Antisense therapyFrom Wikipedia, the free encyclopediaJump to: navigation, searchAntisense therapy is a form of treatment ...
AVI-6003 demonstrated reproducible and high rates of survival in non-human primateschallenged with a lethal infection of t...
oligonucleotides in high purity. Typically, synthetic oligonucleotides are single-strandedDNA or RNA molecules around 15–2...
set, very early textbooks disagreed on the DNA strands called "sense" and "antisense."[citationneeded]In biology and resea...
(identical to the noncoding                                                    (template/antisense) DNA strand.)A note on ...
RNA sense in virusesIn virology, the genome of an RNA virus can be said to be either positive-sense, also knownas a "plus-...
need for RNase H recognition, this can include chemistries such as 2-O-alkyl, peptidenucleic acid (PNA), locked nucleic ac...
Antisense therapy in Modern medicineAntisense therapy is one of the types of treatment for genetic disorders. The therapy ...
complementary to the mRNA, produced from the target gene. The antisense, then binds to themRNA and stop the translation an...
Laboratory of Molecular Design                           Antisense Therapy    If a particular gene has a role in disease, ...
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  1. 1. Antisense therapyFrom Wikipedia, the free encyclopediaJump to: navigation, searchAntisense therapy is a form of treatment for genetic disorders or infections. When thegenetic sequence of a particular gene is known to be causative of a particular disease, it ispossible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that willbind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectivelyturning that gene "off". This is because mRNA has to be single stranded for it to betranslated. Alternatively, the strand might be targeted to bind a splicing site on pre-mRNAand modify the exon content of an mRNA.[1]This synthesized nucleic acid is termed an "anti-sense" oligonucleotide because its basesequence is complementary to the genes messenger RNA (mRNA), which is called the"sense" sequence (so that a sense segment of mRNA " 5-AAGGUC-3 " would be blocked bythe anti-sense mRNA segment " 3-UUCCAG-5 ").Antisense drugs are being researched to treat cancers (including lung cancer, colorectalcarcinoma, pancreatic carcinoma, malignant glioma and malignant melanoma), diabetes,Amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy and diseases such asasthma, arthritis and pouchitis with an inflammatory component. Most potential therapieshave not yet produced significant clinical results[citation needed], though two antisense drugs havebeen approved by the U.S. Food and Drug Administration (FDA), fomivirsen (marketed asVitravene) as a treatment for cytomegalovirus retinitis and mipomersen (marketed asKynamro] for homozygous familial hypercholesterolemia.Example antisense therapiesSome 40 antisense oligonucleotides and siRNAs are in clinical trials, including over 20 inadvanced clinical trials (Phase II or III).[2][3]Cytomegalovirus retinitisFomivirsen (marketed as Vitravene), was approved by the U.S. FDA in Aug 1998 as atreatment for cytomegalovirus retinitis.Hemorrhagic fever virusesIn early 2006, scientists studying the Ebola hemorrhagic fever virus at USAMRIIDannounced a 75% recovery rate after infecting four rhesus monkeys and then treating themwith an antisense Morpholino drug developed by Sarepta Therapeutics (formerly named AVIBioPharma), a U.S. biotechnology firm.[4] The usual mortality rate for monkeys infected withEbola virus is 100%. In late 2008, AVI BioPharma successfully filed Investigational NewDrug (IND) applications with the FDA for its two lead products for Marburg and Ebolaviruses. These drugs, AVI-6002 [5] and AVI-6003 are novel analogs based on AVIs PMOantisense chemistry in which anti-viral potency is enhanced by the addition of positively-charged components to the morpholino oligomer chain. Preclinical results of AVI-6002 and
