Development of efficient transient transfection systems for introducing antisense oligonucleotides into human epithelial skin cells

Systemic treatment with antisense oligonucleotides is confounded by the dual problems of potential cytotoxicity of antisense oligonucleotides and carrier molecules such as cationic lipids. Treatment of pathologic conditions affecting the skin may avoid these problems to a large degree due to local application. The success of antisense strategies has been limited by the poor uptake of the transfection reagent and inadequate intracellular compartmentalization. Human skin epithelial cells, therefore, are attractive experimental tools for testing both in vitro and in vivo antisense therapies. In the present study, we determined commercially available liposomes which reproducibly induced a nontoxic increase of oligonucleotide uptake in cultured SZ95 sebocytes and keratinocytes. The final protocol for SZ95 sebocytes was a 4-hour incubation with DOTAP in a 2:1 (w/w) lipid/oligonucleotide ratio in serum-free medium. The fluorescein-labeled (ATCG)(5) random oligonucleotide molecules were detected within the nucleus. The optimum transfection system for primary keratinocytes was poly-L-ornithine (12 microg/ml) in a medium without bovine pituitary extract over 4 hours. The uptake of the oligonucleotide increased in the presence of the polycation and oligonucleotide molecules were localized in the cytoplasm of keratinocytes. Oligonucleotide transfection with the help of cationic lipids did not affect the expression of androgen receptor and of the house-keeping gene beta-actin. Thus, cationic lipids are useful for delivery of antisense oligonucleotides into skin cells in vitro and may be used for topical application on animal and human skin.     

Responsiveness to androgens and effectiveness of antisense oligonucleotides against the androgen receptor on human epidermal keratinocytes is dependent on the age of the donor and the location of cell origin.

Local androgen excess has been associated with attenuation of wound healing in elderly individuals and with a decline in permeability barrier homeostasis in adult human skin. In this study we have applied specific antisense oligonucleotides, whose activity has already been investigated in SZ95 sebocytes, to inactivate transiently the androgen receptor in a reconstituted epidermis model and in primary human epidermal keratinocytes of different origin (breast, abdomen, foreskin) and donor age (females, 30- and 60-year-old). Further a possible interaction between blockage of androgen receptor and the expression of tissue inhibitors of matrix metalloproteinases was investigated. Androgen receptor levels were similar in pooled keratinocytes of the two age groups. Cell transfection with antisense oligonucleotides against the androgen receptor resulted in decreasing protein levels detected in all epidermal keratinocytes tested, whereas cells of aged donors (60-year-old) exhibited a stronger response than cells of young individuals (30-year-old). Keratinocytes from aged donors also responded to androgens with a stronger regulation of proliferation than keratinocytes of young individuals. The pattern of the androgen-induced response was dependent on the skin region of keratinocyte origin. The expression levels of tissue inhibitor of matrix metalloproteinase-1 were not age-related. Our results demonstrate an enhanced androgen sensitivity of keratinocytes from aged individuals associated with an origin-specific type of response.     

Potent inhibitory activity of chimeric oligonucleotides targeting two different sites of human telomerase

Suppression of telomerase activity in tumor cells has been considered as a new anticancer strategy. Here, we present chimeric oligonucleotides (chimeric ODNs) as a new type of telomerase inhibitor that contains differently modified oligomers to address two different sites of telomerase: the RNA template and a suggested protein motif. We have shown previously that phosphorothioate-modified oligonucleotides (PS ODNs) interact in a length-dependent rather than in a sequence-dependent manner, presumably with the protein part of the primer-binding site of telomerase, causing strong inhibition of telomerase. In the present study, we demonstrate that extensions of these PS ODNs at their 3'-ends with an antisense oligomer partial sequence covering 11 bases of the RNA template cause significantly increased inhibitory activity, with IC(50) values between 0.60 and 0.95 nM in a Telomeric Repeat Amplification Protocol (TRAP) assay based on U-87 cell lysates. The enhanced inhibitory activity is observed regardless of whether the antisense part is modified (phosphodiester, PO; 2'-O-methylribosyl, 2'-OMe/PO; phosphoramidate, PAM). However, inside intact U-87 cells, these modifications of the antisense part proved to be essential for efficient telomerase inhibition 20 hours after transfection. In particular, the chimeric ODNs containing PAM or 2'-OMe/PO modifications, when complexed with lipofectin, were most efficient telomerase inhibitors (ID(50) = 0.04 and 0.06 microM, respectively). In conclusion, ODNs of this new type emerged as powerful inhibitors of human telomerase and are, therefore, promising candidates for further investigations of the anticancer strategy of telomerase inhibition.     

