Antisense studies

Recent antisense studies have shown that knock down of Gi2 in mice-blocked morphine-induced analgesia, suggesting that morphine binds to fi receptors to activate G 2 to modulate neuronal circuits involved in analgesia [68, 84]. Sufentanil-induced analgesia was not diminished by Gi2 knock down suggesting that a different G protein mediated its behavioral effects [84]. 

Other locked nucleic acids have been described the bicyclic analogue (30) has been prepared in both the ribose and arabinose conformations where it was found that the ribose analogue was destabilising with complementary DNA and RNA, whilst the arabinose derivative was Tm neutral. Tbe tricyclic analogue (31, R=H), which has been previously described, bas been used in an antisense study compared with 2 -0-methojyethyl-DNA, where it was observed that oligomers of (31, R = H) exhibited better antisense activity. Tbe effect of substitution at C6 (31, R = esters) bas been reported where it was found to favourably stabilise duplex DNA. ... 

Many human diseases are caused when certain proteins are either over- or underexpressed. Eor example, breast cancer can be induced by overexpressing certain cellular oncogenes within mammary tissue. To study the disease, researchers produce a line of transgenic mice that synthesize an abnormal amount of the same protein. This leads to symptoms of the disease in mice that are similar to what is found in humans. A protein can be overexpressed by inserting a DNA constmct with a strong promotor. Conversely, underexpression of a protein can be achieved by inserting a DNA constmct that makes antisense RNA. This latter blocks protein synthesis because the antisense RNA binds and inactivates the sense mRNA that codes for the protein. Once a line of mice is developed, treatments are studied in mice before these therapies are appHed to humans. 

Modification of the Phosphodiester Backbone. Oligonucleotides having modified phosphate backbones have been extensively studied (46). Because altering the backbone makes derivatives generally more resistant to degradation by cellular nucleases, these materials have the potential to be more resilient antisense dmgs. 

For ISIS 301012, a second-generation antisense inhibitor of apoB-100 in November 2006. Isis announced results from two Phase 2 clinical trials of ISIS 301012. In the first study repotted, patients with high cholesterol on stable doses of statins were treated with ISIS 301012 for 5 weeks. Patients who received 300 mg/week of ISIS 301012 in this study achieved a 51% reduction in LDL-cholesterol (LDL), a 42% reduction in total cholesterol (TC), and a 4l% reduction in triglycerides (TG) beyond the levels achieved with statins alone. 

Moore J, Seiter K, Kolitz J et al (2006) A Phase II study of Bcl-2 antisense (oblimersen sodium) combined with gemtuzumab ozogamicin in older patients with acute myeloid leukemia in first relapse. Leuk Res 30(7) 777-783... 

Kleimnan et al. 2008). In addition, synthetic siRNAs are also subject to degradation in vivo by nuclease activity. Besides side effects and instability, the efficient and specific delivery of the RNAi indncers to the target cell still requires optimization. Here we snmmarize the cnrrent statns of nncleic acid-based antiviral therapentics. The focns will be on antiviral strategies nsing antisense and RNAi technology. Additionally, antiviral ribozymes and aptamers will be discussed briefly, with a focus on recent studies. Gene therapy approaches and delivery systems are the subject of Chapter 11 of this book. 

The development of nucleic acid-based therapeutics is not as straightforward as researchers had initially anticipated. Stability, toxicity, specificity, and delivery of the compounds continue to be challenging issues that need further optimization. In recent years, researchers have come up with intricate solutions that have greatly improved the efficacy of potential antisense, ribozyme, as well as RNAi-based therapeutics. Clinical trials for all these types of nucleic acid-based therapeutics are underway. So far, data from several trials and studies in animal models look promising, in particular, the therapies that trigger the RNAi pathway. However, history has shown that compounds that do well in phase I or phase II clinical trials may still fail in phase III. A striking example is the nonspecific suppression of angiogenesis by siRNA via toII-Iike receptor 3 (Kleinman et al. 2008). It will become clear in the near future which compounds will make it as a new class of antiviral therapeutics. 

A few ex vivo and in vivo studies have been published claiming an antigene (and antisense) effect of mixed purine/pyrimidine sequence PNA [48, 49, 78-80]. However, as pointed out by us in recent reviews [81, 82] these studies lack fundamental controls such as the inclusion of relevant internal standards as a control for sequence-specific non-antigene/antisense effects, thus confirmatory studies are warranted. The in vivo antigene studies from Richelsoris group [79, 83] completely lack a rational basis for the claimed effects. First of all there is no evidence that... 

Zamecnik P.C, Stephensen M.L., Proc. Natl Acad. Sci. USA 1978, 75, 280-284. Antisense technology Part A General methods, methods of delivery and RNA studies. In Phillips M.I. (Ed.), Methods in Enzymology Vol. 313. Academic Press San Diego, CA, 2000. 

BIRD C R, RAY J A, FLETCHER J D, BONIWELL J M, BIRD A S, TEULIERES C, BLAIN I, BRAMLEY P M and SCHUCH w (1991) Using antisense RNA to study gene function inhibition of carotenoid biosynthesis in transgenic tomatoes , BioTechnology, 9, 635-9. 

In a very recent study in potatoes, inhibition of LCYE accumulation was accomplished by an antisense LcyE driven by the patatin promoter and allowed rechanneling of lycopene toward the P-carotene branch of the pathway to produce up to 14-fold increased levels of P-carotene as well as up to 2.5-fold increased total carotenoids. RNAi and TILLING for manipulation of carotenogenesis have yet to be reported, but these new techniques for suppression of function and generation and selection of allelic diversity are likely to impact future research and production of varieties with enhanced pigment accumulation. 

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