  2. 2. AVI-6003 demonstrated reproducible and high rates of survival in non-human primateschallenged with a lethal infection of the Ebola and Marburg viruses, respectively.[6]CancerAlso in 2006, German physicians reported on a dose-escalation study for the compound AP12009 (a phosphorothioate antisense oligodeoxynucleotide specific for the mRNA of humantransforming growth factor TGF-beta2) in patients with high grade gliomas. At the time ofthe report, the median overall survival had not been obtained and the authors hinted at apotential cure.[7]HIV/AIDSStarting in 2004, researchers in the US have been conducting research on using antisensetechnology to combat HIV.[8]In February 2010 researchers reported success in reducing HIV viral load using patient T-cells which had been harvested, modified with an RNA antisense strand to the HIV viralenvelope protein, and re-infused into the patient during a planned lapse in retroviral drugtherapy.[9]Familial HypercholesterolemiaIn January 2013 mipomersen (marketed as Kynamro] was approved by the FDA for thetreatment of homozygous familial hypercholesterolemia.[Oligonucleotide synthesisFrom Wikipedia, the free encyclopediaJump to: navigation, searchOligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleicacids with defined chemical structure (sequence). The technique is extremely useful incurrent laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired sequence. Whereas enzymes synthesize DNA and RNAin a 5 to 3 direction, chemical oligonucleotide synthesis is carried out in the opposite, 3 to 5direction. Currently, the process is implemented as solid-phase synthesis usingphosphoramidite method and phosphoramidite building blocks derived from protected 2-deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, and U), or chemicallymodified nucleosides, e.g. LNA.To obtain the desired oligonucleotide, the building blocks are sequentially coupled to thegrowing oligonucleotide chain in the order required by the sequence of the product (seeSynthetic cycle below). The process has been fully automated since the late 1970s. Upon thecompletion of the chain assembly, the product is released from the solid phase to solution,deprotected, and collected. The occurrence of side reactions sets practical limits for the lengthof synthetic oligonucleotides (up to about 200 nucleotide residues) because the number oferrors accumulates with the length of the oligonucleotide being synthesized.[1] Products areoften isolated by high-performance liquid chromatography (HPLC) to obtain the desired
  3. 3. oligonucleotides in high purity. Typically, synthetic oligonucleotides are single-strandedDNA or RNA molecules around 15–25 bases in length.Oligonucleotides find a variety of applications in molecular biology and medicine. They aremost commonly used as antisense oligonucleotides, small interfering RNA, primers for DNAsequencing and amplification, probes for detecting complementary DNA or RNA viamolecular hybridization, tools for the targeted introduction of mutations and restriction sites,and for the synthesis of artificial genes.DNA senseSchematic showing how antisense DNA strands can interfere with protein translation.Molecular biologists call a single strand of DNA sense (or positive (+) ) if an RNA versionof the same sequence is translated or translatable into protein. Its complementary strand iscalled antisense (or negative (-) sense). Sometimes the phrase coding strand is encountered;however, protein coding and non-coding RNAs can be transcribed similarly from bothstrands, in some cases being transcribed in both directions from a common promoter region,or being transcribed from within introns, on both strands (see "ambisense" below).[1][2][3]Antisense DNAThe two complementary strands of double-stranded DNA (dsDNA) are usually differentiatedas the "sense" strand and the "antisense" strand. The DNA sense strand looks like themessenger RNA (mRNA) and can be used to read the expected protein code by human eyes(e.g. ATG codon = Methionine amino acid). However, the DNA sense strand itself is notused to make protein by the cell. It is the DNA antisense strand which serves as the source forthe protein code, because, with bases complementary to the DNA sense strand, it is used as atemplate for the mRNA. Since transcription results in an RNA product complementary to theDNA template strand, the mRNA is complementary to the DNA antisense strand. The mRNAis what is used for translation (protein synthesis).Hence, a base triplet 3-TAC-5 in the DNA antisense strand can be used as a template whichwill result in an 5-AUG-3 base triplet in mRNA (AUG is the codon for Methionine, the startcodon). The DNA sense strand will have the triplet ATG which looks just like AUG but willnot be used to make Methionine because it will not be used to make mRNA. The DNA sensestrand is called a "sense" strand not because it will be used to make protein (it wont be), butbecause it has a sequence that looks like the protein codon sequence.Students may find this confusing if they misunderstand the meaning of "sense" and if theymisunderstand complementation. To make things more confusing, before the convention was