Inhibition of 5alpha-reductase activity in SZ95 sebocytes and HaCaT keratinocytes in vitro.

Inhibition of 5alpha-reductase type 1 has been considered to be a promising target for treatment of androgen-dependent skin disorders, however, currently published clinical results on acne treatment are rather disappointing. In this study, the influence of selective inhibitors of 5alpha-reductase on testosterone metabolism within SZ95 sebocytes and HaCaT keratinocytes IN VITRO was investigated. In both cell types, the isotype 1 inhibitor MK386 completely inhibited the conversion of testosterone to 5alpha-dihydrotestosterone in concentrations higher than 10 (-9) M. Inhibitors of the isotype 2 such as finasteride, dihydrofinasteride, and turosteride, were >100-fold less active, while, as expected, androgen receptor inhibitors did not affect the 5alpha-reductase activity. MK386, but not finasteride, reduced testosterone-stimulated proliferation and slightly reduced the testosterone-induced increase in the amount of SZ95 sebocyte proteins. The androgen receptor inhibitor cyproterone acetate exhibited no effect on testosterone-induced proliferation, but inhibited the 5alpha-dihydrotestosterone-induced sebocyte proliferation. Our experimental findings and the existing clinical results indicate that the inhibition of 5alpha-reductase activity alone may be insufficient to reduce overall sebocyte activity and improve acne lesions.     

Antisense Oligodeoxynucleotide Complementary to CXCR4 mRNA Block Replication of HIV-1 in COS Cells

Abstract

CXCR4 is both a chemokine receptor and an entry co-receptor for the T-cell line-adapted human immunodeficiency virus type 1 (HIV-1). To find a more efficacious therapeutic treatement of acquied immunodeficiency syndrome, we exmined the effects of antisense oligonucleotides on CXCR4 production. COS cells, stably expressing CXCR4 and CD4, were incubated with several kinds of oligonucleotides. Total human p24 antigen production was determined using an enzyme-linked immunosorbent assay system. An antisense phosphorothioate-modified oligonucleotide, complementary to the translation region of the CXCR4 mRNA, showed minimal inhibition of p24 antigen production at the high concentration of 2μM. On the other hand, the antisense phosphorothioate oligonucleotide, when used with transfection reagents, showed high efficiency at low concentrations, and confirmed the sequence-specific action. Interestingly, the oligonucleotide with the natual phosphodiester backbone, when used with the transfection reagents, also had high functional effects, comparable to the modified oligonucleotide. This defines the prerequisite criteria necessary for the design and the application of antisense oligonucleotides against HIV-1 in vivo.     

Fas ligand-independent, FADD-mediated activation of the Fas death pathway by anticancer drugs

