Identification and functional validation of PNAs that inhibit murine C
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《核酸研究医学期刊》
Department of Medicinal Chemistry, ISIS Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, CA, 92008, USA
*To whom correspondence should be addressed. Tel: +1 760 603 3852; Fax: +1 760 603 4654; Email: aeldrup@isisph.com
ABSTRACT
Cognate recognition between the CD40 receptor and its ligand, CD154, is thought to play a central role in the initiation and propagation of immune responses. We describe the specific down regulation of cell surface associated CD40 protein expression by use of a peptide nucleic acid (PNA) antisense inhibitor, ISIS 208529, that is designed to bind to the 3' end of the exon 6 splice junction within the primary CD40 transcript. Binding of ISIS 208529 was found to alter constitutive splicing, leading to the accumulation of a transcript lacking exon 6. The resulting protein product lacks the transmembrane domain. ISIS 208529-mediated CD40 protein depletion was found to be sequence specific and dose dependent, and was dependent on the length of the PNA oligomer. CD40-dependent induction of IL-12 in primary murine macrophages was attenuated in cells treated with ISIS 208529. Oligolysine conjugation to the PNA inhibitor produced an inhibitor, ISIS 278647, which maintained its specificity and displayed efficacy in BCL1 cells and in primary murine macrophages in the absence of delivery agents. These results demonstrate that PNA oligomers can be effective inhibitors of CD40 expression and hence may be useful as novel immuno-modulatory agents.
INTRODUCTION
CD40 is a cell membrane protein that plays a key role in the initiation and propagation of immune responses (1–3). CD40, a member of the tumor necrosis factor (TNF) receptor family of proteins, is expressed on antigen-presenting cells such as B lymphocytes and dendritic cells as well as additional cell types such as macrophages, endothelial cells, smooth muscle cells and epithelial cells. The ‘ligand’ for CD40 is CD154 (also known as CD40L, or gp39), a member of the TNF family and is expressed mainly on activated T cells and platelets (4–6).
Perhaps the best characterized response to signaling through CD40 on immune cells is B-lymphocyte activation, which results in increased expression of B7 molecules (CD80 and CD86), protection against apoptosis, and Ig class switching (2,6–8). In addition to activation of B lymphocytes, binding to CD40 expressed on other antigen-presenting cells leads to the secretion of cytokines such as IL-1?, TNF- and IL-12, the production of chemokines, as well as expression of adhesion molecules such as ICAM-1 and VCAM-1. Numerous studies have demonstrated that interference with CD40–CD154 signaling has profound effects on immune responses in cellular and animal model systems and suggest that inhibition of CD40 function could offer therapeutic benefit for inflammatory and autoimmune diseases (1,3).
Antisense technology represents a novel approach to the modulation of CD40–CD154 cognate signaling. There are multiple antisense mechanisms that operate in mammalian cells (9). The best characterized mechanism is RNase H dependent, in which oligonucleotides containing DNA or DNA analogs bind to the target RNA and recruit RNase H, resulting in degradation of the target RNA. There is one approved antisense drug and over 20 drugs in various stages of clinical trials that work by this mechanism of action. RNA interference is a recently characterized antisense mechanism that utilizes double-stranded RNA oligonucleotides that dissociate with the antisense strand binding to target RNA and promoting degradation by an unknown RNase (10,11). There are also antisense mechanisms that do not promote degradation of target RNA, such as translation arrest and inhibition of splicing (9). Oligonucleotides that work by the latter mechanisms bind to the target RNA with high affinity and do not support RNase activities. Examples of such modified oligonucleotides include uniform 2' modifications such as 2'-O-methyl and 2'-O-methoxyethyl, morpholino or peptide nucleic acid (PNA).
The pharmacological, toxicological and pharmacokinetic behavior of second generation, 2'-O-methyl and 2'-O-methoxyethyl phosphorothioate oligonucleotides (MOEs) are well characterized (12–16). These antisense oligonucleotides primarily distribute to the liver, kidney, spleen and mesenteric lymph nodes (12). PNAs are an example of a third generation antisense oligonucleotide analog with the potential to exhibit different pharmacological, toxicological and pharmacokinetic properties than 2'-modified oligonucleotides due to differences in physical chemical properties. PNAs lack the negatively charged backbone characteristic to most other nucleic acid analogs, but instead incorporate a neutral amide linkage. Preliminary studies examining the pharmacokinetics of unmodified PNA suggest that they exhibit limited tissue distribution and are rapidly excreted in the urine (17) (C. F. Bennett, unpublished results). These findings have led investigators to seek modifications that confer a more favorable pharmacokinetic profile. Furthermore, progress in developing PNA oligomers has been impeded by the difficulty in intracellular delivery of PNA in cultured cells. In contrast to charged oligonucleotides, PNA molecules are not readily amenable to simple cationic lipid-mediated delivery (18). Considerable effort has been directed towards peptide conjugation of PNA as both a means to enhance cellular uptake and as a way to direct delivery to certain tissue types (19). Mixed results have been obtained through the conjugation of amphipathic peptides like penetratin (which is derived from the homeodomain of Antennapedia) (20). Recently, it has been reported that a conjugate of (lys)4 to PNAs exhibit antisense effects when delivered to cells in culture and displays antisense effects in a variety of tissues following injection into mice (21,22).
Here we demonstrate that uniformly modified MOE and PNA oligomers specifically decrease cell surface expression of the CD40 receptor on BCL1 cells. PNA-mediated reduction of cell surface CD40 expression is shown to occur through an RNase-independent mechanism of action, resulting in an alternatively spliced CD40 transcript. This transcript encodes a truncated CD40 protein lacking its transmembrane domain. PNA-mediated depletion of cell-associated CD40 protein is demonstrated to be sequence specific, dose dependent and dependent on oligomer length. Treatment of primary murine macrophages with the PNA inhibitor results in a decrease in CD40-mediated IL-12 production. Thus, this antisense agent reduces the expression of cell-associated CD40, leading to the inhibition of signaling through CD40 and ultimately to the reduction of IL-12 cytokine production. Moreover, N-terminal conjugation of eight lysines onto the PNA is shown to lead to a compound that inhibits CD40 expression in BCL1 cells and in macrophages in the absence of facilitated delivery methodologies.
MATERIALS AND METHODS
Cells
Murine B-cell lymphoma cells, BCL1 cells, were obtained from the American Type Culture Collection and grown in normal growth medium . Cells were incubated in a humidified chamber at 37°C, containing 5% CO2. Antisense agents were delivered to cells by electroporation (200 V, 13 , 1000 μFa) using 2 mm gap width cuvettes and a BTX 600 Electro Cell Manipulator. Cells were plated in normal growth medium and incubated for the indicated times prior to harvest.
Primary thioglycollate-elicited macrophages were isolated by peritoneal lavage from 6- to 8-week-old female C57Bl/6 mice that had been injected with 1 ml of 3% thioglycollate broth 4 days previously. PNAs were delivered to unpurified peritoneal cells by a single 6 ms pulse, 90 V, on a BTX square wave electroporator in 1 mm cuvettes. After electroporation, the cells were plated for 1 h in serum-free RPMI 1640 (supplemented with 10 mM HEPES) at 37°C, 5% CO2 to allow the macrophages to attach. Non-adherent cells were then washed away and the media was replaced with complete RPMI 1640 (10% FBS, 10 mM HEPES). Primary macrophages were activated by treatment with 100 ng/ml rIFN- (R&D Systems) for 4 h, followed by 10 μg/ml anti-CD40 antibody (clone 3/23; BD Pharmingen) for the indicated timepoints.
Flow cytometry analysis
Cells were detached from culture plates with 0.25% trypsin. Trypsin was neutralized with an equal volume of normal growth medium and cells were pelleted. Cell pellets were resuspended in 200 μl of staining buffer . For antibody staining, staining buffer containing 1 μg of either FITC-labeled isotype control antibody or FITC-labeled anti-CD40 antibody (clone HM40-3; BD Biosciences) was used, and cells were stained for 1 h, washed once with staining buffer, and resuspended in PBS. CD40 surface expression level was determined using a FACScan flow cytometer (Becton Dickinson).
Western blot
Cells were harvested in RIPA buffer (PBS containing 1% NP-40, 0.1% SDS and 0.5% sodium deoxycholate). Total protein concentrations were determined by Lowry assay (Bio-Rad) and equal quantities were precipitated with cold acetone by centrifugation. Protein pellets were vacuum dried and resuspended in load dye (Invitrogen) containing 5% ?-mercaptoethanol. Samples were heated to 92°C for 10 min prior to gel loading. Protein samples were separated on 10% PAGE Tris-glycine gels and transferred to PVDF membranes. Membranes were blocked with blocking solution (TBS-T containing 5% non-fat dry milk) and blotted with appropriate antibody. The polyclonal CD40 antibody was obtained from Calbiochem. G3PDH monoclonal antibody was obtained from Advanced Immunochemical, TRADD antibody was obtained from Cell Signaling, and HRP-conjugated secondary antibodies were obtained from Jackson Immunoresearch. Protein bands were visualized using ECL-Plus (Amersham-Pharmacia).
ELISA assay
Levels of mouse IL-12 in the supernatants of activated macrophages were measured with mouse IL-12 p40 + p70 ELISA kit (Biosource), according to the manufacturer’s instructions.