  4. 4. set, very early textbooks disagreed on the DNA strands called "sense" and "antisense."[citationneeded]In biology and research, short antisense molecules can interact with complementary strandsof nucleic acids, modifying expression of genes. See the section on "antisenseoligonucleotides" below.Example with double-stranded DNADNA strand 1: antisense strand (copied to)→ RNA strand (sense)DNA strand 2: sense strandSome regions within a double strand of DNA code for genes, which are usually instructionsspecifying the order of amino acids in a protein along with regulatory sequences, splicingsites, noncoding introns, and other complicating details. For a cell to use this information, onestrand of the DNA serves as a template for the synthesis of a complementary strand of RNA.The template DNA strand is called the transcribed strand with antisense sequence and themRNA transcript is said to be sense sequence (the complement of antisense). Because theDNA is double-stranded, the strand complementary to the antisense sequence is called non-transcribed strand and has the same sense sequence as the mRNA transcript (though T basesin DNA are substituted with U bases in RNA). DNA antisense strand5CGCTATAGCGTTTCAT 3 (template/noncoding), Used as a template for transcription. Watson strand DNA sense strand3GCGATATCGCAAAGTA 5 (nontemplate/coding), Complementary to the template strand. Crick strand RNA strand that is transcribed from the noncoding (template/antisense) strand. Note1: Except for the fact that all thymines are now uracils (T-->U), it is complementary3GCGAUAUCGCAAAGUA 5 mRNA Sense transcript to the noncoding (template/antisense) DNA strand (identical to the coding (nontemplate/sense) DNA strand). Note2 There is an AUG start codon at the 5 end (although written backwards here). RNA strand that is transcribed from the mRNA Antisense coding (nontemplate/sense) strand. Note:5CGCUAUAGCGUUUCAU 3 Except for the fact that all thymines are now transcript uracils (T-->U), it is complementary to the coding (nontemplate/sense) DNA strand
  5. 5. (identical to the noncoding (template/antisense) DNA strand.)A note on the confusion between "sense" and "antisense" strands: The strand names actuallydepend on which direction you are writing the sequence that contains the information forproteins (the "sense" information), not on which strand is on the top or bottom (that isarbitrary). The only real biological information that is important for labeling strands is thelocation of the 5 phosphate group and the 3 hydroxyl group because these ends determinethe direction of transcription and translation. A sequence 5 CGCTAT 3 is equivalent to asequence written 3 TATCGC 5 as long as the 5 and 3 ends are noted. If the ends are notlabeled, convention is to assume that the sequence is written in the 5 to 3 direction. Goodrule of thumb for figuring out the "sense" strand: Look for the start codon ATG (AUG inmRNA). In the table example, the sense mRNA has the AUG codon at the end (rememberthat translation proceeds in the 5 to 3 direction).AmbisenseA single-stranded genome that contains both positive-sense and negative-sense is said to beambisense. Bunya viruses have 3 single-stranded RNA (ssRNA) fragments containing bothpositive-sense and negative-sense sections; arenaviruses are also ssRNA viruses with anambisense genome, as they have 2 fragments that are mainly negative-sense except for part ofthe 5 ends of the large and small segments of their genome.Antisense RNAMain article: Antisense RNAAntisense RNA is an RNA transcript that is complementary to endogenous mRNA. In otherwords, it is a non-coding strand complementary to the coding sequence of RNA; this issimilar to negative-sense viral RNA. Introducing a transgene coding for antisense RNA is atechnique used to block expression of a gene of interest. Radioactively-labelled antisenseRNA can be used to show the level of transcription of genes in various cell types. Somealternative antisense structural types are being experimentally applied as antisense therapy,with at least one antisense therapy approved for use in humans.When mRNA forms a duplex with a complementary antisense RNA sequence, translation isblocked. This process is related to RNA interference.Antisense nucleic acid molecules have been used experimentally to bind to mRNA andprevent expression of specific genes. Antisense therapies are also in development; in theUSA, the Food and Drug Administration (FDA) has approved phosphorothioate antisenseoligos fomivirsen (Vitravene) and mipomersen (Kynamro)[4] for human therapeutic use.Cells can produce antisense RNA molecules naturally, which interact with complementarymRNA molecules and inhibit their expression.
