Indian Journal of Biotechnology Vol 2, April 2003, pp 175-183 Immunostimulation: The Sense in Antisense Technology Archana Pandey', Satyendra Mishra/ and Krishna Misra*2,3 'Chemistry Department, C M P Degree College, Allahabad 211 002, India 2Nucleic Acids Research Laboratory, Chemistry Department and 3Centre for Biotechnology, University of Allahabad, Allahabad 211 002, India Received 25 March 2002; accepted 21 June 2002 The post-human genomic era has led to the development of therapies, which specifically target molecular pathways responsible for diseases. The original concept of antisense therapy was to simply turn off gene's activity by a short synthetic DNA sequence, having sequence complementary to mRNA and thus block the production of undesirable protein. However, during the last two decades this concept has undergone miraculous change. Today antisense therapy is on crossroads. The observation that oligodeoxynucleotides containing CpG dinucleotides (CpG DNA) exhibit immunostimulatory effect has lead to their use as therapeutic agents and adjuvants for various diseases. Knowledge gained from studies of the medicinal chemistry of CpG DNA has provided a base for designing the second generation of CpG DNA agents with immunostimulatory activity. The present article reviews the recent developments, which have caused the revival of antisense therapy. Keywords: antisense, immunostimulation, vaccine, oligonucleotide therapy Introduction Antisense therapeutics once appeared a wonderful path to biotech glory. Its allure rested in its simplicity and specificity. It was expected to be an elegant, systematic approach to drug target validation and gene function. The concept of antisense therapy initiated by Zamecnik and Stephenson in 1978 was straightforward; design an oligonucleotide to bind to a gene's mRNA, inhibit its translation, prevent protein expression, thus turn off gene's activity and thereby exert a therapeutic effect. All that needed was to know the gene's or mRNA's sequence. More than two decade's of elusive chase, after the idea of a lightening quick drug discovery method has met with some hard ground. The history of antisense drug development although filled with disappointments, surely has a bright future. Its finest hours are yet to come. The toughest years of antisense development now seem to be ending in this post-genomic era, since the antisense tools are tailor-made. The unraveling of human genome sequence has certainly enabled companies to develop therapies that specifically target molecular pathways responsible for disease. Antisense technology makes it possible not only to identify the role of individual genes in disease but * Author for correspondence: Tel: 0532-2460816; Fax: 0532-2623221 E-mail: [email protected]also to develop specific drugs to treat particular diseases. "The question is not any longer will antisense deliver a drug", says Frank Bennett (Principal Scientist of Antisense Research at Carlsbad, CA based ISIS Pharmaceuticals Inc, San Diego, USA) with confidence "It's what will be the next antisense drug". However, what no one knows and therefore, cannot answer is what would be the impact of antisense drugs on medicine in future. Today antisense technology is on cross-roads. Mechanism of Antisense Therapy In the pathway from gene to protein there are several possible regulatory steps. The process of genetic expression consists of the transcription of antisense strand of DNA (acting like a template) to single stranded mRNA, which now becomes a sense strand followed by mRNA binding to cellular factors (ribosomes) where specific proteins dictated by the genes are made (translation). Many diseases result when either foreign DNA or RNA or inappropriate expression of host DNA generates a disease associated protein. The insertion of viral DNAIRNA sequences into the host cell by retrovirus, results in assembly of new viral particles (Fig. 1). In order to block production of undesirable protein, short synthetic DNA sequences approximately 10-20 nucleotides long having sequence complementary to
Transcript
Indian Journal of BiotechnologyVol 2, April 2003, pp 175-183
Immunostimulation: The Sense in Antisense Technology
Archana Pandey', Satyendra Mishra/ and Krishna Misra*2,3'Chemistry Department, C M P Degree College, Allahabad 211 002, India
2Nucleic Acids Research Laboratory, Chemistry Department and3Centre for Biotechnology, University of Allahabad, Allahabad 211 002, India
Received 25 March 2002; accepted 21 June 2002
The post-human genomic era has led to the development of therapies, which specifically target molecularpathways responsible for diseases. The original concept of antisense therapy was to simply turn off gene's activity bya short synthetic DNA sequence, having sequence complementary to mRNA and thus block the production ofundesirable protein. However, during the last two decades this concept has undergone miraculous change. Todayantisense therapy is on crossroads. The observation that oligodeoxynucleotides containing CpG dinucleotides (CpGDNA) exhibit immunostimulatory effect has lead to their use as therapeutic agents and adjuvants for variousdiseases. Knowledge gained from studies of the medicinal chemistry of CpG DNA has provided a base for designingthe second generation of CpG DNA agents with immunostimulatory activity. The present article reviews the recentdevelopments, which have caused the revival of antisense therapy.