RT–PCR
Total RNA was isolated using an RNeasy Mini Kit (Qiagen). Two-step RT–PCR was performed using primers complementary to sequences of the CD40 gene (GenBank accession no. M83312 ). Reverse transcription was performed using a reverse primer (5'-TGATATAGAGAAACACCCCGAAA ATGG-3') complementary to sequence in exon 7. The resulting cDNA was subjected to 35 cycles of PCR using a forward primer consisting of a sequence span identical to that found in exon 5 of the gene (5'-GCCACTGAGACCA CTGATACCGTCTGT-3') as well as the reverse primer used for cDNA generation. The resulting PCR products were separated on a 1.6% agarose gel. PCR products were excised and the DNA purified. The resulting products were sequenced using primers used in PCR. Real-time quantitative RT–PCR was performed on total RNA from BCL1 or primary macrophages using an ABI Prism? 7700. Primer and dual-labeled probe sequences were as follows: mouse IL-12 p40: forward 5'-GCCAGTACACCTGCCACAAA-3', reverse 5'-GACCAAATTCCATTTTCCTTCTTG-3', probe 5'-FAM- AGGCGAGACTCTGAGCCACTCACATCTG-TAMRA-3'; mouse CD18: forward 5'-CTGCATGTCCGGAGGAAATT-3', reverse 5'-AGCCATCGTCTGTGGCAAA-3', probe 5'-FAM-CTGGCGCAATGTCACGAGGCTG-TAMRA-3'; mouse CD40, type 1: forward 5'-CACTGATACCGTC TGTCATCCCT-3', reverse 5'-AGTTCTTATCCTCACAG CTTGTCCA-3', probe 5'-FAM-AGTCGGCTTCTTCTC CAATCAGTCATCACTT-TAMRA-3'; mouse CD40, type 2: forward 5'-CACTGATACCGTCTGTCATCCCT-3', reverse 5'-CCACATCCGGGACTTTAAACCTTGT-3', probe 5'-FAM-CCAGTCGGCTTCTTCTCCAATCAGTCA-TAMRA-3'; mouse CD40: forward 5'-TGTGTTACGTGCAGTGACA AACAG-3', reverse 5'-GCTTCCTGGCTGGCACAA-3', probe 5'-FAM-CCTCCACGATCGCCAGTGCTGTG-TAM RA-3'; mouse cyclophilin: forward 5'-TCGCCGCTTGCT GCA-3'; reverse 5'-ATCGGCCGTGATGTCGA-3', probe 5'-FAM-CCATGGTCAACCCCACCGTGTTC-TAMRA-3'.
PNA synthesis, purification and characterization
PNAs were synthesized manually using a LabMate 24 parallel synthesizer (Advanced Chemtech) as previously described for single compound synthesis (23,24). Synthesis was performed on solid phase, in 10 μmol scale using a MBHA resin LL (NovaBiochem, 01-64-0006), which was preloaded to 0.1–0.2 mmol/g with Boc-Lys(2-Cl-Z)-OH, and commercially available tert-butyloxycarbonyl/benzyloxycarbonyl (Boc/Cbz) protected PNA monomers (Perseptive Biosystems; GEN063010, GEN063011, GEN063012, GEN063013). Completion of coupling was verified by randomized sampling and the qualitative Kaiser test. An additional coupling step was included when the Kaiser test was inconclusive. PNAs were deprotected and cleaved in parallel using methods previously applied to single compound synthesis (23,24). Purification was performed on a Gilson HPLC system (215 liquid handler, 155 UV/VIS and 321 pump), by reversed-phase high-performance liquid chromatography (RP–HPLC), using a DELTA PAK (C-18, 15 μm, 300 ?, 300 x 7.8 mm, 3 ml/min). A linear gradient from solvent A to B was used as the liquid phase. Purity was determined by analytical HPLC (0.1% trifluoroacetic acid in acetonitrile) and composition confirmed by mass spectrometry. A purity level of >95% was generally accomplished. Samples were lyophilized on a FreezeZone 6 (LABCONCO, equipped with a chamber to accommodate racks) and stored at –20°C.
Synthesis, purification and characterization of oligolysine conjugated PNAs
PNA–lysine conjugates were synthesized in 10 μmol scale in parallel on a LabMate 24 parallel synthesizer (Advanced Chemtech) using a solid support bound PNA that was synthesized as previously described (23,24). The quality of the PNA synthesis was checked prior to peptide conjugation by cleavage and QC of a fraction of the PNA from the support as previously described. Peptide synthesis was performed by a standard solid-phase tert-butoxycarbonyl (Boc) strategy on support bound PNA, leading to lysine conjugation at the N-terminal end of the PNA. In addition to the N-terminal lysine conjugate, each PNA also contained a single lysine unit at the C-terminus due to the fact that synthesis was performed on Boc-Lys(Z-Cl-Z)OH MBHA resin. The PNA–peptide constructs were synthesized, deprotected and cleaved in parallel. Purification was performed by reversed-phase high-performance liquid chromatography (RP–HPLC), as described above. Purity and composition was determined/confirmed as described above. A purity level of >95% was generally accomplished. Samples were lyophilized on a FreezeZone 6 (LABCONCO, equipped with a chamber to accommodate racks) and stored at –20°C.
Synthesis, purification and characterization of MOE oligonucleotides
Synthesis of MOEs was performed on a 2 μmol scale on a DNA/RNA synthesizer 380B (Applied Biosystems) using standard phosphoramidite methodology according to the manufacturer’s recommendations and 3H-1,2-benzodithiol-3-one 1,1-dioxide (0.05 M in CH3CN) as the sulfur transfer reagent. After HPLC purification the compounds were analyzed by electrospray ionization mass spectrometry, lyophilized and stored at –20°C.
RESULTS AND DISCUSSION
Identification of specific PNA and MOE inhibitors of CD40 expression
A panel of oligomers containing either MOE or PNA backbones was synthesized (Table 1). These oligomers were designed to regions of the murine CD40 pre-mRNA where binding could potentially alter splicing or inhibit translation, both of which are validated non-RNase-dependent mechanisms (22,25–28). The MOE and PNA oligomers were delivered by electroporation into BCL1 cells, a mouse B-cell line that constitutively expresses high levels of CD40. Following a 48 h incubation period, cells were harvested and analyzed for surface expression of CD40 by flow cytometry. The activities of the PNA oligomers were compared to those of the MOE oligomers of identical sequence and length (Fig. 1). There was a strong correlation between the activities of PNA and MOE oligomers designed to the same target sites, as demonstrated by both paired sample t-test and Spearman rank correlation (P < 0.001, in both cases). These results demonstrate that the sequence dependence of CD40 inhibitory activity is similar for MOE- and PNA-based inhibitors. The fact that oligomers of different backbone chemistry display similar activity when targeted to identical RNA sequences, supports the notion that PNA and MOE oligomers inhibit CD40 cell surface expression by the same antisense mechanism.
Table 1. Sequences of PNA and MOE compounds targeting the murine CD40 pre-mRNA
Figure 1. Activity of MOE and PNA antisense oligomers as measured by flow cytometry. BCL1 cells were electroporated with 20 μM oligomer and incubated for 48 h. Cells were analyzed by flow cytometry for CD40 cell surface expression levels. Values shown represent the relative mean fluorescence intensities obtained from each treatment group. Error bars represent the standard deviations (n = 3) for each group. Results were obtained from cells treated with MOE (A) or sequence-matched PNA (B) oligomers. ISIS 29848 and ISIS 117886 were included in each screen as negative and positive controls, respectively, for RNase H-mediated CD40 inhibition.
A PNA targeted towards the 3' end of exon 6, ISIS 208529, was found to be the most active PNA oligomer. The corresponding MOE sequence, ISIS 208353, was also found to be the most active oligomer within the series of MOE compounds. To further assess the specificity of ISIS 208529, CD40 levels were measured by western blot using protein extracts prepared from BCL1 cells electroporated with either the parent PNA (ISIS 208529), a PNA containing a four-base mismatch (ISIS 256644), or one of two PNAs of unrelated sequences (ISIS 256645 and ISIS 256646) (Fig. 2). In each case, protein was harvested and analyzed 48 h after electroporation. Using an antibody specific for the C-terminal region of the CD40 protein, western blot analysis showed that none of the three mismatched PNAs affected CD40 expression, whereas the inhibition of CD40 expression by ISIS 208529 was confirmed.
Figure 2. Sequence specificity of PNA-mediated inhibition of CD40 protein expression. BCL1 cells were electroporated with no compound (NT), 10 μM lead PNA (ISIS 208529) or 10 μM mismatch control PNAs (ISIS 256644, ISIS 256645, ISIS 256646). Protein was harvested 48 h later and analyzed for CD40 expression by western blot. G3PDH protein levels were visualized to verify equal loading.
Mode of action of the PNA inhibitor ISIS 208529
Pre-mRNA splicing represents an important pathway by which the cells affect post-transcriptional regulation of gene expression. The target sequence for ISIS 208529 is located on the 3' end of exon 6 of the primary murine CD40 transcript, abutting the splice junction. Previous studies have documented alternatively spliced forms of murine CD40 (29). The type 1 transcript, which retains exon 6, is the predominant form. Its translation product is the canonical membrane-bound, signaling-competent CD40 protein (Fig. 3A and B). The type 2 transcript is lower in abundance and does not contain exon 6. The omission of exon 6 causes a frame-shift in codons contained in exons 7, 8 and 9, and leads to mistranslation of the sequence encoding for the transmembrane domain and truncation of the protein due to an in-frame stop codon in exon 8 (29). In order to verify the mechanism by which ISIS 208529 reduces the expression of cell-surface CD40 expression, RT–PCR was performed on RNA isolated from both treated and untreated cells using primers seated in exons 5 and 7. A sequence-specific, PNA-mediated shift in the relative abundance of the two CD40 splice forms was observed upon treatment with ISIS 208529. No change in the relative abundance of the splice forms was observed in cells treated with the four-base mismatched PNA, ISIS 256644 (Fig. 3B). The identities of the splice forms were examined by sequencing of the two RT–PCR products and confirmed to be the type 1 and type 2 CD40 transcripts. While no attempt was made to investigate the detailed mechanism by which ISIS 208529 caused the omission of exon 6, the positioning of the binding site at the 3' end of exon 6 points to either steric interference with snRNP recognition of the splice donor site or interference with spliceosome assembly as possible modes of action. Whereas increased levels of the type 2 transcript were clearly detectable by RT–PCR, we were unable to detect the corresponding protein in the supernatant from BCL1 cells treated with ISIS 208529 (data not shown). The possibility exists, however, that a translated product from the type 2 transcript accumulated within the cell at levels below the detection limit by western blot. Regardless, the truncated CD40 protein encoded by the type 2 transcript would lack its signaling ability due to the omission of its transmembrane domain.