Trimerization of the Fas receptor (CD95, APO-1), a membrane bound protein, triggers cell death by apoptosis. The main death pathway activated by Fas receptor involves the adaptor protein FADD (for Fas-associated death domain) that connects Fas receptor to the caspase cascade. Anticancer drugs have been shown to enhance both Fas receptor and Fas ligand expression on tumor cells. The contribution of Fas ligand-Fas receptor interactions to the cytotoxic activity of these drugs remains controversial. Here, we show that neither the antagonistic anti-Fas antibody ZB4 nor the Fas-IgG molecule inhibit drug-induced apoptosis in three different cell lines. The expression of Fas ligand on the plasma membrane, which is identified in untreated U937 human leukemic cells but remains undetectable in untreated HT29 and HCT116 human colon cancer cell lines, is not modified by exposure to various cytotoxic agents. These drugs induce the clustering of Fas receptor, as observed by confocal laser scanning microscopy, and its interaction with FADD, as demonstrated by co-immunoprecipitation. Overexpression of FADD by stable transfection sensitizes tumor cells to drug-induced cell death and cytotoxicity, whereas down-regulation of FADD by transient transfection of an antisense construct decreases tumor cell sensitivity to drug-induced apoptosis. These results were confirmed by transient transfection of constructs encoding either a FADD dominant negative mutant or MC159 or E8 viral proteins that inhibit the FADD/caspase-8 pathway. These results suggest that drug-induced cell death involves the Fas/FADD pathway in a Fas ligand-independent fashion.     

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes.

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

Corticotropin-releasing hormone skin signaling is receptor-mediated and is predominant in the sebaceous glands.

There is increasing evidence that the sebaceous gland expresses receptors for several neuropeptides and is involved in responses to stress. Among them, corticotropin-releasing hormone (CRH) was currently found to be produced also in the skin. In this study, the distribution of CRH, CRH receptors 1 and 2 (CRH-R1 and CRH-R2), and CRH binding protein (CRH-BP) in cultured human (SZ95) sebocytes was further characterized. Moreover, the effects of CRH and CRH-like peptides on proliferation and inflammatory signaling of CRH receptor-expressing SZ95 sebocytes IN VITRO were investigated. Urocortin (Uct), urotensin and sauvagine are recently described members of the family of structurally related CRH-like peptides, whereas Uct shares a 45% homology with CRH. CRH and Uct inhibited SZ95 sebocyte proliferation with CRH also stimulating interleukin-6 (IL-6) and interleukin-8 (IL-8) release from SZ95 sebocytes. However, CRH had no effect on interleukin-1alpha and interleukin-1beta production in these cells. alpha-Helical-CRF, a CRH antagonistic peptide, annulled the CRH effect on SZ95 sebocyte proliferation and interleukin secretion, while the non-peptidic CRH-R1 selective antagonist antalarmin inhibited the increased production of neutral lipids caused by CRH. In conclusion, CRH, and to a lesser extent Uct, may be involved in signaling of stress pathophysiology in the skin. However, further investigations into the downstream effects of CRH and Uct are required to elucidate the mechanism by which these neuropeptides could establish a stress-related pathophysiological condition in the skin.     

Inhibition of translation of hepatitis C virus RNA by 2-modified antisense oligonucleotides.

Inhibition of hepatitis C virus (HCV) gene expression by antisense oligonucleotides was investigated using both a rabbit reticulocyte lysate in vitro translation assay and a transformed human hepatocyte cell expression assay. Screening of overlapping oligonucleotides complementary to the HCV 5' noncoding region and the core open reading frame (ORF) identified a region susceptible to translation inhibition between nucleotides 335 and 379. Comparison of 2'-deoxy-, 2'-O-methyl-, 2'-O-methoxyethyl-, 2'-O-propyl-, and 2'-fluoro-modified phosphodiester oligoribonucleotides demonstrated that increased translation inhibition correlated with both increased binding affinity and nuclease stability. In cell culture assays, 2'-O-methoxyethyl-modified oligonucleotides inhibited HCV core protein synthesis with comparable potency to phosphorothioate oligodeoxynucleotides. Inhibition of HCV core protein expression by 2'-modified oligonucleotides occurred by an RNase H-independent translational arrest mechanism.     