IntroductionAntisense therapeutics once appeared a wonderful
path to biotech glory. Its allure rested in its simplicityand specificity. It was expected to be an elegant,systematic approach to drug target validation andgene function. The concept of antisense therapyinitiated by Zamecnik and Stephenson in 1978 wasstraightforward; design an oligonucleotide to bind to agene's mRNA, inhibit its translation, prevent proteinexpression, thus turn off gene's activity and therebyexert a therapeutic effect. All that needed was toknow the gene's or mRNA's sequence. More than twodecade's of elusive chase, after the idea of alightening quick drug discovery method has met withsome hard ground. The history of antisense drugdevelopment although filled with disappointments,surely has a bright future. Its finest hours are yet tocome. The toughest years of antisense developmentnow seem to be ending in this post-genomic era, sincethe antisense tools are tailor-made. The unraveling ofhuman genome sequence has certainly enabledcompanies to develop therapies that specifically targetmolecular pathways responsible for disease.Antisense technology makes it possible not only toidentify the role of individual genes in disease but
*Author for correspondence:Tel: 0532-2460816; Fax: 0532-2623221E-mail: [email protected]
also to develop specific drugs to treat particulardiseases.
"The question is not any longer will antisensedeliver a drug", says Frank Bennett (PrincipalScientist of Antisense Research at Carlsbad, CAbased ISIS Pharmaceuticals Inc, San Diego, USA)with confidence "It's what will be the next antisensedrug". However, what no one knows and therefore,cannot answer is what would be the impact ofantisense drugs on medicine in future. Todayantisense technology is on cross-roads.
Mechanism of Antisense TherapyIn the pathway from gene to protein there are
several possible regulatory steps. The process ofgenetic expression consists of the transcription ofantisense strand of DNA (acting like a template) tosingle stranded mRNA, which now becomes a sensestrand followed by mRNA binding to cellular factors(ribosomes) where specific proteins dictated by thegenes are made (translation). Many diseases resultwhen either foreign DNA or RNA or inappropriateexpression of host DNA generates a diseaseassociated protein. The insertion of viral DNAIRNAsequences into the host cell by retrovirus, results inassembly of new viral particles (Fig. 1).
In order to block production of undesirable protein,short synthetic DNA sequences approximately 10-20nucleotides long having sequence complementary to
I I 'DNA dependent DNA polymerase3 --------- S'DNA' .5' < 'lI)NA [Provirus]
JoCircularform of Viral DNA
-------t-------
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:c:==) Target forsense oligos
Target for~aJ)tisellSC oligus
. _~""IntCgrated DNA
Triplex'~L~ •Nucleus . -----:)) .
ITranscript~')n~ 'I I L-lntron • [~~5. 5exon I I' . 3_ JPrecursor
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Mutation ofmRNA
.Ribosome Binding site Start5/r~Cap
.LCapsid protein)
Mes age sico
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Reverse Trnnscriptase
Fig. l-Life cycle of a retrovirus inside the host cell.
certain conserved regions of viral mRNA weredesigned and prepared (Milligan et al, 1993). Thissynthetic DNA is a mirror image (antisense) to aportion of the mRNA (sense). The antisense DNAbinds to mRNA because it is designed to be its exactmirror image. This binding (hybridisation) inhibits theproduction of the disease associated protein. The
\
Several newparticles
antisense concept is effectively applied for regressionof cancerous growth by selectively binding to thetelomere sequences (Misra et al, 2002).