Figure 3. Redirection of murine CD40 splicing by ISIS 208529. (A) The two predominant splice forms of murine CD40 are shown. The binding site for the lead PNA (208529), as depicted, is located at the 3' end of exon 6. (B) BCL1 cells were electroporated in the presence of no compound or 10 μM either 256644 (four-base mismatch control) or 208529. RNA was harvested 48 h later and subsequently analyzed by RT–PCR using primers positioned in exons 5 and 7. RT–PCR products were separated on a 1.6% agarose gel and visualized by ethidium bromide staining.
Evaluation of PNAs targeting sequences surrounding the binding site for ISIS 208529
Further optimization of inhibitor binding was performed by designing additional PNA oligomers targeted to sites overlapping with or adjacent to the ISIS 208529 binding site. The PNA oligomers were designed to bind to CD40 RNA within a range of 10 nt upstream and downstream of the ISIS 208529 binding site (Table 2 and Fig. 4A). The activities of the resulting 10 PNAs, as well as that of ISIS 208529, were evaluated in parallel by western blot (Fig. 4B). Eight of the 10 PNAs demonstrated a level of activity similar to that of ISIS 208529. The number of active PNAs identified in this second screen is in contrast to the limited number of active PNAs identified in the initial screen, which may indicate that the 3' end of exon 6 of the primary CD40 transcript is generally accessible for inhibitor binding. Alternatively, splicing factors may be more sensitive to the perturbations in the RNA structure caused by PNA binding at this 20 nt region of the transcript. Two PNAs, ISIS 256636 and ISIS 256637, positioned slightly upstream from the 3' exon 6 splice site, failed to inhibit CD40 expression. This finding was verified by flow cytometry (data not shown). Examination of the primary sequences of the two inactive PNAs did not reveal any obvious features, such as a high guanosine content, that might promote the formation of undesirable secondary structure. Likewise, the RP–HPLC elution profiles for these two compounds did not indicate a tendency for self-aggregation. Furthermore, examination of the target RNA sequence did not reveal a propensity for secondary structure formation that might limit target accessibility. Hence, the underlying cause of the observed anomalous inactivity of these two PNAs remain elusive.
Table 2. Sequences of PNAs targeting sequences surrounding the binding site for ISIS 208529 and sequences designed to examine the effect of PNA length
Figure 4. Subscreen of PNAs targeting sequences surrounding the binding site for ISIS 208529. (A) Schematic representation of the binding sites for PNAs targeting the region around that of the lead PNA, ISIS 208529 (represented by dotted line). PNAs were designed to target the region 10 bases upstream and downstream, relative to the identified lead sequence, ISIS 208529. All PNAs were 15 units in length. (B) Inhibitory activity of PNAs on CD40 cell surface expression as assessed by western blot. BCL1 cells were electroporated with no compound (NT) or 10 μM of the various targeted PNAs (ISIS 256634 to ISIS 256643). The mismatch control PNA (ISIS 256644) was included. Protein was harvested 48 h later and analyzed for CD40 expression by western blot. G3PDH protein levels were visualized to verify equal loading.
The effect of PNA length on CD40 inhibitory activity
The effect of PNA length on activity was assessed by systematic variation of the length of the PNA inhibitor from seven to 20 monomer units. For the initial examination of length effects, 13 PNAs were designed and synthesized (Table 2). The first set, consisting of PNAs of seven to 14 units in length, were all targeted to portions of the binding site of ISIS 208529 (Fig. 5A). Each of these compounds as well as the 15mer parent, ISIS 208529, were electroporated into BCL1 cells at a final concentration of 10 μM. Three days following delivery, the cells were harvested and analyzed by western blot for CD40 levels (Fig. 5B). G3PDH protein levels were also measured to verify equal protein loading. While no apparent reduction in CD40 levels was observed in cells treated with compounds ranging from seven to 11 units in length, inhibition of CD40 expression was observed with compounds ranging from 12 to 15 units in length. The efficacy of the PNA inhibitors was found to increase with increasing length, up to a PNA length of 14 units, where efficacy reached a level similar to that displayed by the lead 15mer PNA, ISIS 208529. Subsequently, PNAs covering a range of 12 to 20 units in length were examined. PNAs were electroporated into BCL1 cells at various concentrations to determine their relative potencies (Fig. 5C). Compounds were evaluated for their ability to inhibit CD40 cell surface expression by flow cytometry. Potency was found to increase with increasing length, reaching a plateau at 14 unit length, beyond which no additional gain in potency was observed. This observation suggests that the potency of ISIS 208529 is not limited by its length, and that potency cannot be improved by increasing the length of the PNA. At this target site, EC50 values were in the range of 0.6 to 0.9 μM for all PNAs of 14 units or longer (Table 3).
Figure 5. Effect of PNA length on potency of CD40 inhibition. (A) Schematic representation of PNA length variation relative to the active ISIS 208529 inhibitor (represented by dotted line). (B) The effect of PNA length on activity as assessed by western blot. BCL1 cells were electroporated with no compound (NT), 10 μM PNAs of varying length (7–15 units). Protein was harvested 72 h later and analyzed for CD40 expression by western blot. G3PDH protein levels were visualized to verify equal loading. (C) CD40 depletion as a function of length and dose. PNAs ranging from 12 to 20 units in length were electroporated into BCL1 cells at various concentrations (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8 μM). Cells were harvested 3 days later and analyzed by flow cytometry for CD40 cell surface expression.
Table 3. Potency of CD40 inhibition as a function of PNA length
Dose and time dependence of CD40 inhibitory activity by ISIS 208529
The dose-dependent reduction of cell surface CD40 protein levels upon treatment of BCL1 cells with ISIS 208529 was evaluated by flow cytometry and was further supported by verification of CD40 protein depletion by western blot (Fig. 6A and B). Inhibition of CD40 protein expression was found to be dose dependent, with an EC50 of 1–2 μM. Specificity was verified by inclusion of a PNA containing a four-base mismatch (ISIS 256644). In order to assess the effect of ISIS 208529 over time, western blot analysis was used to study the effect of a single dose (10 μM) of ISIS 208529 for 8 days following electroporation (Fig. 7). Maximal inhibition of CD40 expression was observed 4 days post-treatment and persisted through the fifth day. A temporal delay in activity was observed that likely reflects the turnover time of the pre-existing CD40 protein. By day 8, the level of CD40 expression had returned to the levels measured in the untreated controls (Fig. 7). No change in CD40 expression levels was observed in cells treated with the four-base mismatched PNA (ISIS 256644).
Figure 6. Effect of dose on activity for the lead PNA inhibitor ISIS 208529. BCL1 cells were electroporated with either no PNA (NT), 16 μM mismatch control PNA (ISIS 256644) or various concentrations (16, 8, 4, 2, 1, 0.5 or 0.25 μM) of ISIS 208529. Cells were harvested three days later and analyzed by either flow cytometry (A) or western blot (B) for CD40 expression levels. For the western blot, the membrane was subsequently reblotted for G3PDH to verify protein loading was equal among samples. A representative blot is shown. The values shown in (A) represent the averages (n = 3 per group) and their corresponding standard deviations.
Figure 7. Time course of activity of lead PNA inhibitor. BCL1 cells were electroporated with no PNA or 10 μM either mismatch control PNA (ISIS 256644) or lead PNA (ISIS 208529). Cells were harvested 1, 2, 3, 4, 5 or 8 days later and protein lysates were analyzed by western blot for CD40 protein expression levels. Protein samples from cells harvested one day following electroporation with no PNA (1*) were included on both blots as a common reference. Blots were analyzed for G3PDH levels to verify equal loading.
Inhibitory activity of ISIS 208529 on CD40-dependent IL-12 production in primary murine macrophages
Macrophages, which express the CD40 receptor, play an important role in modulation of the immune response. Interaction of CD40 with CD154 triggers the secretion of numerous cytokines, such as IL-12. Hence, measurement of IL-12 levels provides a way to assess the functional loss of the CD40 receptor. The functional consequences of PNA-induced alternative splicing in primary murine macrophages were examined by electroporation of thioglycollate-elicited mouse peritoneal cells with various doses of ISIS 208529 or with the PNA containing a four-base mismatch, ISIS 256644. After electroporation, macrophages were selected by adherence to tissue culture plates, treated with IFN- for 4 h to induce CD40 cell surface expression, then stimulated with an activating CD40 antibody for 24 h. The level of IL-12 in the supernatant of PNA-treated macrophages after CD40 activation was examined by an ELISA assay. Treatment of macrophages with ISIS 208529 resulted in a dose-dependent reduction in IL-12 production (Fig. 8A). Treatment of macrophages with ISIS 208529 at a concentration of 3 μM resulted in 75% inhibition of IL-12 production compared to macrophages electroporated with no PNA, while 85% inhibition in IL-12 production was observed upon treatment with 10 μM ISIS 208529. Macrophages treated with the mismatch control PNA (ISIS 256644) showed no decrease in IL-12. Examination of CD40 levels by western blot demonstrated a dose-dependent reduction in CD40 protein levels following treatment with ISIS 208529, which correlated well with the decrease observed in IL-12 production (Fig. 8B). No reduction in CD40 protein was found after treatment with the mismatch control ISIS 256644. Examination of the CD40 splice forms by quantitative RT–PCR showed a 70% decrease in the predominant type 1 splice form, and a 2-fold increase in the alternative type 2 splice form, at 3 μM ISIS 208529 (Fig. 8C). The four-base mismatch control, ISIS 256644, had no significant effect on the relative abundance of the CD40 splice forms, indicating that inhibitory activity observed for ISIS 208529 was sequence dependent.
