The Treatment of Cystic Fibrosis using gene therapy, in particular Adeno-Associated Viruses

Sample details Pages: 25 Words: 7507 Downloads: 4 Date added: 2017/06/26 Category Statistics Essay Did you like this example? In this dissertation we shall consider the field of gene therapy in specific relation to cystic fibrosis. We examine the different delivery vector mechanisms that have already been explored and concentrate primarily on the adeno-associated vectors. We examine the current state of research and consider the advantages and drawbacks of the various methods considered. Don’t waste time! Our writers will create an original "The Treatment of Cystic Fibrosis using gene therapy, in particular Adeno-Associated Viruses | Physics" essay for you Create order We conclude with a discussion and analysis of our findings and make anumber of assumptions relating to the future direction of the researchin the field. The rate of progress in the field of gene therapy has been enormous. We must remind ourselves that the first clinical gene transfer experiment took place in 1989 when a patient with malignant melanoma received genetically modified auto logous T cells. (Rosenberg SA et al1990) Gene therapy encompasses two major areas. The in vivo field, where genes are incorporated into the target cells of the living body and the ex-vivo field where the target cells themselves are genetically modified outside the body and then re-implanted. Medical science has been using the basic techniques of gene transfer for a long time. The technique has been exploited when viral genes are introduced to human cells when a viral vaccine is administered. The key technologies that allowed the transition from vaccination to gene therapy were the evolution of methods that allowed the genes to be isolated and replicated (cloned) and manipulated (engineered) prior to transfer into human cells (Freeman SM et al 1996) The key principle in this process is the efficient transfer of the manipulated therapeutic genes into the nuclei of target cells usually be means of various vectors. This dissertation will be considering the utilisation of these vectors in some detail. In broad terms, the new or modified genetic material is able to produce new proteins which can restore deficient or abnormal functions of genetically diseased tissues, to generate tissues that have entirely new properties or to create transplantable tissues for the controlled release of therapeutic proteins. (Russell SJ1997) In terms of viral vectors, prior to 1996 science was dependent on the use of modified retroviral vectors (eg.MMLV) to effect gene transfer into the chromosomes of a target cell and the adenovirus vectors when such integration was not needed. Neither vector was particularly successful as the intact nuclear membrane (in then on-dividing state) was a major barrier for chromosomal gene integration. (Sikorski R et al 1998). A breakthrough came with the realisation that lentiviruses (e.g. HIV) have the same ability to transfer genetic coding into the cellular genome but could do this in the non-dividing or dormant phase cells. (Amado R G et al 1999) In vitro, lentiviruses have been shown to change the target cells expression of proteins for up to six months. importantly, they can be used for terminally differentiated cells such as respiratory epithelium. The only cells that the lentivirus cannot penetrate the nucleus are those in the quiescent (G0) state as this blocks the reverse transcription stages of protein synthesis. (Amado R G et al1999) Cystic fibrosis Cystic fibrosis is the most commonly lethal inherited recessive inthe caucasian population. It affects about 1 per 2,500 livebirths. Thetreatment of cystic fibrosis has improved enormously in the last fiftyyears with the life expectancy increasing from an average 10 years to30-40 years now. The prime cause of death in affected individuals is the repeatedcycle of infection, inflammation and fibrosis of the respiratory tractwhich eventually culminates in respiratory failure and death. The disease itself is caused by mutations in the single gene forthe cystic fibrosis transmembrane conductance regulator (CFTR) whichproduces a protein found in sweat and pancreatic ducts, gut,seminiferous tubules, lungs and many other tissues. The mutationsresult in an abnormal protein which, when expressed in the lungs,produces thick viscous and dehydrated secretions. This does not allow for the efficient expulsion of bacterial pathogensfrom the lungs and a number of highly resistant forms of bacteria arecommonly found in cystic fibrosis (viz pseudomonas aeruginosa)(Porteous DJ et al 1997). An individual must receive a defective copy of the cystic fibrosisgene from each parent in order to develop the clinical picture ofcystic fibrosis. Following normal genetic principles, if two carriersconceive a child, there is a 25% chance that it will have cysticfibrosis, a 50% chance that it will be a carrier and a 25% chance thatit will have two normal cystic fibrosis genes. Viral and non viral vectors Viruses have an ability to enter a host cell and combine their owngenetic material with that of the host cell. This is the basicrationale behind the science of gene transfer therapy. As we shalldiscuss in some detail in this dissertation, there are a number ofpotential viral vectors that have been explored, evaluated andexploited in the search for an efficient and safe form of therapy.Viruses are not the only vector that can be utilised . Simply placingDNA in the nasal mucosa will produce some incorporation into theepithelial cells (Knowles MR et al 1998). This absorption can bedemonstrably enhanced further by the combination of the DNA withvarious plasmid or lipid complexes(Zabner et al 1997) The advantages of lipid or plasmid assisted transfer mechanisms arethat they do not appear to generate the immunological responses thatare seen with the viral vectors. They can also be used to facilitatethe transport of much larger pieces of DNA which would otherwise belimited by the packaging consideration incumbent on the viral vectors.