The activity of antisense oligonucleotide IS
enhanced by another mechanism, which involves anenzyme called "ribonuclease H" or "RNase H"(Agrawal & Kandimala, 1999). The normal activity of
PANDEY et al: ANTISENSE TECHNOLOGY
this enzyme is to mop up the unwanted copies ofmRNA. If an antisense oligonucleotide binds to themRNA strand, this RNase H dismantles the strands ofmRNA which bear the antisense oligonucleotidesleaving the antisense oligonucleotide as suchuntouched. As a result, this antisense oligonucleotidemay bind to another mRNA one after the other and soon. Consequently, a lower dose of sucholigonucleotide would be as effective as a higher doseof oligonucleotide that do not team up with RNase H(Fig. 2).
Mechanism of Antisense-inhibitionThe following probabilities have been suggested
for antisense inhibition:1. Transient inhibition by prevention of ribosome
binding to the RNA by masking its binding siteon the mRNA.
2. Permanent inhibition of the digestion of the RNA-part of the DNA by RNase H, an enzyme that canbe found in most cells.
3. Permanent inhibition by cross-linking theoligonucleotide to the target RNA.
The interference with complementaryoligonucleotides can be accomplished at any point inthe genetic information flow i.e. at storage level(antigene therapy) by use of triple helix formingoligonucleotide (at DNA level), at informationprocessing level (anti-messenger therapy) i.e.oligonucleotide directed against mRNA (cytoplasm)or its precursor (nucleolus).
Approaches to Gene RegulationAlthough various means of gene regulation are
available, the transcriptional approach (anti-messenger therapy) is probably the most versatile. Itcan be effected by, 1 Natural antisense transcripts; 2Ribozymes; and 3 Synthetic oligonucleotides.
-ceo_~o:o-o~eo"
a::e _~liTranslation into protein
R b
/
No Translationioosome /
"f 1l1I1l1JlJ Antisense DNA
. /" ~~ .lI.J..I. m RNA breaks
Normal.>" ~'?).s
11111 •• -( rrumrtI-Messenger With Antisense
RNA Oligonucleotide
Fig. 2-Dismantling of m-RNA with antisense-oligonucleotides.
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1.Natural Antisense TranscriptsAntisense was first described as a naturally
occurring phenomenon in which cells transcribe anantisense RNA complementary to a cellular rnRNA(Weintraub, 1990; Helene & Toulme, 1990). Thisantisense RNA was found to be a repressor of geneexpression, hybridizing to a target mRNA, inhibitingits translation, and decreasing the cellular levels ofproteins. Antisense RNAs inhibit gene expressionthrough the activity of a cellular enzyme, whichmodifies double stranded RNAs (Bass & Weintraub,1988). This enzyme recognizes the RNA:DNAduplex, disrupts the base pairing and changes many ofthe adenosine residues to inosine (Bass & Weintraub,1988; Nishikura et al, 1989). Gene expression isinhibited since the modified mRNA is no longercompetent for translation. Nishikura et al (1990) havereported that mammalian cells possess a dsRN Aunwinding and modifying activity capable ofspecifically dissociating RNA duplexes. It has beensuggested that several cases of biased hypermutationof RNA viruses are caused by the dsRNAunwinding/modifying activity. During the last 10-15years, artificial antisense transcripts have beenextensively used to control the expression of differentproteins.