Figure 8. Dose-dependent effect of lead PNA inhibitor on CD40 expression and CD40-mediated IL-12 production in primary murine macrophages. Freshly isolated thioglycollate-elicited mouse peritoneal cells were electroporated with either the lead PNA (ISIS 208529) or the four-base mismatch control (ISIS 256644). Macrophages were purified by adhering to tissue culture plate, treated with 100 ng/ml mIFN- for 4 h, then with 10 μg/ml anti-CD40 antibody for 24 h. (A) ELISA assay measuring IL-12 p40+p70 in supernatant of PNA-treated macrophages after 24 h of CD40 activation. (B) Western blot for CD40 protein. The blot was reprobed for G3PDH protein levels to verify equal protein loading. (C) Real-time quantitative RT–PCR for type 1 and type 2 CD40 splice forms.
Effect of ISIS 208529 peptide conjugation on CD40 cell surface expression in BCL1 cells and in macrophages
Lysine conjugation of PNA has previously been found to improve PNA uptake into cells. Additional evidence demonstrating the utility of lysine-conjugated PNAs was provided by experiments in a transgenic mouse model, where a lysine-conjugated PNA demonstrated efficacy in a broad range of tissues (21). Hence, a series of conjugates were synthesized that contained one to eight lysine units at the N-terminus of ISIS 208529 (data not shown). These conjugates were evaluated as inhibitors of CD40 cell surface expression by flow cytometry without the use of transfection agents. The results indicated that the eight-lysine derivative, ISIS 278647, was a more effective inhibitor of CD40 expression when delivered by free uptake compared to its shorter analogs while maintaining similar potency as its unconjugated parent, ISIS 208529, when delivered by electroporation (data not shown). We interpreted this result to signify that the eight-lysine conjugate was more effectively transported across the cellular and nuclear membranes compared to the shorter analogs. Flow cytometry was used to quantitate the levels of fluorescently labeled PNA derivatives accumulating in cells. BCL1 cells accumulated both unconjugated parent and eight-lysine conjugate in a dose-dependent manner (Fig. 9). Cells accumulated >10-fold more conjugated PNA compared to unconjugated oligomer following 1 h incubation.
Figure 9. Oligolysine conjugated PNA promotes greater cellular compound accumulation. BCL1 cells were exposed to various concentrations (4, 2, 1, 0.5 or 0.25 μM) either ISIS 208529 or ISIS 278647, labeled with Oregon Green on the C-terminal lysing of each compound. Cells were washed twice with whole media, once with PBS, and subsequently lifted from the plates and analyzed for MFI as described. Propidium iodide exclusion was employed to verify cell membranes remained intact. Values shown represent the means and standard deviations from three replicates per treatment.
ISIS 278647 was further evaluated for its effect on CD40 expression in BCL1 cells without transfection or electroporation (‘free uptake’) (Fig. 10). Compared to untreated cells, BCL1 cells treated with ISIS 278647 at 10 μM, displayed reduced levels of the CD40 type 1 transcript and increased amounts of the type 2 transcript as determined by both standard RT–PCR (Fig. 10A) and real-time quantitative RT–PCR (Fig. 10B). ISIS 278647 caused an 85% decrease in the type 1 transcript and a >3-fold increase in the type 2 transcript. Neither the unconjugated lead PNA (ISIS 208529), nor an eight-lysine conjugated, four-base mismatched PNA (ISIS 287294), had any effect on the abundance of either splice variant or on total CD40 transcript relative to the untreated control. These results demonstrate that redirection of splicing by ‘free uptake’ and subsequent loss of the CD40 protein encoded by the type 1 transcript variant are dependent on both PNA sequence and inclusion of the eight-lysine carrier.
Figure 10. Free uptake of lysine-conjugated PNAs in BCL1 cells. BCL1 cells were either mock treated (NT) or treated with 10 μM either unconjugated lead PNA (ISIS 208529), eight-lysine-conjugated four-base mismatch control PNA (ISIS 287294) or eight-lysine-conjugated PNA (ISIS 278647) for 3 days in normal growth media. (A) Cellular RNA was purified and analyzed by standard RT–PCR for changes in the relative abundance of CD40 transcript variants. (B) Total RNA was also subjected to quantitative RT–PCR using primer/probe sets capable of individually quantifying each of the two transcript variants as well as one recognizing both splice forms. (C) Protein lysates were analyzed by western blot for changes in CD40 expression level. TRADD protein levels were also visualized to verify equal sample loading.
Analysis of the protein lysates by western blot, showed that ISIS 278647 promoted depletion of CD40 protein levels, whereas treatment with the unconjugated PNA, ISIS 208529, or the four-base mismatch control, ISIS 287294, had no effect on CD40 protein levels (Fig. 10C). In a separate experiment, the slight decrease in total CD40 transcript levels (Type 1 & 2) resulting from treatment with ISIS 278647 was confirmed by northern blot (data not shown).
The effect of the lysine conjugation of ISIS 208529 on CD40 expression and on the relative abundance of the type 1 and type 2 transcripts was also examined in primary murine macrophages (Fig. 11). Adherent peritoneal macrophages were incubated with various concentrations of unconjugated or conjugated PNA for 16 h, and treated with IFN- to induce CD40 protein expression. Expression of CD40 protein was then examined by western blot. No reduction in CD40 protein was observed after treatment with ISIS 208529 (Fig. 11A), while a modest reduction in CD40 protein levels was observed in macrophages treated with the four-lysine-conjugated PNA (ISIS 278643). In contrast, treatment with the eight-lysine-conjugated CD40 PNA (ISIS 278647) resulted in a dose-dependent decrease in CD40 protein levels (Fig. 11A). Treatment with ISIS 278647 at 10 μM resulted in undetectable CD40 protein levels as determined by western blot, indicating that the eight-lysine-conjugated PNA was readily taken up by the primary macrophages and that carrier conjugation did not prevent the PNA from binding to its target and attenuating CD40 protein expression. Treatment with an eight-lysine-conjugated four-base mismatch control PNA (ISIS 287294) did not affect CD40 protein levels, indicating that the observed effect on CD40 protein expression is sequence specific. Analysis of the CD40 splice forms by quantitative RT–PCR demonstrated that the eight-lysine-conjugated CD40 PNA (ISIS 278647) caused a substantial reduction in CD40 type 1 mRNA levels with a concomitant 5-fold induction of the CD40 type 2 transcript levels (Fig. 11B). The eight-lysine-conjugated four-base mismatch PNA (ISIS 287294) had no significant effect on the relative levels of the type 1 and type 2 splice forms.
Figure 11. Free uptake of lysine-conjugated PNAs in primary murine macrophages. Adherent thioglycollate-elicited peritoneal macrophages were incubated with the indicated CD40 PNAs for 16 h. The cells were treated with 100 ng/ml mIFN- for 4 h, then with 10 μg/ml anti-CD40 antibody (clone 3/23) for 6 h. (A) Western blot for CD40 protein. The blot was re-probed for G3PDH protein levels to verify equal protein loading. (B) Real-time quantitative RT–PCR for type 1 and type 2 CD40 splice forms. (C) Real-time quantitative RT–PCR for mCD18.
In summary, expression of the IL-12 cytokine in primary murine macrophages, was reduced in a sequence-specific, dose-dependent manner as a consequence of ISIS 208529 delivery by electroporation. Moreover, the reduction in IL-12 levels were found to correlate with the reduced levels of full-length CD40 protein in the macrophages. As observed in the BCL1 cells, the reduction in type 1 transcript levels in primary murine macrophages was found to coincide with an increase in the amount of the type 2 transcript. Reduction of CD40 expression by free uptake in macrophages was found to rely on lysine conjugation of the PNA. A modest reduction in CD40 protein was observed upon treatment with a four-lysine-conjugated inhibitor (ISIS 278643), suggesting that four lysines will not promote efficient cellular uptake. In contrast, treatment with the eight-lysine-conjugated PNA (ISIS 278647) led to complete depletion of CD40 protein levels. The dramatic improvement in efficacy of the eight-lysine-conjugated PNA relative to that of the unconjugated ISIS 208529 stresses the importance of identification and optimization of suitable carriers for PNA antisense molecules. The observed improved efficacy is likely to reflect improved cellular uptake for the eight-lysine-conjugated PNA. The possibility cannot be ruled out, however, that the peptide conjugate facilitates improved PNA activity by some other means, e.g. improved trafficking or improved endosomal release.
Significant interest has evolved in CD40–CD154 cognate recognition and subsequent downstream signaling, due to the central role of the CD40–CD154 interaction in autoimmune and/or inflammatory disease. Interference with CD40–CD154 signaling appears to be a promising approach for the development of therapeutics. The results described here demonstrating the depletion of CD40 protein and reduction of IL-12 production in macrophages, validates antisense-mediated interference with CD40–CD154 cognate interaction as a means to control downstream signaling. Development of specific antisense inhibitors of CD40 cell surface expression may provide a therapeutic approach to effectively modulate CD40–CD154-mediated signaling.