Characterization of the vitamin D endocrine system in human sebocytes in vitro

Sebocytes are sebum-producing cells that form the sebaceous glands. We investigated the role of sebocytes as target cells for vitamin D metabolites and the existence of an enzymatic machinery for the local synthesis and metabolism of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃, calcitriol], the biologically active vitamin D metabolite, in these cell types. Expression of vitamin D receptor (VDR), vitamin D-25-hydroxylase (25OHase), 25-hydroxyvitamin D-1α-hydroxylase (1αOHase), and 1,25-dihydroxyvitamin D-24-hydroxylase (24OHase) was detected in SZ95 sebocytes in vitro using real time quantitative polymerase chain reaction. Splice variants of 1αOHase were identified by nested touchdown polymerase chain reaction. We demonstrated that incubation of SZ95 sebocytes with 1,25(OH)₂D₃ resulted in a cell culture condition-, time-, and dose-dependent modulation of cell proliferation, cell cycle regulation, lipid content and interleukin-6/interleukin-8 secretion in vitro. RNA expression of VDR and 24OHase was upregulated along with vitamin D analogue treatment. Although several other splice variants of 1αOHase were detected, our findings indicate that the full length product represents the major 1αOHase gene product in SZ95 cells. In conclusion, SZ95 sebocytes express VDR and the enzymatic machinery to synthesize and metabolize biologically active vitamin D analogues. Sebocytes represent target cells for biologically active metabolites. Our findings indicate that the vitamin D endocrine system is of high importance for sebocyte function and physiology. We conclude that sebaceous glands represent potential targets for therapy with vitamin D analogues or for pharmacological modulation of 1,25(OH)₂D₃ synthesis/metabolism.     

Expression of ssDNA in mammalian cells

Antisense therapy involves the use of antisense oligonucleotides for altering targeted gene function. However, the low efficiency of cell delivery of antisense oligonucleotides has limited the efficacy of antisense therapeutic approaches. RNA-based antisense or ribozyme oligonucleotides can be either synthesized endogenously (e.g., by a viral vector) or delivered exogenously. However, there is presently no vector delivery system available for DNA-based oligonucleotides. Recently, a novel ssDNA expression vector that can generate intracellularly any ssDNA molecule, such as antisense oligonucleotide or DNA enzyme, has been developed in our laboratory. Here we describe an improved expression vector based on the first-generation two-vector system. To test this new expression vector, we chose to express a single-stranded "10-23" DNA enzyme targeting c-raf mRNA in the human lung carcinoma A549 cell line. After introduction into cells by transient transfection, c-raf-cleaving DNA enzymes produced by this expression vector can significantly suppress the expression of c-raf mRNA. Furthermore, the expressed c-raf DNA enzymes induced cell apoptosis, as indicated by genomic DNA fragmentation assay. Our study further demonstrates the feasibility of using this novel ssDNA expression technology to produce intracellularly any sequence of interest, including antisense oligonucleotides and DNA enzyme molecules.     

Propionibacterium acnes and lipopolysaccharide induce the expression of antimicrobial peptides and proinflammatory cytokines/chemokines in human sebocytes

Acne is a common skin disorder of the pilosebaceous unit. In addition to genetic, hormonal and environmental factors, abnormal colonization by Propionibacterium acnes has been implicated in the occurrence of acne via the induction of inflammatory mediators. To gain more insight into the role that sebocytes play in the innate immune response of the skin, particularly in acne, we compared the antimicrobial peptide and proinflammatory cytokine/chemokine expression at mRNA and protein levels, as well as the viability and differentiation of SZ95 sebocytes in response to co-culture with representative isolates of P. acnes type IA and type IB as well as Escherichia coli-derived lipopolysaccharide (LPS). We found that, in vitro, P. acnes type IA and IB isolates and LPS induced human beta-defensin-2 and proinflammatory cytokine/chemokine expression, and influenced sebocyte viability and differentiation. Our results provide evidence that sebocytes are capable of producing proinflammatory cytokines/chemokines and antimicrobial peptides, which may have a role in acne pathogenesis. Furthermore, since P. acnes types IA and IB differentially affect both the differentiation and viability of sebocytes, our data demonstrate that different strains of P. acnes vary in their capacity to stimulate an inflammatory response within the pilosebaceous follicle.     