2. Transacting Ribozymes (Catalytic RNA's)Cech and his colleagues discovered that certain
splicing reactions are catalyzed by RNA. It wasunequivocally demonstrated that certain interveningsequences (Group I) were inherently capable ofcatalyzing RNA splicing reactions to give rise tomature RNA. Cech termed such RNA moleculespossessing enzymatic activity 'ribozymes' (Cech etal, 1982). These ribozymes exert their inhibitoryaction in a highly specific manner and are notexpected to be detrimental to the cell function.Therefore, the concept of exploiting the ribozymes,catalytic centres for cleaving a specific target RNAtranscript is now emerging as a potential therapeuticor preventive strategy in human diseases andagriculture. Ribozymes are isolated from plants,animals, bacteria and fungi. Hammerhead ribozymesfrom plant virus RNA genomes, discovered bySymons (1992) offer an attractive example fortailoring ribozyme activity and specificity. It is a goodcandidate for incorporation into antisense transcripts.It was found that hammerhead ribozymes can disarmAIDS virus in tissue culture cells (Bertrand & Rossi,1996). One of the principal advantages of the use of
178 INDIAN J BIOTECHNOL, APRIL 2003
ribozymes as antisense agent is their ability to cleavemultiple targets (Fig. 3) (Rossi & Sarver, 1990). Thisproperty of turnover is a function of both length andbase composition of the flanking sequences used forbase pairing of the ribozymes with its substrate (Misraet at, 2002).
3. Synthetic OligonucleotidesDuring the last one decade, tremendous progress
has been achieved in designing the appropriateantisense oligonucleotides. The sequence of theantisense oligonucleotides should be reverse andcomplementary to a putative single stranded region onthe RNA. As a control molecule the senseoligonucleotide meaning the oligonucleotide, whichhas the same sequence and orientation as the target,should be chosen. The target region should be chosenwith regard to the mechanism of the antisenseapproach. Regions around the start codons fortranslation may lead to success by preventing theribosome binding to RNA, but other regions work aswell because of the degradation by RNase H. The keyto success may be to find a region of highaccessibility for both the oligonucleotide and RNaseH. The length of the oligonucleotide is compromisedbetween specificity and accessibility to the target. A13 or 14 mer sequence discriminates between twopoint mutations and with regard to the population ofmRN A's in a cell, this sequence should appear onlyonce. Oligonucleotides with 16 or more nucleotidesare too long, leading to less accessibility (Hersch lag,1991).
Limitation for Antisense OligonucleotidesFor the antisense oligonucleotide to reach its target
and be effective, several barriers have to beovercome. The oligonucleotide has to be internalizedby the cell at first to cross the cell membrane andsecondly to escape the degradative enzymes and lastlyto bind specifically to its RNA target. The polyionic-unmodified oligos are not capable of crossing the cellwall by passive diffusion. The naturaloligonucleotides have been shown to accumulate overa period of 3-4 hrs inside the cell probably enteringthe cell by receptor mediated endocytosis. However,the process sometimes produces unexpected results.How the molecule escapes the endocytotic vesicle isstill an unanswered question. Liposome encapsulationcan prove to be most efficient method of deliveringthe oligonucleotides inside the cells. Delivery ofantisense oligonucleotides may be carried by one ofthe following methods of administration:
Syntuesis ofSubstrate'S' andCatalytic 'c' RNAs
5'P~~ ~C _' OH, HO'" ~P5
I 1I K: 5'P s I
. 1-10 ~ = :::;OH [-S ComplexlI C P 5'I,I _, Sip l K, 0' PI)P~_' J
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I
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Fig. }--Diagrammatic representation of ribozyrne-substrateinteraction and cleavage of substrate.
In human clinical trials, mainly intravenous and/orintravitreal injections are used.