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*To whom correspondence should be addressed. Tel: +1 760 603 3852; Fax: +1 760 603 4654; Email: aeldrup@isisph.com
ABSTRACT
Cognate recognition between the CD40 receptor and its ligand, CD154, is thought to play a central role in the initiation and propagation of immune responses. We describe the specific down regulation of cell surface associated CD40 protein expression by use of a peptide nucleic acid (PNA) antisense inhibitor, ISIS 208529, that is designed to bind to the 3' end of the exon 6 splice junction within the primary CD40 transcript. Binding of ISIS 208529 was found to alter constitutive splicing, leading to the accumulation of a transcript lacking exon 6. The resulting protein product lacks the transmembrane domain. ISIS 208529-mediated CD40 protein depletion was found to be sequence specific and dose dependent, and was dependent on the length of the PNA oligomer. CD40-dependent induction of IL-12 in primary murine macrophages was attenuated in cells treated with ISIS 208529. Oligolysine conjugation to the PNA inhibitor produced an inhibitor, ISIS 278647, which maintained its specificity and displayed efficacy in BCL1 cells and in primary murine macrophages in the absence of delivery agents. These results demonstrate that PNA oligomers can be effective inhibitors of CD40 expression and hence may be useful as novel immuno-modulatory agents.
INTRODUCTION
CD40 is a cell membrane protein that plays a key role in the initiation and propagation of immune responses (1–3). CD40, a member of the tumor necrosis factor (TNF) receptor family of proteins, is expressed on antigen-presenting cells such as B lymphocytes and dendritic cells as well as additional cell types such as macrophages, endothelial cells, smooth muscle cells and epithelial cells. The ‘ligand’ for CD40 is CD154 (also known as CD40L, or gp39), a member of the TNF family and is expressed mainly on activated T cells and platelets (4–6).
Perhaps the best characterized response to signaling through CD40 on immune cells is B-lymphocyte activation, which results in increased expression of B7 molecules (CD80 and CD86), protection against apoptosis, and Ig class switching (2,6–8). In addition to activation of B lymphocytes, binding to CD40 expressed on other antigen-presenting cells leads to the secretion of cytokines such as IL-1?, TNF- and IL-12, the production of chemokines, as well as expression of adhesion molecules such as ICAM-1 and VCAM-1. Numerous studies have demonstrated that interference with CD40–CD154 signaling has profound effects on immune responses in cellular and animal model systems and suggest that inhibition of CD40 function could offer therapeutic benefit for inflammatory and autoimmune diseases (1,3).
Antisense technology represents a novel approach to the modulation of CD40–CD154 cognate signaling. There are multiple antisense mechanisms that operate in mammalian cells (9). The best characterized mechanism is RNase H dependent, in which oligonucleotides containing DNA or DNA analogs bind to the target RNA and recruit RNase H, resulting in degradation of the target RNA. There is one approved antisense drug and over 20 drugs in various stages of clinical trials that work by this mechanism of action. RNA interference is a recently characterized antisense mechanism that utilizes double-stranded RNA oligonucleotides that dissociate with the antisense strand binding to target RNA and promoting degradation by an unknown RNase (10,11). There are also antisense mechanisms that do not promote degradation of target RNA, such as translation arrest and inhibition of splicing (9). Oligonucleotides that work by the latter mechanisms bind to the target RNA with high affinity and do not support RNase activities. Examples of such modified oligonucleotides include uniform 2' modifications such as 2'-O-methyl and 2'-O-methoxyethyl, morpholino or peptide nucleic acid (PNA).
The pharmacological, toxicological and pharmacokinetic behavior of second generation, 2'-O-methyl and 2'-O-methoxyethyl phosphorothioate oligonucleotides (MOEs) are well characterized (12–16). These antisense oligonucleotides primarily distribute to the liver, kidney, spleen and mesenteric lymph nodes (12). PNAs are an example of a third generation antisense oligonucleotide analog with the potential to exhibit different pharmacological, toxicological and pharmacokinetic properties than 2'-modified oligonucleotides due to differences in physical chemical properties. PNAs lack the negatively charged backbone characteristic to most other nucleic acid analogs, but instead incorporate a neutral amide linkage. Preliminary studies examining the pharmacokinetics of unmodified PNA suggest that they exhibit limited tissue distribution and are rapidly excreted in the urine (17) (C. F. Bennett, unpublished results). These findings have led investigators to seek modifications that confer a more favorable pharmacokinetic profile. Furthermore, progress in developing PNA oligomers has been impeded by the difficulty in intracellular delivery of PNA in cultured cells. In contrast to charged oligonucleotides, PNA molecules are not readily amenable to simple cationic lipid-mediated delivery (18). Considerable effort has been directed towards peptide conjugation of PNA as both a means to enhance cellular uptake and as a way to direct delivery to certain tissue types (19). Mixed results have been obtained through the conjugation of amphipathic peptides like penetratin (which is derived from the homeodomain of Antennapedia) (20). Recently, it has been reported that a conjugate of (lys)4 to PNAs exhibit antisense effects when delivered to cells in culture and displays antisense effects in a variety of tissues following injection into mice (21,22).
Here we demonstrate that uniformly modified MOE and PNA oligomers specifically decrease cell surface expression of the CD40 receptor on BCL1 cells. PNA-mediated reduction of cell surface CD40 expression is shown to occur through an RNase-independent mechanism of action, resulting in an alternatively spliced CD40 transcript. This transcript encodes a truncated CD40 protein lacking its transmembrane domain. PNA-mediated depletion of cell-associated CD40 protein is demonstrated to be sequence specific, dose dependent and dependent on oligomer length. Treatment of primary murine macrophages with the PNA inhibitor results in a decrease in CD40-mediated IL-12 production. Thus, this antisense agent reduces the expression of cell-associated CD40, leading to the inhibition of signaling through CD40 and ultimately to the reduction of IL-12 cytokine production. Moreover, N-terminal conjugation of eight lysines onto the PNA is shown to lead to a compound that inhibits CD40 expression in BCL1 cells and in macrophages in the absence of facilitated delivery methodologies.
MATERIALS AND METHODS
Cells
Murine B-cell lymphoma cells, BCL1 cells, were obtained from the American Type Culture Collection and grown in normal growth medium . Cells were incubated in a humidified chamber at 37°C, containing 5% CO2. Antisense agents were delivered to cells by electroporation (200 V, 13 , 1000 μFa) using 2 mm gap width cuvettes and a BTX 600 Electro Cell Manipulator. Cells were plated in normal growth medium and incubated for the indicated times prior to harvest.
Primary thioglycollate-elicited macrophages were isolated by peritoneal lavage from 6- to 8-week-old female C57Bl/6 mice that had been injected with 1 ml of 3% thioglycollate broth 4 days previously. PNAs were delivered to unpurified peritoneal cells by a single 6 ms pulse, 90 V, on a BTX square wave electroporator in 1 mm cuvettes. After electroporation, the cells were plated for 1 h in serum-free RPMI 1640 (supplemented with 10 mM HEPES) at 37°C, 5% CO2 to allow the macrophages to attach. Non-adherent cells were then washed away and the media was replaced with complete RPMI 1640 (10% FBS, 10 mM HEPES). Primary macrophages were activated by treatment with 100 ng/ml rIFN- (R&D Systems) for 4 h, followed by 10 μg/ml anti-CD40 antibody (clone 3/23; BD Pharmingen) for the indicated timepoints.
Flow cytometry analysis
Cells were detached from culture plates with 0.25% trypsin. Trypsin was neutralized with an equal volume of normal growth medium and cells were pelleted. Cell pellets were resuspended in 200 μl of staining buffer . For antibody staining, staining buffer containing 1 μg of either FITC-labeled isotype control antibody or FITC-labeled anti-CD40 antibody (clone HM40-3; BD Biosciences) was used, and cells were stained for 1 h, washed once with staining buffer, and resuspended in PBS. CD40 surface expression level was determined using a FACScan flow cytometer (Becton Dickinson).
Western blot
Cells were harvested in RIPA buffer (PBS containing 1% NP-40, 0.1% SDS and 0.5% sodium deoxycholate). Total protein concentrations were determined by Lowry assay (Bio-Rad) and equal quantities were precipitated with cold acetone by centrifugation. Protein pellets were vacuum dried and resuspended in load dye (Invitrogen) containing 5% ?-mercaptoethanol. Samples were heated to 92°C for 10 min prior to gel loading. Protein samples were separated on 10% PAGE Tris-glycine gels and transferred to PVDF membranes. Membranes were blocked with blocking solution (TBS-T containing 5% non-fat dry milk) and blotted with appropriate antibody. The polyclonal CD40 antibody was obtained from Calbiochem. G3PDH monoclonal antibody was obtained from Advanced Immunochemical, TRADD antibody was obtained from Cell Signaling, and HRP-conjugated secondary antibodies were obtained from Jackson Immunoresearch. Protein bands were visualized using ECL-Plus (Amersham-Pharmacia).
ELISA assay
Levels of mouse IL-12 in the supernatants of activated macrophages were measured with mouse IL-12 p40 + p70 ELISA kit (Biosource), according to the manufacturer’s instructions.