Glial cell line-derived neurotrophic factor (GDNF) is a proliferation factor for rat C6 glioma cells: evidence from antisense experiments

Growth factors play an important role in proliferation and differentiation of malignant brain gliomas in humans. Glial cell line-derived neurotrophic factor (GDNF) has been shown recently to be highly expressed in human glioblastomas and in rat glial cell lines B49 and C6. The aim of the present study was to knockdown GDNF, its receptor GFR-alpha1, and the related family member persephin by using antisense oligonucleotides and to observe the effects on cell proliferation. To enhance cellular uptake into C6 glioma cells, 15-mer phosphorothioate oligonucleotides were complexed with the cationic lipid Lipofectamine. The complex was applied for 3 x 12 hours to C6 glioma cells, and cells were allowed to recover for 24 hours after each transfection and then analyzed. This protocol markedly reduced GDNF and GFR-alpha1 protein levels in C6 glioma cells compared with control oligonucleotides. Knockdown of C6 cells with GDNF and GFR-alpha1 but not with persephin antisense oligonucleotides significantly decreased the number of C6 glioma cells and also inhibited the incorporation of bromodeoxyuridine as a sign of reduced DNA synthesis. In conclusion, it is shown that GDNF but not persephin is a potent proliferation factor for rat glioma cells. Knockdown of GDNF using antisense oligonucleotides complexed with lipids as carriers may be useful in gene therapeutic approaches in vitro and possibly also in vivo.     

A peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNA.

We have combined a peptide nucleic acid (PNA) with the SV40 core nuclear localization signal (NLS), to create a bifunctional PNA-NLS peptide. The PNA-NLS peptide increased the nuclear uptake of oligonucleotides and enhanced the transfection efficacy of plasmids. Gene expression from an enhanced green fluorescent protein plasmid and a lacZ plasmid was preserved when hybridized to PNA-NLS. In combination with the transfection agent polyethyleneimine, we have improved both the nuclear translocation of fluorescence-marked oligonucleotides, and the efficacy of plasmid transfection, up to eightfold. The technique obviates the use of cumbersome coupling procedures of the vector due to DNA-PNA duplex formation or displacement of the antisense plasmid DNA strand by a PNA molecule.     

A novel and rapid prediction assay for the effectiveness of IL-6 receptor specific antisense oligonucleotides by proliferation inhibition of an interleukin-6 dependent cell line

Interleukin-6 (IL-6) belongs to a family of cytokines that use receptors consisting of a common signal-transducing chain (gp130). Baf/3 cells transfected with the human IL-6 receptor (IL-6R) and gp130 (Baf/3-gp130/IL-6R) can only grow in medium containing IL-6. We attempted to interrupt the signal transducing pathway of IL-6 with the help of antisense oligonucleotides (ASOs) designed against the IL-6R. We used 18 different kinds of antisense oligonucleotides of overlapping sequences around the translational start codon of the human IL-6R. Sense ASOs were used as a control. The proliferation of cells was analysed by H-thymidine incorporation. Cell surface expression of the IL-6R was assessed by FACS analysis. We identified three ASOs which strongly inhibited the proliferation of IL-6 dependent transfected Baf/3 cells. Flow cytometric studies on the suppression of surface expression of IL-6R by ASOs showed a similar pattern. These results should help to clarify the structural requirements of functionally effective ASOs in the inhibition of IL-6R.     