  6. 6. RNA sense in virusesIn virology, the genome of an RNA virus can be said to be either positive-sense, also knownas a "plus-strand", or negative-sense, also known as a "minus-strand". In most cases, theterms sense and strand are used interchangeably, making such terms as positive-strandequivalent to positive-sense, and plus-strand equivalent to plus-sense. Whether a virusgenome is positive-sense or negative-sense can be used as a basis for classifying viruses.Positive-sensePositive-sense (5 to 3) viral RNA signifies that a particular viral RNA sequence may bedirectly translated into the desired viral proteins. Therefore, in positive-sense RNA viruses,the viral RNA genome can be considered viral mRNA, and can be immediately translated bythe host cell. Unlike negative-sense RNA, positive-sense RNA is of the same sense asmRNA. Some viruses (e.g., Coronaviridae) have positive-sense genomes that can act asmRNA and be used directly to synthesize proteins without the help of a complementary RNAintermediate. Because of this, these viruses do not need to have an RNA polymerasepackaged into the virion.Negative-senseNegative-sense (3 to 5) viral RNA is complementary to the viral mRNA and thus must beconverted to positive-sense RNA by an RNA polymerase prior to translation. Negative-senseRNA (like DNA) has a nucleotide sequence complementary to the mRNA that it encodes.Like DNA, this RNA cannot be translated into protein directly. Instead, it must first betranscribed into a positive-sense RNA that acts as an mRNA. Some viruses (Influenza, forexample) have negative-sense genomes and so must carry an RNA polymerase inside thevirion.Antisense oligonucleotidesGene silencing can be achieved by introducing into cells a short "antisense oligonucleotide"that is complementary to an RNA target. This experiment was first done by Zamecnik andStephenson in 1978[5] and continues to be a useful approach, both for laboratory experimentsand potentially for clinical applications (antisense therapy).[6]If the antisense oligonucleotide contains a stretch of DNA or a DNA mimic(phosphorothioate DNA, 2F-ANA, or others) it can recruit RNase H to degrade the targetRNA. This makes the mechanism of gene silencing catalytic. Double-stranded RNA can alsoact as a catalytic, enzyme-dependent antisense agent through the RNAi/siRNA pathway,involving target mRNA recognition through sense-antisense strand pairing followed by targetmRNA degradation by the RNA-induced silencing complex (RISC). The R1 plasmid hok/soksystem provides yet another example of an enzyme-dependent antisense regulation process,through enzymatic degradation of the resulting RNA duplex.Other antisense mechanisms are not enzyme-dependent, but involve steric blocking of theirtarget RNA (e.g. to prevent translation or induce alternative splicing). Steric blockingantisense mechanisms often use oligonucleotides that are heavily modified. Since there is no
  7. 7. need for RNase H recognition, this can include chemistries such as 2-O-alkyl, peptidenucleic acid (PNA), locked nucleic acid (LNA), and Morpholino oligomers.Antisense RNAFrom Wikipedia, the free encyclopedia (Redirected from Antisense mRNA)Jump to: navigation, searchAntisense RNA (asRNA) is a single-stranded RNA that is complementary to a messengerRNA (mRNA) strand transcribed within a cell. Antisense RNA may be introduced into a cellto inhibit translation of a complementary mRNA by base pairing to it and physicallyobstructing the translation machinery.[1] This effect is therefore stoichiometric. An exampleof naturally occurring mRNA antisense mechanism is the hok/sok system of the E. coli R1plasmid. Antisense RNA has long been thought of as a promising technique for diseasetherapy; the only such case to have reached the market is the drug fomivirsen. Onecommentator has characterized antisense RNA as one of "dozens of technologies that aregorgeous in concept, but exasperating in [commercialization]".[2] Generally, antisense RNAstill lack effective design, biological activity, and efficient route of administration.[3]Historically, the effects of antisense RNA have often been confused with the effects of RNAinterference (RNAi), a related process in which double-stranded RNA fragments called smallinterfering RNAs trigger catalytically mediated gene silencing, most typically by targetingthe RNA-induced silencing complex (RISC) to bind to and degrade the mRNA. Attempts togenetically engineer transgenic plants to express antisense RNA instead activate the RNAipathway, although the processes result in differing magnitudes of the same downstreameffect, gene silencing. Well-known examples include the Flavr Savr tomato and two cultivarsof ringspot-resistant papaya.[4][5]Transcription of longer cis-antisense transcripts is a common phenomenon in the mammaliantranscriptome.[6] Although the function of some cases have been described, such as theZeb2/Sip1 antisense RNA, no general function has been elucidated. In the case ofZeb2/Sip1,[7] the antisense noncoding RNA is opposite the 5 splice site of an intron in the5UTR of the Zeb2 mRNA. Expression of the antisense ncRNA prevents splicing of an intronthat contains a ribosome entry site necessary for efficient expression of the Zeb2 protein.Transcription of long antisense ncRNAs is often concordant with the associated protein-coding gene,[8] but more detailed studies have revealed that the relative expression patterns ofthe mRNA and antisense ncRNA are complex.[9