RT–PCR
Total RNA was isolated using an RNeasy Mini Kit (Qiagen). Two-step RT–PCR was performed using primers complementary to sequences of the CD40 gene (GenBank accession no. M83312 ). Reverse transcription was performed using a reverse primer (5'-TGATATAGAGAAACACCCCGAAA ATGG-3') complementary to sequence in exon 7. The resulting cDNA was subjected to 35 cycles of PCR using a forward primer consisting of a sequence span identical to that found in exon 5 of the gene (5'-GCCACTGAGACCA CTGATACCGTCTGT-3') as well as the reverse primer used for cDNA generation. The resulting PCR products were separated on a 1.6% agarose gel. PCR products were excised and the DNA purified. The resulting products were sequenced using primers used in PCR. Real-time quantitative RT–PCR was performed on total RNA from BCL1 or primary macrophages using an ABI Prism? 7700. Primer and dual-labeled probe sequences were as follows: mouse IL-12 p40: forward 5'-GCCAGTACACCTGCCACAAA-3', reverse 5'-GACCAAATTCCATTTTCCTTCTTG-3', probe 5'-FAM- AGGCGAGACTCTGAGCCACTCACATCTG-TAMRA-3'; mouse CD18: forward 5'-CTGCATGTCCGGAGGAAATT-3', reverse 5'-AGCCATCGTCTGTGGCAAA-3', probe 5'-FAM-CTGGCGCAATGTCACGAGGCTG-TAMRA-3'; mouse CD40, type 1: forward 5'-CACTGATACCGTC TGTCATCCCT-3', reverse 5'-AGTTCTTATCCTCACAG CTTGTCCA-3', probe 5'-FAM-AGTCGGCTTCTTCTC CAATCAGTCATCACTT-TAMRA-3'; mouse CD40, type 2: forward 5'-CACTGATACCGTCTGTCATCCCT-3', reverse 5'-CCACATCCGGGACTTTAAACCTTGT-3', probe 5'-FAM-CCAGTCGGCTTCTTCTCCAATCAGTCA-TAMRA-3'; mouse CD40: forward 5'-TGTGTTACGTGCAGTGACA AACAG-3', reverse 5'-GCTTCCTGGCTGGCACAA-3', probe 5'-FAM-CCTCCACGATCGCCAGTGCTGTG-TAM RA-3'; mouse cyclophilin: forward 5'-TCGCCGCTTGCT GCA-3'; reverse 5'-ATCGGCCGTGATGTCGA-3', probe 5'-FAM-CCATGGTCAACCCCACCGTGTTC-TAMRA-3'.
PNA synthesis, purification and characterization
PNAs were synthesized manually using a LabMate 24 parallel synthesizer (Advanced Chemtech) as previously described for single compound synthesis (23,24). Synthesis was performed on solid phase, in 10 μmol scale using a MBHA resin LL (NovaBiochem, 01-64-0006), which was preloaded to 0.1–0.2 mmol/g with Boc-Lys(2-Cl-Z)-OH, and commercially available tert-butyloxycarbonyl/benzyloxycarbonyl (Boc/Cbz) protected PNA monomers (Perseptive Biosystems; GEN063010, GEN063011, GEN063012, GEN063013). Completion of coupling was verified by randomized sampling and the qualitative Kaiser test. An additional coupling step was included when the Kaiser test was inconclusive. PNAs were deprotected and cleaved in parallel using methods previously applied to single compound synthesis (23,24). Purification was performed on a Gilson HPLC system (215 liquid handler, 155 UV/VIS and 321 pump), by reversed-phase high-performance liquid chromatography (RP–HPLC), using a DELTA PAK (C-18, 15 μm, 300 ?, 300 x 7.8 mm, 3 ml/min). A linear gradient from solvent A to B was used as the liquid phase. Purity was determined by analytical HPLC (0.1% trifluoroacetic acid in acetonitrile) and composition confirmed by mass spectrometry. A purity level of >95% was generally accomplished. Samples were lyophilized on a FreezeZone 6 (LABCONCO, equipped with a chamber to accommodate racks) and stored at –20°C.
Synthesis, purification and characterization of oligolysine conjugated PNAs
PNA–lysine conjugates were synthesized in 10 μmol scale in parallel on a LabMate 24 parallel synthesizer (Advanced Chemtech) using a solid support bound PNA that was synthesized as previously described (23,24). The quality of the PNA synthesis was checked prior to peptide conjugation by cleavage and QC of a fraction of the PNA from the support as previously described. Peptide synthesis was performed by a standard solid-phase tert-butoxycarbonyl (Boc) strategy on support bound PNA, leading to lysine conjugation at the N-terminal end of the PNA. In addition to the N-terminal lysine conjugate, each PNA also contained a single lysine unit at the C-terminus due to the fact that synthesis was performed on Boc-Lys(Z-Cl-Z)OH MBHA resin. The PNA–peptide constructs were synthesized, deprotected and cleaved in parallel. Purification was performed by reversed-phase high-performance liquid chromatography (RP–HPLC), as described above. Purity and composition was determined/confirmed as described above. A purity level of >95% was generally accomplished. Samples were lyophilized on a FreezeZone 6 (LABCONCO, equipped with a chamber to accommodate racks) and stored at –20°C.
Synthesis, purification and characterization of MOE oligonucleotides
Synthesis of MOEs was performed on a 2 μmol scale on a DNA/RNA synthesizer 380B (Applied Biosystems) using standard phosphoramidite methodology according to the manufacturer’s recommendations and 3H-1,2-benzodithiol-3-one 1,1-dioxide (0.05 M in CH3CN) as the sulfur transfer reagent. After HPLC purification the compounds were analyzed by electrospray ionization mass spectrometry, lyophilized and stored at –20°C.
RESULTS AND DISCUSSION
Identification of specific PNA and MOE inhibitors of CD40 expression
A panel of oligomers containing either MOE or PNA backbones was synthesized (Table 1). These oligomers were designed to regions of the murine CD40 pre-mRNA where binding could potentially alter splicing or inhibit translation, both of which are validated non-RNase-dependent mechanisms (22,25–28). The MOE and PNA oligomers were delivered by electroporation into BCL1 cells, a mouse B-cell line that constitutively expresses high levels of CD40. Following a 48 h incubation period, cells were harvested and analyzed for surface expression of CD40 by flow cytometry. The activities of the PNA oligomers were compared to those of the MOE oligomers of identical sequence and length (Fig. 1). There was a strong correlation between the activities of PNA and MOE oligomers designed to the same target sites, as demonstrated by both paired sample t-test and Spearman rank correlation (P < 0.001, in both cases). These results demonstrate that the sequence dependence of CD40 inhibitory activity is similar for MOE- and PNA-based inhibitors. The fact that oligomers of different backbone chemistry display similar activity when targeted to identical RNA sequences, supports the notion that PNA and MOE oligomers inhibit CD40 cell surface expression by the same antisense mechanism.
Table 1. Sequences of PNA and MOE compounds targeting the murine CD40 pre-mRNA
Figure 1. Activity of MOE and PNA antisense oligomers as measured by flow cytometry. BCL1 cells were electroporated with 20 μM oligomer and incubated for 48 h. Cells were analyzed by flow cytometry for CD40 cell surface expression levels. Values shown represent the relative mean fluorescence intensities obtained from each treatment group. Error bars represent the standard deviations (n = 3) for each group. Results were obtained from cells treated with MOE (A) or sequence-matched PNA (B) oligomers. ISIS 29848 and ISIS 117886 were included in each screen as negative and positive controls, respectively, for RNase H-mediated CD40 inhibition.
A PNA targeted towards the 3' end of exon 6, ISIS 208529, was found to be the most active PNA oligomer. The corresponding MOE sequence, ISIS 208353, was also found to be the most active oligomer within the series of MOE compounds. To further assess the specificity of ISIS 208529, CD40 levels were measured by western blot using protein extracts prepared from BCL1 cells electroporated with either the parent PNA (ISIS 208529), a PNA containing a four-base mismatch (ISIS 256644), or one of two PNAs of unrelated sequences (ISIS 256645 and ISIS 256646) (Fig. 2). In each case, protein was harvested and analyzed 48 h after electroporation. Using an antibody specific for the C-terminal region of the CD40 protein, western blot analysis showed that none of the three mismatched PNAs affected CD40 expression, whereas the inhibition of CD40 expression by ISIS 208529 was confirmed.
Figure 2. Sequence specificity of PNA-mediated inhibition of CD40 protein expression. BCL1 cells were electroporated with no compound (NT), 10 μM lead PNA (ISIS 208529) or 10 μM mismatch control PNAs (ISIS 256644, ISIS 256645, ISIS 256646). Protein was harvested 48 h later and analyzed for CD40 expression by western blot. G3PDH protein levels were visualized to verify equal loading.
Mode of action of the PNA inhibitor ISIS 208529
Pre-mRNA splicing represents an important pathway by which the cells affect post-transcriptional regulation of gene expression. The target sequence for ISIS 208529 is located on the 3' end of exon 6 of the primary murine CD40 transcript, abutting the splice junction. Previous studies have documented alternatively spliced forms of murine CD40 (29). The type 1 transcript, which retains exon 6, is the predominant form. Its translation product is the canonical membrane-bound, signaling-competent CD40 protein (Fig. 3A and B). The type 2 transcript is lower in abundance and does not contain exon 6. The omission of exon 6 causes a frame-shift in codons contained in exons 7, 8 and 9, and leads to mistranslation of the sequence encoding for the transmembrane domain and truncation of the protein due to an in-frame stop codon in exon 8 (29). In order to verify the mechanism by which ISIS 208529 reduces the expression of cell-surface CD40 expression, RT–PCR was performed on RNA isolated from both treated and untreated cells using primers seated in exons 5 and 7. A sequence-specific, PNA-mediated shift in the relative abundance of the two CD40 splice forms was observed upon treatment with ISIS 208529. No change in the relative abundance of the splice forms was observed in cells treated with the four-base mismatched PNA, ISIS 256644 (Fig. 3B). The identities of the splice forms were examined by sequencing of the two RT–PCR products and confirmed to be the type 1 and type 2 CD40 transcripts. While no attempt was made to investigate the detailed mechanism by which ISIS 208529 caused the omission of exon 6, the positioning of the binding site at the 3' end of exon 6 points to either steric interference with snRNP recognition of the splice donor site or interference with spliceosome assembly as possible modes of action. Whereas increased levels of the type 2 transcript were clearly detectable by RT–PCR, we were unable to detect the corresponding protein in the supernatant from BCL1 cells treated with ISIS 208529 (data not shown). The possibility exists, however, that a translated product from the type 2 transcript accumulated within the cell at levels below the detection limit by western blot. Regardless, the truncated CD40 protein encoded by the type 2 transcript would lack its signaling ability due to the omission of its transmembrane domain.
