding of FADD/MORT1.

NF-kB activation initiates a response that blunts the apoptotic action of several agonists of cell death, including TNFRl (Van Antwerp et al., Science 274:787-9 (1996); Liu et al, Cell 87:565-76 (1996); Beg and

Baltimore, Science 274:782-4 (1996); Wang et al, Science 274:784-7 (1996); and Wu et al., Science 281 :998-1001 (1998)). A diminished NF-kB activation might facilitate apoptosis through TNFR2. However Jurkat cells expressing

TNFR2 triggered a strong NF-kB response upon TNF treatment (Figure 3A), indicating that the NF-kB-activated protective response failed to protect against

TNFR2-induced apoptosis.

Activation of NF-kB by TNFRl has been shown to require RIP

(Ting et al., EMBO J. 15:6189-96 (1996); and Kelliher et al., Immunity 8:297-

303 (1998)) and the RIP null cells used here showed an absolute block in TNFRl -mediated NF-kB induction (Ting et al, EMBO J. 15:6189-96 (1996)).

Unexpectedly, TNF was able to activate NF-kB in RIP(-) cells expressing

TNFR2 (Figure 3B), indicating that TNFR2 is capable of activating NF-kB by a pathway that intersects the TNFRl -initiated pathway somewhere downstream of RIP. As a result, apoptosis and NF-kB activation induced by TNFR2 are uncoupled at the level of RIP.

These findings imply that, in T cells, RIP may be able to determine the outcome of TNF signaling: in the absence of RIP, only TNFR2 would be functional, triggering NF-kB activation; and both apoptosis and the induction of NF-kB through TNFRl would be blocked. In the presence of RIP, TNF-induced apoptosis should proceed efficiently through a TNFR2-dependent pathway, despite induction of NF-kB activity through both receptors. This differential signaling mimics naturally occurring events in T cell activation. Activated human and murine T cells principally express TNFR2 (Zheng et al., Nature 377:348-51 (1995); and Ware et al., J. Immunol. 147:4229-38 (1991)), and TNFR2 has been found to mediate TNF-induced proliferation in the initial phase of the T cell activation response (Gehr et al., J. Immunol. 149:911-7 (1992); Tartaglia et al., J. Immunol. 151 :4637-41 (1993); and Grell et al., Eur. J. Immunol. 28:257-63 (1998)), by upregulating the expression levels of the IL-2 receptor α chain (CD25) in an NF-kB-dependent manner (Scheurich et al., J. Immunol. 138:1786-90 (1987); and Lowenthal et al., J. Immunol. 142:3121-8 (1989)). Later in the process, T cells become sensitive to apoptosis induced by TNF (Sarin et al., J. Immunol. 155:3716-8 (1995)), and when activated murine CD8-positive cells are restimulated through the T cell receptor, TNFR2 mediates activation-induced cell death triggered by the autocrine secretion of TNF (Zheng et al., Nature 377:348-51 (1995); Alexander-Miller et al., J. Exp. Med. 188: 1391-9 (1998); and Herbein et al. Nature 395: 189-94 (1998)). To determine whether developmental regulation of RIP controls susceptibility to cell death in vivo, we studied TNF sensitivity and RIP expression in activated T cells from multiple human donors. Both CD4- and CD8-positive cells were resistant to TNF-induced apoptosis 2 days after phytohemagglutinin (PHA) stimulation (Figure 4A). At this time NF-kB activation, measured as nuclear translocation of the p65 (RelA) subunit of NF-kB, was intact (Figure 4B), and the cells expressed TNFR2 (Figure 4C). However, after the cells were cultured 4 to 10 more days in the presence of IL-2, they became susceptible to TNF-induced cell death (Figure 4A), while retaining unchanged their ability to activate NF-kB (Figure 4B). The transition to a death-sensitive state occurred without significant variation in the expression of TNFR2 (Figure 4C) or FADD (Figure 4D), and TNFRl expression remains undetectable throughout (not shown). Consistent with previous reports (Sarin et al., J. Immunol. 155:3716-8 (1995)), the presence of IL-2 during TNF treatment inhibited cell death (Figure 4A).