  8. 8. Antisense therapy in Modern medicineAntisense therapy is one of the types of treatment for genetic disorders. The therapy aims atworking at the mRNA level and switching off the translation of the protein from the mRNAof a mutated gene. Hence, the antisense drugs are responsible for the silencing of the generesponsible for the disease and thereby have great potential to cure many incurable, geneticdiseases. Extensive research is going on in this field to develop antisense drugs for HIV,Cancer, Asthma, etc.Antisense refers to a stretch of oliognucleotides, which may be DNA or RNA, that is
  9. 9. complementary to the mRNA, produced from the target gene. The antisense, then binds to themRNA and stop the translation and expression of the protein from the mRNA, therebysilencing the target gene, although the exact mechanism by which the gene silencing takesplace is not certain. It is proposed that maybe the mRNA and antisense oligonucleotides orASO form a duplex structure, thereby mediating the cleavage of mRNA by RNAase H. Someother models have also been proposed like the mRNA transport to the cytoplasm beingprevented, the formation of triple helix structure by the binding of the ASO with the duplexDNA, inhibiting DNA transcription, inhibition of the splicing of the mRNA, etc. Theconstruction of a proper ASO is very essential and is possible only after the proper study ofthe genes responsible for the disease and the sequence of the mRNA formed from thetranscription of the gene. The site available for the hybridization of the ASO on the mRNAmust also be known as then only the ASO can bind to the mRNA and switch off theexpression of the mRNA. The in-vivo stability of the ASO is crucial as it has to reach thetarget mRNA within the cell without getting degraded. The ASO drugs are developed byproper chemical modification to their backbone structure such that they are resistant to thedegradation by nuclease and have proper tissue distribution within the body along with goodin-vivo half life.The use of ASO drugs over other drugs is advantageous as the latter usually target theproteins formed during the expression of the disease, while the former works at the genelevel. The ASOs being made of nucleotides are much easier to prepare as only the sequenceof the mRNA is needed. The ASO target is only of one domain, compared to multipledomains in case of protein related drugs. Hence, the sensitivity of the ASO drugs can beeasily measured by scanning or the southern and northern blotting. The manifestation of thediseases in case of the ASO drugs is much less as the mRNA is itself silenced, hence toovercome this silencing the clonal expansion of the cells that is needed takes a long time. Thebinding of the ASO with the mRNA is by means of hydrogen bonds, which is much morestronger than any other types of forces like Van der Waals force, etc which occurs in case ofthe binding of the drugs to proteins.The research on the ASO therapeutics has moved from the pre-clinical models to the clinicaltrials. Antisense technology has proved to be a formidable tool for the discovery and study ofthe various physiological and pathological processes within the body. The research going onwill refine the drug delivery methods, specificity, and affinity of the antisense therapeutics,which would be a better tool for the treatment of the patients considering the progress in theuse of various gene therapies for treating incurable diseases. ASOs are being researched uponfor the treatment of different types of cancer, diabetes, Duchanne muscular dystrophy,obesity, different inflammatory diseases like Asthma, autoimmune diseases like HIV/AIDS,different cardiovascular diseases and many other diseases.Antisense technology has proved to be better method of treatment considering short drugdevelopment time and lesser failure in clinical trials compared to other traditional drugs. Theapproach of the antisense technology is in accord with the latest, emerging technology in thedrug development process, technologies based on genome and the integration of thetherapeutics with diagnostics. Hence, these advantages put the ASO therapy on a higher scalethan the other drugs targeting proteins, which gives scope for further research on the topic.Antisense technology has provided a good base for the research of more new and highlyspecific therapeutics.
  10. 10. Laboratory of Molecular Design Antisense Therapy If a particular gene has a role in disease, and the genetic code of that gene is known, onecould use this knowledge to stop that gene specifically. Genes are made of double-helicalDNA. When a gene is turned on, the genetic code in that segment of DNA is copied out as asingle strand of RNA, called messenger RNA. The messenger RNA is called a "sense"sequence, because it can be translated into a string of amino acids to form a protein. Theopposite strand in a DNA double helix (A opposite T, T opposite A, C opposite G, G opposite C)is called the "antisense" strand. We use the antisense coding sequence of a disease gene tomake short antisense DNAs in our laboratory. These antisense DNA drugs work by bindingto messenger RNAs from disease genes, so that the genetic code in the RNA cannot be read,stopping the production of the disease-causing protein.Click here to return to Wickstrom Lab Homepage.