(Felgner P 1997). The use of retroviral vectors is far from straightforward. The heavilypublicised case in April 2000 brought some of the problems to theattention of the media. A retroviral manipulation of a case of X-SCID(X linked severe combined immunodeficiency) was treated by gene therapywith an apparent degree of success (BBC 2002). This particular diseaseprocess is caused by a mutation on the gene which codes for the C chainof the cytokine receptors which is situated on the X chromosome andvital for the functional development of T Killer lymphocytes which aretherefore completely absent in the condition A multinational team used a retroviral vector to insert a functionalcopy of the gene into bone marrow stem cells which were thenre-transfused back into the patient. (Cavazzana-Calvo M et al 2000).This particular case resulted in a return to normal levels of T cellsin a comparatively short period of time. This was hailed in both thepopular media and the peer reviewed journals as a major success and itcan indeed be considered a landmark as it pioneered the successful useof an ex-vivo procedure that avoided direct in vivo transfer of thevector. The reason for specifically highlighting this particular case isthat following the initial optimism of the clinical team, two of thefirst ten patients with this condition who were treated in the same waysubsequently developed a leukaemia-like illness. Genetic analysis ofthe malignant cells suggested that the retroviral vector used in thetransfer had also activated an oncogene LIM-only2 (LMO2) which is knownto be associated with some forms of leukaemia. The clinicians reviewingthe situation felt that, although it was not the only cause of themalignancy it was one of the events that triggered it. Similar concernshave been raised in respect of other clinical trials. (Lehrman S 1999) The prime reason for presenting these events is to demonstrate thefact that there is both a theoretical and practical risk of insertionalmutagenesis. Reduction of the risk requires greater specificity of thetargeting of the genetic deficit perhaps by directing the expressionof the therapeutic genes to various specific tissues utilising bothtransductional and transcriptional targeting. Relph K et al 2004), In terms of specific considerations of the arguments in favour of theuse of retroviral vectors, one can cite the fact that they have ahighly efficient mechanism of gene transfer together with lowimmunogenicity. It is a well researched and well studied system and isknown to selectively infect actively dividing cells. The conversearguments reflect their disadvantages including their ability todisturb or activate oncogenes, the fact that they are difficulty tospecifically target and it is difficult to obtain high titres in theclinical situation (after Olsen, J. C. 1998). In broad terms, the principles behind the use of retroviral vectorsare that they must be modified in order not to be able to transmit anyovertly pathological coding. This involves the deletion of viral helpergenes such as gag, pol and env to render the replication processinvalid. This is done by utilisation of a producer or packaging cellline. (Nichols, E. K 1998). An example of a commonly utilised and extensively researched vector isthe MoMuLV. It is an engineered vector which can store 8 kb of RNAwithout compromising packaging efficiency. It is a hybrid cell lineeasily grown in mouse fibroblast cells There is a subdivision of the retroviral vectors known as thelentivirus, which is the only retroviral vector capable of integratinginto the chromosomes of non-dividing cells. This has been effectivelydemonstrated in vitro (Naldini L et al 1996). The biggest problem with the lentivirus vectors is that theyappeared to only produce very low titres. Some recent researchsuggested that a modification to a amphotropic envelope protien wascapable of allowing higher titre levels. (Rolls M et al 1999) At about the same time that the scientific press was learning aboutthe problems with retroviral transfer (see above) other investigatorswere working with adeno-associated viruses (AAVs). A similar processwas invoked using adeno-associated viruses to correct a genetic defectinvolving coagulation factor IX. The adeno-associated viruses were usedas they were considered to be amongst the safest candidates for genetransfer. They do not naturally cause disease processes in humans andhave only rarely been found to incorporate in a random fashion into thehuman genome. Although it is noted that adenoviruses do cause oncogeneactivation in rodents although it has not been found in humans(Blacklow NR 1988). The trial had a very positive outcome. (Kay MA et al 