Antisense RIP Approach

RIP expression was strongly induced in human T cells between day 2 and day 10 of activation and IL2-dependent proliferation (Figure 4D), with good expression apparent by day 5 (not shown). To evaluate whether RIP expression might be necessary for apoptosis in this context, we explored antisense approaches based on retroviral transfer. We inserted a green fluorescent protein (GFP) coding sequence upstream of antisense RIP in a murine leukemia virus-based vector (Riviere et al., Proc. Natl. Acad. Sci. USA 92:6733-7 (1995)). An improved efficiency of the GFP-RIP antisense transcript over conventional antisense RNA was apparent in the inhibition of cell death provoked by overexpression of RIP in 293 T cells (Figure 5A). Retroviral transduction of the GFP-antisense RIP transcription unit into CD8 T cell blasts from 4 different donors decreased susceptibility to TNF-induced cell death at day 10 after activation, as established by comparison with cells not expressing construct or cells expressing GFP only (Figure 5B). Similar constructs bearing an irrelevant antisense segment downstream of GFP lacked death-protective activity, indicating that the effect was specific to antisense RIP (Figure 5C).

Thus the findings in primary human T cells were consistent with observations from experiments with Jurkat cells. In human T cells RIP is needed for TNFR2 induction of death but not NF-kB activation. In resting T cells, or activated T cells that have not been exposed to IL2, TNF activates NF-kB through TNFR2 and a TRAF2-dependent pathway (Rothe et al., Science 269: 1424-7 (1995)). In activated T cells that have been exposed to IL2, RIP is induced and is proposed to bridge the TNFR2-TRAF2 complex to

FADD/MORT1 and the apoptosis machinery. Although in human T cells RIP is needed for TNFRl -mediated induction of both death (this work) and NF-kB activation (Ting et al., EMBO J. 15:6189-96 (1996)), its action downstream of TNFRl clearly depends on cell type. Mouse fibroblasts are normally resistant to TNFRl -induced apoptosis, but are sensitive when RIP is absent (Kelliher et al., Immunity 8:297-303 (1998)), suggesting that in these cells RIP is required for the NF-kB protective response, but not for TNFRl -mediated cell death. Thus there are likely lineage-specific differences in the TNFRl signaling pathway as well. An implicit assumption in many signal transduction studies has been that a single consensus sequence of events suffices to account for the majority of changes in cellular state that follow exposure to an extracellular mediator. However there is abundant evidence to suggest that in cell death pathways, and particularly in the immune system, this assumption has limited power to explain the observed phenomena. In T cells, antigen receptor activation and proliferation are different programs. Antigen receptor engagement does not lead directly to cell cycle transition, but induces competency for proliferation by activation of both cytokine and cytokine receptor promoters. The data presented here indicate that the death susceptibility program is associated with proliferation instead of activation, as RIP induction occurs during the IL-2-driven proliferation phase, not the early activation response. Because RIP is required for both the death and NF-kB induction activities mediated by TNFRl in T cells, TNFRl, if present, is unlikely to contribute significantly to TNF signaling prior to RIP induction.

Methods

The experiments described above were carried out using the following methods. Cells and transfections. Jurkat cells used in this study and electroporation methods have been described in detail elsewhere (Ting et al., EMBO J. 15:6189-96 (1996)). Adherent cells (293 derivatives) were transfected using the calcium phosphate precipitate method, as previously described (Ausubel et al., Current Protocols in Molecular Biology 1-2 v., Greene Publishing Associates, J. Wiley, Brooklyn, NY (1987)).

Flag tagged IkB-α S32A, S36A (Brockman et al., Mol. Cell Biol. 15:2809-18 (1995)) and TNFR2 (Smith et al., Science 248: 1019-23 (1990)) cDNAs were subcloned into a mammalian expression vector which contained a puromycin resistance marker. To create stable transfectants, cells were electroporated with the indicated plasmids and subjected to puromycin (Sigma, St. Louis, MO) selection (1 μg/ml) for 10 days. Cells were further purified by limiting dilution to obtain clonal populations.

Peripheral blood mononuclear cells (PBMCs) were purified by centrifugation onto a Ficoll/diatrizoate cushion (Organon Teknika, Durham, NC) and activated with 2 μg/ml of PHA (Sigma). Purification of CD4 and CD8-positive cells was performed using Dynabeads and the Detachabead system (Dynal, Oslo, Norway). Purity, activation, and TNFR expression were determined by flow cytometry (Coulter Epics XL) using anti-CD3, -CD4, -CD8, -CD25, -CD69 (PharMingen, San Diego, CA) or anti-TNFR2 (R & D Systems, Minneapolis, MN) antibodies. Purity was always greater than 95%. After purification, as indicated, cells were further cultured in the presence of 30 U/ml IL-2 (Sigma). rrnmunoblotting and coimmunoprecipitation studies. To measure TNFR2, IkB.DN, or RIP expression in the Jurkat stable transfectants, equal number of cells were harvested and directly lysed in 2X reducing SDS-sample buffer. To measure RIP and FADD expression levels in human primary T cells, cells were lysed in a standard lysis buffer containing 1% NP-40 (Sigma). To evaluate NF-kB activation in primary T cells, nuclear extracts were obtained after TNF treatment as previously described (Schreiber et al., Nucleic Acids Res. 17:6419 (1989)). Protein concentrations were measured by the bicinchoninic acid method (Pierce, Rockford, IL). For the coimmunoprecipitation studies, 293EBNA/T cells were transfected with the indicated DNAs and lysed in a buffer containing 0.1% NP40 as described previously (Hsu et al., Cell 81 :495-504 (1995)). The Ig.TNFR2intracellular domain chimera were immunoprecipitated with protein A-sepharose (Sigma), for 3 hours at 4°C. Precipitates were washed twice with lysis buffer, twice with lysis buffer containing 0.8 M NaCl, and twice more with lysis buffer.