Figure 3. Redirection of murine CD40 splicing by ISIS 208529. (A) The two predominant splice forms of murine CD40 are shown. The binding site for the lead PNA (208529), as depicted, is located at the 3' end of exon 6. (B) BCL1 cells were electroporated in the presence of no compound or 10 μM either 256644 (four-base mismatch control) or 208529. RNA was harvested 48 h later and subsequently analyzed by RT–PCR using primers positioned in exons 5 and 7. RT–PCR products were separated on a 1.6% agarose gel and visualized by ethidium bromide staining.
Evaluation of PNAs targeting sequences surrounding the binding site for ISIS 208529
Further optimization of inhibitor binding was performed by designing additional PNA oligomers targeted to sites overlapping with or adjacent to the ISIS 208529 binding site. The PNA oligomers were designed to bind to CD40 RNA within a range of 10 nt upstream and downstream of the ISIS 208529 binding site (Table 2 and Fig. 4A). The activities of the resulting 10 PNAs, as well as that of ISIS 208529, were evaluated in parallel by western blot (Fig. 4B). Eight of the 10 PNAs demonstrated a level of activity similar to that of ISIS 208529. The number of active PNAs identified in this second screen is in contrast to the limited number of active PNAs identified in the initial screen, which may indicate that the 3' end of exon 6 of the primary CD40 transcript is generally accessible for inhibitor binding. Alternatively, splicing factors may be more sensitive to the perturbations in the RNA structure caused by PNA binding at this 20 nt region of the transcript. Two PNAs, ISIS 256636 and ISIS 256637, positioned slightly upstream from the 3' exon 6 splice site, failed to inhibit CD40 expression. This finding was verified by flow cytometry (data not shown). Examination of the primary sequences of the two inactive PNAs did not reveal any obvious features, such as a high guanosine content, that might promote the formation of undesirable secondary structure. Likewise, the RP–HPLC elution profiles for these two compounds did not indicate a tendency for self-aggregation. Furthermore, examination of the target RNA sequence did not reveal a propensity for secondary structure formation that might limit target accessibility. Hence, the underlying cause of the observed anomalous inactivity of these two PNAs remain elusive.
Table 2. Sequences of PNAs targeting sequences surrounding the binding site for ISIS 208529 and sequences designed to examine the effect of PNA length
Figure 4. Subscreen of PNAs targeting sequences surrounding the binding site for ISIS 208529. (A) Schematic representation of the binding sites for PNAs targeting the region around that of the lead PNA, ISIS 208529 (represented by dotted line). PNAs were designed to target the region 10 bases upstream and downstream, relative to the identified lead sequence, ISIS 208529. All PNAs were 15 units in length. (B) Inhibitory activity of PNAs on CD40 cell surface expression as assessed by western blot. BCL1 cells were electroporated with no compound (NT) or 10 μM of the various targeted PNAs (ISIS 256634 to ISIS 256643). The mismatch control PNA (ISIS 256644) was included. Protein was harvested 48 h later and analyzed for CD40 expression by western blot. G3PDH protein levels were visualized to verify equal loading.
The effect of PNA length on CD40 inhibitory activity
The effect of PNA length on activity was assessed by systematic variation of the length of the PNA inhibitor from seven to 20 monomer units. For the initial examination of length effects, 13 PNAs were designed and synthesized (Table 2). The first set, consisting of PNAs of seven to 14 units in length, were all targeted to portions of the binding site of ISIS 208529 (Fig. 5A). Each of these compounds as well as the 15mer parent, ISIS 208529, were electroporated into BCL1 cells at a final concentration of 10 μM. Three days following delivery, the cells were harvested and analyzed by western blot for CD40 levels (Fig. 5B). G3PDH protein levels were also measured to verify equal protein loading. While no apparent reduction in CD40 levels was observed in cells treated with compounds ranging from seven to 11 units in length, inhibition of CD40 expression was observed with compounds ranging from 12 to 15 units in length. The efficacy of the PNA inhibitors was found to increase with increasing length, up to a PNA length of 14 units, where efficacy reached a level similar to that displayed by the lead 15mer PNA, ISIS 208529. Subsequently, PNAs covering a range of 12 to 20 units in length were examined. PNAs were electroporated into BCL1 cells at various concentrations to determine their relative potencies (Fig. 5C). Compounds were evaluated for their ability to inhibit CD40 cell surface expression by flow cytometry. Potency was found to increase with increasing length, reaching a plateau at 14 unit length, beyond which no additional gain in potency was observed. This observation suggests that the potency of ISIS 208529 is not limited by its length, and that potency cannot be improved by increasing the length of the PNA. At this target site, EC50 values were in the range of 0.6 to 0.9 μM for all PNAs of 14 units or longer (Table 3).
Figure 5. Effect of PNA length on potency of CD40 inhibition. (A) Schematic representation of PNA length variation relative to the active ISIS 208529 inhibitor (represented by dotted line). (B) The effect of PNA length on activity as assessed by western blot. BCL1 cells were electroporated with no compound (NT), 10 μM PNAs of varying length (7–15 units). Protein was harvested 72 h later and analyzed for CD40 expression by western blot. G3PDH protein levels were visualized to verify equal loading. (C) CD40 depletion as a function of length and dose. PNAs ranging from 12 to 20 units in length were electroporated into BCL1 cells at various concentrations (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8 μM). Cells were harvested 3 days later and analyzed by flow cytometry for CD40 cell surface expression.
Table 3. Potency of CD40 inhibition as a function of PNA length
Dose and time dependence of CD40 inhibitory activity by ISIS 208529
The dose-dependent reduction of cell surface CD40 protein levels upon treatment of BCL1 cells with ISIS 208529 was evaluated by flow cytometry and was further supported by verification of CD40 protein depletion by western blot (Fig. 6A and B). Inhibition of CD40 protein expression was found to be dose dependent, with an EC50 of 1–2 μM. Specificity was verified by inclusion of a PNA containing a four-base mismatch (ISIS 256644). In order to assess the effect of ISIS 208529 over time, western blot analysis was used to study the effect of a single dose (10 μM) of ISIS 208529 for 8 days following electroporation (Fig. 7). Maximal inhibition of CD40 expression was observed 4 days post-treatment and persisted through the fifth day. A temporal delay in activity was observed that likely reflects the turnover time of the pre-existing CD40 protein. By day 8, the level of CD40 expression had returned to the levels measured in the untreated controls (Fig. 7). No change in CD40 expression levels was observed in cells treated with the four-base mismatched PNA (ISIS 256644).
Figure 6. Effect of dose on activity for the lead PNA inhibitor ISIS 208529. BCL1 cells were electroporated with either no PNA (NT), 16 μM mismatch control PNA (ISIS 256644) or various concentrations (16, 8, 4, 2, 1, 0.5 or 0.25 μM) of ISIS 208529. Cells were harvested three days later and analyzed by either flow cytometry (A) or western blot (B) for CD40 expression levels. For the western blot, the membrane was subsequently reblotted for G3PDH to verify protein loading was equal among samples. A representative blot is shown. The values shown in (A) represent the averages (n = 3 per group) and their corresponding standard deviations.
Figure 7. Time course of activity of lead PNA inhibitor. BCL1 cells were electroporated with no PNA or 10 μM either mismatch control PNA (ISIS 256644) or lead PNA (ISIS 208529). Cells were harvested 1, 2, 3, 4, 5 or 8 days later and protein lysates were analyzed by western blot for CD40 protein expression levels. Protein samples from cells harvested one day following electroporation with no PNA (1*) were included on both blots as a common reference. Blots were analyzed for G3PDH levels to verify equal loading.
Inhibitory activity of ISIS 208529 on CD40-dependent IL-12 production in primary murine macrophages
Macrophages, which express the CD40 receptor, play an important role in modulation of the immune response. Interaction of CD40 with CD154 triggers the secretion of numerous cytokines, such as IL-12. Hence, measurement of IL-12 levels provides a way to assess the functional loss of the CD40 receptor. The functional consequences of PNA-induced alternative splicing in primary murine macrophages were examined by electroporation of thioglycollate-elicited mouse peritoneal cells with various doses of ISIS 208529 or with the PNA containing a four-base mismatch, ISIS 256644. After electroporation, macrophages were selected by adherence to tissue culture plates, treated with IFN- for 4 h to induce CD40 cell surface expression, then stimulated with an activating CD40 antibody for 24 h. The level of IL-12 in the supernatant of PNA-treated macrophages after CD40 activation was examined by an ELISA assay. Treatment of macrophages with ISIS 208529 resulted in a dose-dependent reduction in IL-12 production (Fig. 8A). Treatment of macrophages with ISIS 208529 at a concentration of 3 μM resulted in 75% inhibition of IL-12 production compared to macrophages electroporated with no PNA, while 85% inhibition in IL-12 production was observed upon treatment with 10 μM ISIS 208529. Macrophages treated with the mismatch control PNA (ISIS 256644) showed no decrease in IL-12. Examination of CD40 levels by western blot demonstrated a dose-dependent reduction in CD40 protein levels following treatment with ISIS 208529, which correlated well with the decrease observed in IL-12 production (Fig. 8B). No reduction in CD40 protein was found after treatment with the mismatch control ISIS 256644. Examination of the CD40 splice forms by quantitative RT–PCR showed a 70% decrease in the predominant type 1 splice form, and a 2-fold increase in the alternative type 2 splice form, at 3 μM ISIS 208529 (Fig. 8C). The four-base mismatch control, ISIS 256644, had no significant effect on the relative abundance of the CD40 splice forms, indicating that inhibitory activity observed for ISIS 208529 was sequence dependent.