2000), but thetrial author (in later research work) published a study which suggestedthat, in study mice, the vector used in the trials actually integrateditself into gene containing regions of DNA more frequently that it didinto non-coding regions (Kay MA et al 2003). The findings were reportedas the fact that new genetic material was randomly distributed amongstall of the chromosomes particularly at sites of gene activity. On thisbasis, there appears to be at least a theoretical basis for thepossibility of similar cellular defects such as occurred in the X-SCIDpatients. Adenoviruses are comparatively simple structures. They arecategorised as double stranded DNA viruses. They have icosahedralcapsids with twelve vertices and seven surface proteins. The virionitself is spherical and non-enveloped and in the region of 70-90 nm insize. Their natural history is that they are spread easily in the naturalstate by the faeco-oral route and also by respiratory inhalation whichclearly has great implications for the treatment of cystic fibrosis. A theoretical analysis would immediately suggest that the adenovirus should be a suitable candidate for gene therapy as they can codefor specific proteins and they do not produce infection pathogenicviral offspring. The early trials into this particular area were reviewed by Griesenbach(Griesenbach U et al 2002) who pointed out that the cystic fibrosisgene was first cloned in 1989 and in the subsequent years, 18different trials were carried out, all with rather low degrees ofsuccess. They collectively trialed three different vectors, namelyadenoviruses, adeno-associated viruses 2 and cationic liposomes, andalmost universally found that each vector had a very low rate ofclinically significant gene transfer and none was sufficient to achieveclinical benefit Plasmid Complexes At its most basic level, a plasmid is a small accessory collection ofDNA which is found in the cytoplasm outside of the nucleus. They arecapable of independent replication and can be manipulated with rathermore ease than nuclear DNA. Early investigations into the field of gene transfer explored thepossibility of plasmid vectors and demonstrated the feasibility of themethod to effect CFTR gene transfer in vitro (Alton EW 1993). Otherteams had demonstrated the fact that, in clinical use theplasmid-liposome is both nontoxic and non-immunogenic (Hyde,SC et al1993). This appeared to raise the possibility that many of theimmunological problems encountered by teams working with viral mediatedgene transfer mechanisms might be circumvented. In vivo work (Yoshimura,K et al. 1992) had demonstrated that genescould be transferred into the cytoplasm by this method and Stribling, R(et al 1992) demonstrated that, once there, they would then replicatenormally. Alton experimented with a CFTR-plasmid preparation in miceand demonstrated that it was capable of correcting the chloride levelsin cystic fibrosis mice back to normal levels (Alton EW 1993) Although the initial results were encouraging, clinical trials weredisappointing as the plasmid complex could not easily penetrate thethick mucous residues in the diseased lungs of patients with cysticfibrosis. (Erickson,R 1993) The plasmids typically have a positively charged head-group which isable to bind to the DNA strand and a hydrophobic tail group whichfacilitates the transfer of the complex across the cellular membranes.Initial studies suggest that between 100-1000 times more DNA isrequired to effect successful gene transfer when this method iscompared to viral vectors. (Santis,G et al 1994). One alternative adaptation has been reported by Stern M (et al 2003)who points out that one of the solution of delivery is to ensure thatthe respiratory epithelium is exposed to the DNA over a long period.Their solution was to encapsulate the CFTR-plasmid in a slow releasebiocompatible polymer. Clinical trials are underway but not yetreported. The adeno-associated vectors appear to have (at least on atheoretical basis) a number of advantages over the vectors that we havealready discussed. They are based on a virus vector that is alreadynon-pathogenic (Berns, KI et al 1995) and has a mechanism that allowsit to be a long-term persistent entity in human cells (Blacklow, NR etal 1989). The adeno-associated vectors are particularly useful indealing with disease process that involve single gene mutations. This,therefore makes it particularly suitable for single gene disorderssuch as cystic fibrosis and alpha 1 antitrypsin deficiency. (Flotte, TRet al 1998). In addition, some workers have also developed vectors which are capableof producing either inducible or constitutive expression of thecytokine, interlukin-10 (IL-10) which is an importantanti-inflammatory protein which, on a theoretical basis, could beuseful not only in cystic fibrosis but in other disease process whichhave chronic inflammation as their prime manifestation (viz Type Idiabetes mellitus or inflammatory bowel disease) (Egan, M et al 1992).These manifestations have been studied and have now reached the stageof early clinical trials (Wagner J et al 2002). With specific reference to the implications of cystic fibrosis, wecan point to trials which have resulted in the expression of cysticfibrosis transmembrane conductance regulator (CFTR) from rAAV(recombinant adeno-associated vectors) in cell cultures (Flotte, TR etal 1993), in animal