Chemical Modification and StabilityOligonucleotides initially used by Zamecnik and
his co-workers (Zamecnik et at, 1986) and Goodchildand his co-workers (Goodchild et al, 1988) to inhibitthe HIV replication in vitro were 20-mers,synthesized by using phosphoramidite approach.These unmodified oligonucleotides are required inhigh dose (5-8 times) resulting in undesirablecytotoxicity. Thus, a highly effective dose ofoligonucleotides is required because theseoligonucleotides are susceptible to extra orintracellular nuclease digestion (Agrawal et at, 1990).These are also known as first generationoligonucleotides. Nuclease resistant modifiedoligonuleotides have been developed by usingdifferent modified backbone (Agrawal, 1996; Akhtar& Agrawal, 1997; Bennett, 1998; Agrawal, 1996;Crooks, 1998; Wickstroin, 1998; Ciba FoundationSymposium, 1997; Stein & Kreig, 1998). Amongstthese modified oligonucleotides, phosphorothioates(Agrawal et al, 1997; Phillips, 1996; Grindel, 1998;Martin, 1998; Glover, 1997; Agrawal & Zhang, 1998;
PANDEY et al: ANTISENSE TECHNOLOGY
Nicklin & Craig, 1998; Agrawal & Zhao, ]998; Zhaoet at, 1996) have proved most useful since in additionto being resistant to nuclease digestion, these arewater soluble, form stable duplexes with specifictarget mRNA's and also catalyse mRNA cleavage ofRNA-DNA duplex by enhancing the activity ofRNase H. Later on some non-sequence specificeffects (aptamer effects) were observed withphosphorothioate analogues. Other hurdles werebinding affinity and cell permeability. Despite thecontroversies, the phosphorothioate oligonucleotideshave been evaluated in clinical trials for the treatmentof diseases like cancer (Henry et al, 1997), Crohn'sdisease, rheumatoid arthritis and in cytomegalo virusinfections (Crooke, 1998). These oligonucleotideswith modified backbone are also known as second-generation antisense oligomers. In order to modify theinternucleotide linkage a number of protecting groupsfor this purpose have. been reported from ourlaboratory (Misra et al, 1980a; Misra et al, 1980b;Misra et al, 1982; Kumar & Misra, 1997; Misra et al,1988).
Extensive modification effects hybridisation anddecreases melting temperature of respective duplexes.This destroys selectivity such as methyl phosphonatesand cx-anomeric nucleosides do not activate RNase H,the most important mechanism of antisense action(Goodchild & Cohen, 1998).
As a consequence MBO's (mixed backbone
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oligonucleotides) (Agrawal & Kandimalla, 2000)were introduced as the third generation antisenseoligomers. These were the combination of the two i.e.oligonucleotides having same units with modifiedbackbone and some having normal i.e. unmodifiedbackbone. These MBO's have high selectivity as wellas the nuclease resistivity. Although it has beenreported that the overall immunostimulatory effects ofphosphodiester and phosphorothioate CpG DNA aresimilar, the latter are commonly used (Sester et at,2000). Some probable target genes for specificdiseases amenable to antisense thereby are tabulatedin Table 1.
Non-antisense Aspect of Antisense TherapeuticsSince early 1990' s the antisense technique has run
into unforeseen problems. The antisense compoundswere not responding the way researchers once thoughtthey should. Although the results of clinical trialswere promising, many workers felt that a lot ofpositive effects reported are not just antisense butother non-antisense mechanisms as well. Surprisingly,researchers found that the oligonucleotides,homopurines and homopyrimidines, they were usingas controls, which could not recognize rev, gag orother genes, either shutdown virus replication orblocked cell proliferation almost as efficiently as theones they were testing as drugs. At first noexplanation could be offered for this amazing
Table J-Some diseases thought to be amenable to oligonucleotide therapy with suggested targetgenes
Therapeutic area Disease
Cancer LymphomaLeukaemiaMelanomaCarcinomaHypertensionCardiovascular
Immune disordersimmunoglobulins
Arthritis
Central nervousSystem (CNS)
Lupus erythromatosusAlzheimer diseasedepression
Metabolic diseases UlcerPsoriasisAIDSMalariaHuman papilloma virusInfluenza A & BCytomegalovirusHerpes ZosterHSV-J, HSV-2
pepsinogentransforming growth-aHIV -TAT and othershaem polymerasevarious-do--do--do-ICP4 and others
180 INDIAN J BIOTECHNOL, APRIL 2003
behaviour. A rheumatologist working at theUniversity of Iowa, USA came out with a solution,which was published in April issue of Nature (Kreiget al, 1995). Krieg figured out that antisenseoligonucleotides mimic bacterial DNA in triggering apotent response by mammalian immune cells. Heshowed that if in a oligonucleotide there is C followedby G and if in Krieg's immunostimulatory CpGoligonucleotides, the right bases were upstream anddownstream, then there is immune activation i.e. sucholigonucleotides activate mammalian B-cells andnatural killer (NK) cells in culture. This occurs whenCpG motif (p-phosphodiester) lacks methyl groups.Such sequences are common in bacterial DNA but notin mammalian DNA where cytosines are usuallymethylated. The immune response may be a way ofdefending against bacterial attack. Antisensemanufacturers do not usually add methyl groups totheir synthetic oligonucleotides. Thus, the mammalianimmune system that encounters such compounds withCpG motif may be tricked into thinking that they havebeen invaded by bacterial aliens and consequentlyspring into action. When Krieg methylated CpG in hisoligonucleotides, their immune stimulation ceased.