Figure 8. Dose-dependent effect of lead PNA inhibitor on CD40 expression and CD40-mediated IL-12 production in primary murine macrophages. Freshly isolated thioglycollate-elicited mouse peritoneal cells were electroporated with either the lead PNA (ISIS 208529) or the four-base mismatch control (ISIS 256644). Macrophages were purified by adhering to tissue culture plate, treated with 100 ng/ml mIFN- for 4 h, then with 10 μg/ml anti-CD40 antibody for 24 h. (A) ELISA assay measuring IL-12 p40+p70 in supernatant of PNA-treated macrophages after 24 h of CD40 activation. (B) Western blot for CD40 protein. The blot was reprobed for G3PDH protein levels to verify equal protein loading. (C) Real-time quantitative RT–PCR for type 1 and type 2 CD40 splice forms.
Effect of ISIS 208529 peptide conjugation on CD40 cell surface expression in BCL1 cells and in macrophages
Lysine conjugation of PNA has previously been found to improve PNA uptake into cells. Additional evidence demonstrating the utility of lysine-conjugated PNAs was provided by experiments in a transgenic mouse model, where a lysine-conjugated PNA demonstrated efficacy in a broad range of tissues (21). Hence, a series of conjugates were synthesized that contained one to eight lysine units at the N-terminus of ISIS 208529 (data not shown). These conjugates were evaluated as inhibitors of CD40 cell surface expression by flow cytometry without the use of transfection agents. The results indicated that the eight-lysine derivative, ISIS 278647, was a more effective inhibitor of CD40 expression when delivered by free uptake compared to its shorter analogs while maintaining similar potency as its unconjugated parent, ISIS 208529, when delivered by electroporation (data not shown). We interpreted this result to signify that the eight-lysine conjugate was more effectively transported across the cellular and nuclear membranes compared to the shorter analogs. Flow cytometry was used to quantitate the levels of fluorescently labeled PNA derivatives accumulating in cells. BCL1 cells accumulated both unconjugated parent and eight-lysine conjugate in a dose-dependent manner (Fig. 9). Cells accumulated >10-fold more conjugated PNA compared to unconjugated oligomer following 1 h incubation.
Figure 9. Oligolysine conjugated PNA promotes greater cellular compound accumulation. BCL1 cells were exposed to various concentrations (4, 2, 1, 0.5 or 0.25 μM) either ISIS 208529 or ISIS 278647, labeled with Oregon Green on the C-terminal lysing of each compound. Cells were washed twice with whole media, once with PBS, and subsequently lifted from the plates and analyzed for MFI as described. Propidium iodide exclusion was employed to verify cell membranes remained intact. Values shown represent the means and standard deviations from three replicates per treatment.
ISIS 278647 was further evaluated for its effect on CD40 expression in BCL1 cells without transfection or electroporation (‘free uptake’) (Fig. 10). Compared to untreated cells, BCL1 cells treated with ISIS 278647 at 10 μM, displayed reduced levels of the CD40 type 1 transcript and increased amounts of the type 2 transcript as determined by both standard RT–PCR (Fig. 10A) and real-time quantitative RT–PCR (Fig. 10B). ISIS 278647 caused an 85% decrease in the type 1 transcript and a >3-fold increase in the type 2 transcript. Neither the unconjugated lead PNA (ISIS 208529), nor an eight-lysine conjugated, four-base mismatched PNA (ISIS 287294), had any effect on the abundance of either splice variant or on total CD40 transcript relative to the untreated control. These results demonstrate that redirection of splicing by ‘free uptake’ and subsequent loss of the CD40 protein encoded by the type 1 transcript variant are dependent on both PNA sequence and inclusion of the eight-lysine carrier.
Figure 10. Free uptake of lysine-conjugated PNAs in BCL1 cells. BCL1 cells were either mock treated (NT) or treated with 10 μM either unconjugated lead PNA (ISIS 208529), eight-lysine-conjugated four-base mismatch control PNA (ISIS 287294) or eight-lysine-conjugated PNA (ISIS 278647) for 3 days in normal growth media. (A) Cellular RNA was purified and analyzed by standard RT–PCR for changes in the relative abundance of CD40 transcript variants. (B) Total RNA was also subjected to quantitative RT–PCR using primer/probe sets capable of individually quantifying each of the two transcript variants as well as one recognizing both splice forms. (C) Protein lysates were analyzed by western blot for changes in CD40 expression level. TRADD protein levels were also visualized to verify equal sample loading.
Analysis of the protein lysates by western blot, showed that ISIS 278647 promoted depletion of CD40 protein levels, whereas treatment with the unconjugated PNA, ISIS 208529, or the four-base mismatch control, ISIS 287294, had no effect on CD40 protein levels (Fig. 10C). In a separate experiment, the slight decrease in total CD40 transcript levels (Type 1 & 2) resulting from treatment with ISIS 278647 was confirmed by northern blot (data not shown).
The effect of the lysine conjugation of ISIS 208529 on CD40 expression and on the relative abundance of the type 1 and type 2 transcripts was also examined in primary murine macrophages (Fig. 11). Adherent peritoneal macrophages were incubated with various concentrations of unconjugated or conjugated PNA for 16 h, and treated with IFN- to induce CD40 protein expression. Expression of CD40 protein was then examined by western blot. No reduction in CD40 protein was observed after treatment with ISIS 208529 (Fig. 11A), while a modest reduction in CD40 protein levels was observed in macrophages treated with the four-lysine-conjugated PNA (ISIS 278643). In contrast, treatment with the eight-lysine-conjugated CD40 PNA (ISIS 278647) resulted in a dose-dependent decrease in CD40 protein levels (Fig. 11A). Treatment with ISIS 278647 at 10 μM resulted in undetectable CD40 protein levels as determined by western blot, indicating that the eight-lysine-conjugated PNA was readily taken up by the primary macrophages and that carrier conjugation did not prevent the PNA from binding to its target and attenuating CD40 protein expression. Treatment with an eight-lysine-conjugated four-base mismatch control PNA (ISIS 287294) did not affect CD40 protein levels, indicating that the observed effect on CD40 protein expression is sequence specific. Analysis of the CD40 splice forms by quantitative RT–PCR demonstrated that the eight-lysine-conjugated CD40 PNA (ISIS 278647) caused a substantial reduction in CD40 type 1 mRNA levels with a concomitant 5-fold induction of the CD40 type 2 transcript levels (Fig. 11B). The eight-lysine-conjugated four-base mismatch PNA (ISIS 287294) had no significant effect on the relative levels of the type 1 and type 2 splice forms.
Figure 11. Free uptake of lysine-conjugated PNAs in primary murine macrophages. Adherent thioglycollate-elicited peritoneal macrophages were incubated with the indicated CD40 PNAs for 16 h. The cells were treated with 100 ng/ml mIFN- for 4 h, then with 10 μg/ml anti-CD40 antibody (clone 3/23) for 6 h. (A) Western blot for CD40 protein. The blot was re-probed for G3PDH protein levels to verify equal protein loading. (B) Real-time quantitative RT–PCR for type 1 and type 2 CD40 splice forms. (C) Real-time quantitative RT–PCR for mCD18.
In summary, expression of the IL-12 cytokine in primary murine macrophages, was reduced in a sequence-specific, dose-dependent manner as a consequence of ISIS 208529 delivery by electroporation. Moreover, the reduction in IL-12 levels were found to correlate with the reduced levels of full-length CD40 protein in the macrophages. As observed in the BCL1 cells, the reduction in type 1 transcript levels in primary murine macrophages was found to coincide with an increase in the amount of the type 2 transcript. Reduction of CD40 expression by free uptake in macrophages was found to rely on lysine conjugation of the PNA. A modest reduction in CD40 protein was observed upon treatment with a four-lysine-conjugated inhibitor (ISIS 278643), suggesting that four lysines will not promote efficient cellular uptake. In contrast, treatment with the eight-lysine-conjugated PNA (ISIS 278647) led to complete depletion of CD40 protein levels. The dramatic improvement in efficacy of the eight-lysine-conjugated PNA relative to that of the unconjugated ISIS 208529 stresses the importance of identification and optimization of suitable carriers for PNA antisense molecules. The observed improved efficacy is likely to reflect improved cellular uptake for the eight-lysine-conjugated PNA. The possibility cannot be ruled out, however, that the peptide conjugate facilitates improved PNA activity by some other means, e.g. improved trafficking or improved endosomal release.
Significant interest has evolved in CD40–CD154 cognate recognition and subsequent downstream signaling, due to the central role of the CD40–CD154 interaction in autoimmune and/or inflammatory disease. Interference with CD40–CD154 signaling appears to be a promising approach for the development of therapeutics. The results described here demonstrating the depletion of CD40 protein and reduction of IL-12 production in macrophages, validates antisense-mediated interference with CD40–CD154 cognate interaction as a means to control downstream signaling. Development of specific antisense inhibitors of CD40 cell surface expression may provide a therapeutic approach to effectively modulate CD40–CD154-mediated signaling.
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