Reversal of epidermal hyperproliferation in psoriasis by insulin-like growth factor I receptor antisense oligonucleotides

Epidermal hyperplasia is a key feature of the common skin disorder psoriasis. Stimulation of epidermal keratinocytes by insulin-like growth factor I (IGF-I) is essential for cell division, and increased sensitivity to IGF-I may occur in psoriasis. We hypothesized that inhibition of IGF-I receptor expression in the psoriasis lesion would reverse psoriatic epidermal hyperplasia by slowing the rate of keratinocyte cell division. Here we report the use of C5-propynyl-dU,dC-phosphorothioate antisense oligonucleotides to inhibit IGF-I receptor expression in keratinocytes. We identified several inhibitory antisense oligonucleotides and demonstrated IGF-I receptor inhibition in vitro through an mRNA targeting mechanism. Repeated injection of these oligonucleotides into human psoriasis lesions, grafted onto nude mice, caused a dramatic normalization of the hyperplastic epidermis. The findings indicate that IGF-I receptor stimulation is a rate-limiting step in psoriatic epidermal hyperplasia and that IGF-I receptor targeting by cutaneous administration of antisense oligonucleotides forms the basis of a potential new psoriasis therapy.     

Enhanced activity of an antisense oligonucleotide targeting murine protein kinase C-alpha by the incorporation of 2'-O-propyl modifications.

We have previously described the characterization of a 20mer phosphorothioate oligodeoxynucleotide (ISIS 4189) which inhibits murine protein kinase C-alpha (PKC-alpha) gene expression, both in vitro and in vivo. In an effort to increase the antisense activity of this oligonucleotide, 2'-O-propyl modifications have been incorporated into the 5'- and 3'-ends of the oligonucleotide, with the eight central bases left as phosphorothioate oligodeoxynucleotides. Hybridization analysis demonstrated that these modifications increased affinity by approximately 8 and 6 degrees C per oligonucleotide for the phosphodiester (ISIS 7815) and phosphorothioate (ISIS 7817) respectively when hybridized to an RNA complement. In addition, 2'-O-propyl incorporation greatly enhanced the nuclease resistance of the oligonucleotides to snake venom phosphodiesterase or intracellular nucleases in vivo. The increase in affinity and nuclease stability of ISIS 7817 resulted in a 5-fold increase in the ability of the oligonucleotide to inhibit PKC-alpha gene expression in murine C127 cells, as compared with the parent phosphorothioate oligodeoxynucleotide. Thus an RNase H-dependent phosphorothioate oligodeoxynucleotide can be modified as a 2'-O-propyl 'chimeric' oligonucleotide to provide a significant increase in antisense activity in cell culture.     

Sequence dependence of C5-propynyl-dU,dC-phosphorothioate oligonucleotide inhibition of the human IGF-I receptor: mRNA,protein, and cell growth

Human keratinocytes are highly responsive to mitogenic and antiapoptotic signaling by the insulin-like growth factor-I receptor (IGF-IR). IGF-IR hyperstimulation is a feature of hyperplastic skin conditions, making the IGF-IR an appealing target for antisense therapeutic intervention. In this study, we used a C5-propynyl-dU,dC-phosphorothioate oligo-2'-deoxyribonucleotide antisense 15-mer to the human IGF-IR mRNA, along with liposome transfection, to inhibit IGF-IR activity in a human keratinocyte cell line and demonstrated potent inhibition of cell growth despite the presence of serum. To investigate the sequence specificity of these effects and to establish the concentration range over which a purely antisense effect could be demonstrated, we introduced 1, 2, 4, 8, and 15 base mismatches into the oligonucleotide and analyzed changes in inhibitory efficacy. In the 10-30 nM concentration range, the introduction of 1 and 2 mismatches into the middle of the 15-mer only modestly affected inhibitory efficacy, whereas >4 mismatches profoundly reduced mRNA, protein, and growth-inhibitory effects. From these results, we conclude that (1) sequence-specific antisense inhibition of IGF-IR activity in keratinocytes is achievable, (2) potent anti-IGF-IR antisense inhibition can be achieved in vitro at concentrations as low as 10 nM, and (3) a sequence-dependent mechanism is likely to underpin the observed in vivo therapeutic effects (Wraight et al. Nat. Biotechnol. 2000;18:521) of these antisense oligonucleotides (AS-ODN) in cutaneous hyperplastic disorders, such as psoriasis.