Coley's Toxin and BCGWilliam B Coley was a New York born surgeon
with interest in cancer. He learnt of a cancer survivorwho coincidentally suffered severe skin infectioncaused by Streptococcus pyogenes. Coley wonderedwhether the bacteria had caused the patient's tumourto regress'. In 1890, he began injecting cancer patientswith the crude bacterial lysate that came to be knownas Coley's toxin. He claimed that 40% patientsachieved lengthy remission. At that time, it wasthought that the active principle of Coley's toxin wasa lipopolysaccharide. However, many tried but failedto repeat his results and the father of immunotherapyendured decades of derision. After more than acentury scientists watching the development ofimmunostimulatory oligonucleotides with keen senseof anticipation are now in a position to understandthat probably the activity of Coley's bacterial lysatewas due to its DNA.
Tokunaga et al (1984) tried to again look for theactive agent in Coley's toxin. Working with fractionof Baciillus Calmette Guerin (BCG) bacteria bestknown in connection with tuberculosis vaccination,they concluded that BCG's anti-tumour activity was aproperty of its DNA.
Krieg explained this observation of Japaneseworkers as the destruction of tumour caught in theimmune response provoked by unmethylated BCGDNA. It was just inconceivable, the way in which theimmune system possibly could differentiate betweenBCG DNA and normal human DNA. The vertebrateimmune system has evolved to recognize specificnucleotide sequence motifs present in bacterialgenome and to elicit immunological reactions tocounteract bacterial infections (Yamamoto et al,2000; Gurunathan et al, 2000).
Therapeutic Potential of Synthetic CpG DNA:Current Status
When Krieg's results were published there was acommotion in the scientific community. However, itwas brief since his results could be verified with micebut not with humans. Krieg was lucky twice, he cameout with another startling finding that sequenceswhich activate human cells are different from thosethat activate mouse cells. He reported that"GACGTT" is a good CpG activator motif in mousecell while GTCGTT or TTCGTT is a very goodactivator for humans (Hartmann et al, 2000).Moreover, human cells degrade DNA much fasterthan mice, therefore, experimental conditions thatworked fine with mouse cells did not yield any resultwith humans. CpG related immune stimulation variesfrom species to species.
Several antisense drugs are in different stages ofclinical trials since early 90's, e.g. GEM 91, GEM 92,GEM l32 of Hybridon, ISIS-2503, ISIS-2302, ISIS-3521, ISIS-5l32, Vitravene (Fomivirsen) of ISISPharmaceuticals Inc, San Diego, USA. G 1128 and G3139 of Genta are now being viewed from differentperspective. Although Vitravene is the only antisensedrug, which has gained approval of FDA in 1998 andhas reached the market, but the question remains i.e.if one drug can inhibit gene function by binding to itsmRNA and blocking protein translation, why otherscannot follow.
At present several drugs already in the marketstimulate the immune system in various ways,prominently the interferons and Colony stimulatingfactors. However, most (CpG motif present inbacterial DNA rapidly induce lymphocytes to secreteinterleukin 6, interleukin 12 and interferon y) of thesehave an artificial disorganized response (Klinman etal, 2000). The CpG oligonucleotides activate immunesystem by detection of bacterial products in a way that
PANDEY et al: ANTISENSE TECHNOLOGY
all activated responses synergize with each other.Therefore, their toxicity is minimal.