Western-blots were probed with specific antibodies against TNFR2 (R & D Systems), IkB-α (Santa Cruz, CA), RIP, FADD/MORT1, or p65 NF-kB (Transduction Laboratories, Lexington, KY).

Apoptosis assays. Annexin-V-FITC staining was carried out using instructions provided by the manufacturers (R&D Systems). DNA fragmentation assays were performed essentially as described (Herrmann et al., Nucleic Acids Res. 22:5506-7 (1994)). Cycloheximide and TNF were purchased from Sigma, and the anti-Fas antibody (IgM clone CH-11) from UBI (Lake Placid, NY). Cell death was analyzed by flow cytometry with a previously optimized program to measure the percentage of cells present in a Forward Scatter/Side Scatter (FS/SS) gate including only propidium impermeant cells. Agonistic and antagonistic anti-TNFRl antibodies were purchased from R&D Systems. In the case of GFP cotransfection (Figure 2B), cells were electroporated and cultured as indicated and directly subjected to flow cytometry. We used the previously described program to measure the percentage of live cells which were GFP-positive in each experimental condition. For CD 14 selection after transfection, cells were electroporated with the indicated plasmids, and CD14-expressing cells were purified using magnetic beads (Dynal) 36 hours later. The percentage of CD14-positive cells was measured by flow cytometry and used to normalize cell numbers in the purified populations. 5xl0⁶ cells per well were cultured as indicated, and processed to detect DNA fragmentation.

Luciferase assays. Cells were electroporated with an NF-kB-luciferase reporter (Ting et al., EMBO J. 15:6189-96 (1996)), either alone or in combination with a stoichiometric excess of a plasmid simultaneously expressing the anti-apoptotic proteins CrmA and Bcl-X_L. Luciferase activity was measured as previously described (Ting et al., EMBO J. 15:6189-96 (1996)). Ionomycin was purchased from Calbiochem (San Diego, CA).

RIP antisense experiments. Mammalian expression vectors were constructed to express a RIP antisense RNA, or antisense RIP downstream from the GFP open reading frame (GFP-RIP antisense), which was created by inserting antisense RIP downstream from the stop codon of GFP. The ability of both antisense constructs to inhibit cell death triggered by RIP overexpression was compared in 293T cells. Subconfluent cells were transfected with the indicated constructs, and the percentage of green cells showing signs of apoptotic cell death versus the total number of green cells was scored 36 hours later.

Retroviral constructs containing GFP, GFP-RIP antisense, GFP-CD59 antisense, or GFP-CD44H antisense were created in the pMMP vector, a derivative version of the pMFG vector (Riviere et al., Proc. Natl. Acad. Sci. USA 92:6733-7 (1995)), and transfected into subconfluent Phoenix-amphotropic packaging cells (Hitoshi et al., Immunity 8:461-71 (1998)). Virus enriched supernatants were collected 48 hours post transfection and diluted 1 : 1 in the PHA conditioned media (total volume of 3 ml). 2xl0⁷ activated PBMCs were resuspended in this mixture and added onto a 6 cm plate that was previously coated with a recombinant fibronectin fragment (RetroNectin, Takara Biomedicals, Shiga, Japan) following the instructions of the manufacturers. Next day, CD8-positive cells were purified. Transduction efficiencies ranged from 15 to 25% in the case of GFP, and from 5 to 15 % in the case of the GFP-RIP, CD59, or CD44H antisense constructs. After the indicated incubations, cell death was measured with Annexin-V-phycoerythrin (PharMingen).

Additional Antisense Experiments In addition to the above, potentiation of antisense activity by inclusion of a protein-coding sequence in an antisense transcript was also demonstrated in the context of another, unrelated target gene, specifically, the Flpe gene, an engineered version of the yeast Flp site specific recombinase. In these experiments, constructs containing antisense Flpe located in the 3' UTR of a blue fluorescent protein (BFP) gene were shown to be more effective in preventing Flpe expression and action than antisense constructs lacking the BFP open reading frame. In addition, in these experiments, constructs containing antisense Flpe located upstream of the BFP open reading frame were also shown to potentiate antisense activity, but at a level which was less effective than when the antisense sequence was positioned downstream. The BFP open reading frame in this latter context was made active through the use of an internal ribosome entry site (IRES), as shown in Figure 6.

