Design And Screening Of Anti-IFN-α2b ODNs

Since Issacs and Lindeman found a cytokine that interfered with viral replication in 1957, interferon (IFN) has been identified and utilized clinically as an effective agent against viruses and neoplasms, especially as immunotherapeutic against hepatitis (type B and type C hepatitis).Interferons are a group of proteins with homologous amino acid sequences and are classified into typeⅠIFN (IFN-α& IFN-β) and typeⅡIFN (IFN-γ). There are a number of subtypes of IFN-αsuch as IFN-α1b, IFN-α2a, IFN-α2b, IFN-α3 and so on. IFN-α2b is studied in this thesis.TypeⅠIFNs are produced by leukocytes (IFN-α) and fibroblasts (IFN-β) as well as other somatic cells responding to IFN inducers such as double stranded RNA, lipopolysaccharide, methylated CpG DNA. All of the inducers can be recognized by different kinds of toll-like receptors (TLRs). Upon the recognition, toll-like receptors are activated, resulting in the production of IFNs that in turn induce the secretion of more IFNs in an autocrine or paracrine way. Instead of inducing by viral motifs, typeⅡinterferon can be produced from T lymphocytes and natural killer cells in response to different stimuli like T lymphocyte specific antigens or other cytokines.After binding of typeⅠIFNs, IFNARs are activated and subsequently start the JAK/STAT signal transduction, leading to producing antiviral proteins including 2'5' OAS /RNase L, PKR, Mx (Mx1 for mouse and MxA for human) that inhibit the replication of the infected virus at different stages of viral lifecycle. Therefore typeⅠIFNs play an important role in innate immunity, while in contrast, typeⅡIFN only bind to IFN-γreceptors (IFNGRs) to activate adaptive immune response rather than innate immunity. Additional studies also demonstrate the immunoregulatory function of type I IFNs may also.In 1988, IFN-αwas approved by FDA for the treatment of condyloma acuminatum. Three years later, it was approved as therapeutics to Hepatitis C. In 1991, IFN-αwas used as regular antiviral cytokine against Hepatitis B viruses, which started the era of treating hepatitis B. Being utilized for more than 20 years, IFN-αhas been proven to be an effective agent against viral infection.Other than antiviral activity, IFN-αhas an antineoplastic history as well. In 1986, the first clinical use of genetically produced IFN-αwas approved by FDA for the treat of hairy leukemia. And now, solid tumors and hematological malignancy could also be managed by IFN-αtherapy, which may depend on antineovascularization mechanism or enhancement of both cellular immunity and tumor cell immunogenicity, resulting in improved anti-tumor immunity. It may as well modulate cell cycles and induce apoptosis to inhibit neoplasia directly.Though multi-role pharmaceutical IFN-αis for so many diseases, significant toxicities arise out of IFN-αtherapy that can be divided into constitutional, neropsychiatric, endocrine and autoimmune effects. Fatigue, without tolerance , is the most common constitutional symptom that 70% patients undergo after receiving IFN-αtherapy. Besides fatigue, chronic side effects conclude nausea, neutrocyto- penia and hypertransaminase, all of which aggravate along with the IFN-αtherapy and the dose increase. Fever, shake, myalgia could happen 3 to 6 hours after injection of IFN-αto most of patients, but would be tolerated gradually. Side effects to central nervous systems include lethargy, confusion, and dazzling that the therapy duration seems more important than the absolute dosage of IFN-α. IFN-αtherapy can as well lead to endocrine disorders that disturbs the hypothalamo- pituitary-gonadal axis (HPGA). And the fatal side effects, although rare, are autoimmune diseases with more tendency to those patients presenting higher autoimmune antibodies previously. Thyroid disorders are the most common autoimmune disease, and the prolonged IFN-αtherapy would increase the incidence of thyroid diseases to 0.6%—2.7%. Rheumatic diseases like lupus and systemic lupus erythematousus can be increased by IFN-α, too.At present there is no effective treatment to the side effects especially autoimmune diseases induced by IFN-αtherapy. Immunosuppressants are still the mainstream drug to autoimmune disorders but induce more severe side effects for the patients. We are trying new ways to solve this via oligonucleotides.Recently, efforts have been made to develop therapeutic oligonucleotides including antisense oligonucleotide, ribozyme, small interfere RNA, unmethylated CpG containing synthesized oligodeoxynucleotides (CpG ODNs) and aptamers. Chemically, aptamers are synthesized RNA, ssDNA or dsDNA that are selected from a oligonucleotides pool by systematic evolution of ligand by exponential enrichment (SELEX). As antibodies, aptamers specifically bind to, with high affinity, targets ranging from inorganic molecules, small organic molecules, proteins even cells and whole organisms. And similar to the combination between antibodies and antigens, aptamers tend to fold into specific conformation to bind to targets in a"lock and key"way to inhibit the activities of the targets respectively.In order to block the activity of IFN-α2b hopefully as to reduce the induced side effects, a variety of ODNs were synthesized based on their predicted secondary structures. VSV protection assay was conducted to select ODNs targeting IFN-α2b. After incubation of IFN-α2b with correspondent amount of ODN, the Vero cells were pretreated with the mixture followed by the challenging of VSV. Once the antiviral activity of IFN-α2b was inhibited it would be considered as an aptamer that bind to the target, IFN-α2b so that other bioactivities might be blocked by it as well, including those side effects. ODNs designed by our lab were synthesized by Takara company. The predicted secondary structures of ODNs consist of stem-loop structures with various number of loops and length of stems.The lowest concentration of IFN-α2b to protect the cells completely from infection of VSV was 64 U/mL according to the dose-effect curve (Figure 3). It was used in the following experiments when mixed with ODNs in 37 oC for a hour. It was confirmed that there are no significant difference between two incubation conditions, one hour at 37 oC and overnight at 4 oC, to prepare the mixtures ,even plus a"binding buffer"(Figure 9).Out of the various concentrations of ODNs, 5μM worked for several ODNs to inhibit the protection from IFNα-2b, such as CB4 and C3. And the two ODNs at 1μM or 0.2μM showed slightly differences compared to the control group. However CB4 at various concentrations showed toxicity to cell growth including 5μM. So the inhibition of protection against VSV might not attribute to the anti-IFNα2b effect alone (Figure 4& Figure 5).It was consistent that 705, either 7.6μM or 5μM, did affect the antiviral activity of IFN-α2b compared to the control group (Figure 4& Figure 7). And inhibition percentage of 705 excluding the interference of toxicity was in (Figure 6). While, interestingly, lower concentration of 705 (0.2μM~1.3μM) improve the activity of IFN-α2b significantly (Figure 4&Figure 7). Apparently the synergization of 705 doesn`t due to increase of anti-VSV activity of 705, because it alone didn`t demonstrate any protection against VSV (Figure 8). So 705 is assumed to help increase the activity of IFN-α2b in cooperation.Comparing the lanes of mixture of ODNs and IFN-α2b to the lanes of ODNs alone, the electrophoretic mobility shift assay showed no band shifts, which suggests ODNs referred to do not bind to IFN-α2b directly, even 705. Therefore 705 perhaps have dual function as a blocker of IFN-α2b like as an aptamer may do and also as a synergizer that could either enhance the binding of IFN-α2b to its receptor or reinforce the JAK/STAT pathway that IFN-α2b activates.Conclusively, we set up an assay to select ODNs to block the antiviral activity of IFN-α2b. A dual functional ODN, 705, was selected. The mechanism of such dual function is supposed to be testified furtherly.