Each CpG oligonucleotide has a profile of its owni.e. of the cell types affected and cytokines stimulatedand usually each leans either towards the Th-1 (cellmediated) or Th-2 (antibody) T helper cell pathway(Fig. 4).
Coley Pharmaceutical's CpG 7909, boosts antibodyand cell mediated responses to pathogens andcancerous cells. CpG 8916 acti vates NK cells, whichcan attack infected cells and NK-sensitive cancerssuch as melanoma. CpG 8954 stimulates interferon-alpha production. It might be used as a prodrug,having low toxicity and a way to stimulate interferonalpha against cancers and viral infections. Its fourthproduct and the lead product i.e. ProMuneTm is inphase IIII trials for non-hodgkin' s lymphoma,melanoma and basal cell carcinoma.
Hybridon is busy learning the effect of changingbases upstream and downstream CpG motif. Buildingon a decade of research as antisense base chemistry,Hybridon CpG oligonucleotides (14-18 mers) mixnatural and synthetic bases. It has several immunestimulatory YpG motifs [pyrimidine analogs in placeof cytosine] and CpR motifs [purine analogs in placeof guanine]. The company has now produced a set ofoligonucleotides driving a range of immune responsedialing it up or down.
Dynavax lead CpG oligonucleotides, now in phaseII treat allergy, where the Th-2 pathway is involved.According to them the inflammation, histamine
Innate Immunity Rapid activation of immune,system defences
181
release and running nose can be treated by shifting theimmune response from Th-2 pathway to the Th-lside.
CpG DNA Recognition by Immune CellsHemmi et al (2000) reported that the recognition of
CpG DNA occurs through a transmembrane protein,which belongs to Toll-like receptor (TLR) family.TLR 9, a recently discovered TLR family proteinrecognizes unmethylated CpG dinucleotides inspecific pathogen-associated molecular contextpresent in bacterial and synthetic DNA. Otherevidences indicate that DNA protein kinase (PK)recognizes CpG DNA and subsequently activates theNF-KB pathway (Chu et al, 2000). It is establishednow that the activation of autoreactive B-cellsinvolves the co-engagement of the B-cell receptor andTLR 9 via antibody/antigen immune complexes. Thisestablishes that endogenous TLR ligands have acritical role in the aberrant activation of the adapti veimmune system (Leadbetter et al, 2002). However,regardless of the fact whether CpG DNA immuneresponses are mediated through the TLR 9 receptor,DNA-PK, or both, the ultimate result is the activationof multiple transcription factors and upregulation ofvarious cytokines (Takeshita et al, 2001; Bauer et al,2001).
ConclusionFrom the results of recent reports it is evident that
CpG DNA is not only a significant tool for
Broad non-specific IIimmunitytherapeutic andprophylactic cancer Iinfectious disease' I,Accelerated immunesystem recovery
Fig. 4--Influence of CpG oligonucleotides on both antibody and cell-mediated immunity and their applications.
182 INDIAN J BIOTECHNOL, APRIL 2003
modulation of immune system, but can be exploited totreat wide variety of diseases quite economically(Scheule, 2000; Hancock et al, 2001; Fiedler et al,2001; Davis et al, 2000). The medicinal chemistry ofepG DNA is still in its infancy but its potential lies inintroducing site specific chemical modificationswhich can also modulate cytokines induction(Agrawal & Kandimalla, 2002). There is a species-dependent selectivity of epG DNA and the optimalepG DNA sequences for many vertebrate species areyet to be worked out. Oligonucleotides can bedesigned to have both antisense (decoy, ribozyme ortriplex) and immunostimulatory components tomodulate a specific gene product and simultaneouslyinduce immune stimulation, resulting in powerfulcombination effects. Thus, this combination strategycan work wonders for treatment of diseases likecancers, asthma and other allergic symptoms.
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