To carry out these experiments, anti-sense Flpe DNAs were generated by PCR using oligonucleotides containing appropriate restriction endonuclease recognition sequences. After restriction enzyme treatment of the PCR products, anti-sense DNA was cloned into the peakl2 vector.

Specifically, the peakl2-aFlpe plasmid was generated by ligating Hind3- and Not 1 -restricted peak 12 vector DNA with a Bsmbl (Hind3 compatible overhang)-Notl restricted aFlpe PCR product. The plasmid containing blue fluorescent protein (BFP) and Flpe antisense sequence (aFlpe) (peakl2-BFP-antiFlpe) was generated by ligating Hind3- and Not 1 -restricted peakl2 vector with a Hind3-Bsrgl fragment containing the BFP coding sequence and a Bsrgl-Notl fragment containing the aFlpe sequence. The aFlpe-IRES-BFP plasmid (peakl2-aFlpe-IRES-BFP) was generated by ligating Hind3- and Not 1 -restricted peak 12 vector DNA with a Bsmbl (having Hind3 compatible overhang)-Notl fragment of the aFlpe PCR product, a Notl-Ncol fragment of an internal ribosome entry sequence (IRES) sequence (generated by PCR using Encephalomyocarditis virus-derived IRES as a template) and a Ncol-Notl fragment of the BFP coding sequence. Each of these constructs is illustrated in Figure 6. 293 cells were then transfected with these vectors using the calcium phosphate method in 6-well plates. The total amount of DNA/well was kept at 3 mg, consisting of 1 mg of Flp substrate plasmid DNA (shown in Figure 7), 1 mg of Flpe plasmid DNA, and 1 mg of either anti-sense plasmid or carrier DNA. GFP-mediated fluorescent intensities of the wells were compared 24 hours post-transfection and thereafter.

The results of these experiments are shown in Figure 8. As discussed above, the activity of the Flpe antisense RNA was potentiated by inclusion of the BFP coding sequence either upstream or downstream of the antisense sequence, with activity being optimally enhanced by positioning of the protein-coding sequence upstream from the antisense sequence.

All publications and patents or patent applications referred to herein are hereby incorporated by reference.

Other embodiments are within the claims.

Claims

1. A method for potentiating antisense RNA activity, said method comprising joining said antisense RNA to a protein-coding nucleic acid sequence.

2. The method of claim 1, wherein said protein-coding sequence encodes a stable protein.

3. The method of claim 1, wherein said antisense RNA is positioned downstream of said protein-coding sequence.

4. A method for generating an antisense RNA, said method comprising expressing a transcript which comprises (i) said antisense RNA and (ii) a protein-coding sequence, whereby said transcript exhibits enhanced antisense activity relative to said antisense RNA in the absence of said protein- coding sequence.

5. The method of claim 4, wherein said protein-coding sequence encodes a stable protein.

6. The method of claim 4, wherein said antisense RNA is positioned downstream of said protein-coding sequence.

7. A nucleic acid comprising an antisense RNA joined to a protein- coding nucleic acid sequence.

8. The nucleic acid of claim 7, wherein said protein-coding nucleic acid sequence encodes a stable protein.

9. The nucleic acid of claim 7, wherein said antisense RNA is positioned downstream of said protein-coding sequence.

10. The nucleic acid of claim 7, wherein said nucleic acid further comprises an mRNA stability sequence.

11. The nucleic acid of claim 7, wherein said nucleic acid further comprises a polyA signal positioned 3' to both the antisense RNA and the protein-coding nucleic acid sequence.

12. The nucleic acid of claim 7, wherein said nucleic acid further comprises an intron sequence.

13. A nucleic acid which, when transcribed, produces an antisense RNA joined to a protein- coding sequence.

14. The nucleic acid of claim 13, wherein said protein-coding sequence encodes a stable protein.

15. The nucleic acid of claim 13, wherein said antisense RNA is positioned downstream of said protein-coding sequence.

16. The nucleic acid of claim 13, wherein said nucleic acid further comprises an mRNA stability sequence.

17. The nucleic acid of claim 13, wherein said nucleic acid further comprises a polyA signal positioned 3' to both the antisense RNA and the protein-coding nucleic acid sequence.

18. The nucleic acid of claim 13, wherein said nucleic acid further comprises an intron sequence.

19. The nucleic acid of claim 13, wherein said nucleic acid further comprises a promoter sequence.