models (primates) (Afione, SA et al 1996), andagain in early phase I clinical trials (Wagner, J et al 1998) The rAAV-IL-10 model has been studied in bronchial cell culturesfrom cystic fibrosis patients, to determine the functional consequencesof CFTR complementation. This has not yet been demonstrated in vivowith humans but in both mice (Song, S et al 1998), and monkeys (Conrad,CK et al 1996) The overall results of these (and other) studies have shown that itis possible to achieve long term gene transfer and functionalexpression of the replaced gene (some studies for as long as 18 months)without any overt pathological findings. The histological findings are something of a surprise however, as,at least in both primate and mouse studies, the vector-introduced DNAin this form does not appear to be assimilated into the geneticmaterial of the chromosome, but persists in log strings or concatemersthat are episomal, which is in complete contrast to what happens whenthe naturally occurring agent infects the cell. There is some evidenceto suggest that host cell intrinsic factors such as DNA-dependentprotein kinase play some role in this process (Song, S 2001). The significance of this finding could be that the exclusion of thefunctional, newly introduced DNA from the rest of the nuclear gene poolmay be less likely to produce effects that could be either potentiallydisruptive to the host cell and less likely to activate oncogenes.Phase I trials have demonstrated significant rises of CFTR levels inboth sinus and lung tissue with no evidence of vector-related toxicity.(Wagner, JA et al 1999) The adeno-associated vectors are constructed from proviraladeno-associated vectors plasmids, which have the Rep and Cap proteinsdeleted and substituting the appropriate gene (CFTR or equivalent)between the rAAV2 inverted terminal repeats together with other signalsequences such as promoter and polyadenylation sequences (Flotte, TR etal 1994) The packaging processes allows for about 5 kb of rAAV genomes to becarried in the vectors which are prepared using a cotransfectiontechnique utilising human embryonic kidney cells (HEK-293) where thevector plasmid is cotransfected into the cells with helper agents(plasmid pDG) being used to encode the rAAV2-rep and -cap genestogether with the adenovirus helper functions (Grimm, D et al 1998).These are incubated for between 48 and 72 hrs. The cells are then lysedand the resultant agents are then separated by ultracentrifugationagainst a density gradient and affinity chromatography (Zolotukhin, Set al 1999). The vectors are thereby amenable to being separated by both theirphysical characteristics and also their biological characteristics(infectious units). They are carefully screened to ensure the absenceof any possible contamination from non-modified (replication competentAAVs) prior to clinical usage. (Muzyczka N 1994) The comparatively small payload of the adeno-associated vectorsis proving to be a significant problem. The vector itself is small whencompared to the comparatively large size of the CFTR gene. (Flotte TRet al 1993) It does not leave any room to manoeuvre to manipulate thevector-specific sequences in the way that we have described with theretroviral and adenoviral groups. (Flotte TR et al 2001). A number of authors have characterised the problem with theobservation that the rAAV is typically about 20 nm across which allowspackaging of about 4.7 kb (kilobases) of transferable modified gene(exogenous DNA). (Dong JY et al 1996), If it is combined with otherenhancers such as the promoter, the polyadenylation signal, thisclearly reduces the capacity for the DNA component. (Duan D et al2000). The Yan paper (Yan Z et al 2000) has outlined a novelexploitation of the unique ability of the rAAV genomes to link togetherin strings which appears to have the ability to bypass this particularlimitation.( Flotte TR 2000). The mechanism itself is the capacity of two distinct rAAV genomes thathappen to simultaneously infect the same target cell to undergo anintermolecular recombination insider the transduced nucleus of thetarget cell. This was a chance finding which arose from work involvingrAAV-derived episomes (Kearns WG et al 1996) in primate airways. It wasfound that some of these episomes were configured as circular head totail concatemers (Duan D et al 1999). This could have been either froma rolling circle replication from a single vector or alternatively,from an intermolecular recombination of material from multiple cellularpenetrations which combined within the palindromic inverted terminalrepeat sequences that are an intrinsic part of the AAV genomestructure. The authors were of the opinion that it was likely to be thelatter eventuality (Duan D et al 1998) It was a logical progression to try to exploit this phenomenon andthereby bypass the limitations imposed by the relatively smallpackaging capacity of rAAV. The adeno-associated vectors capsid onlyhas a capacity of about 5 kb. If we consider that the 145 nucleotidestretch of the AAV-ITR (inverted terminal repeat) sequence has to be inplace at both ends of the single-strand DNA for the vector DNA to beboth replicated and packaged, this only leaves in the region of 4.7 kbof genetically active material in each rAAV particle. As we have cited earlier in relation to the Dong paper (Dong JY et al1996) the CFTR gene accounts for about 4.5 kb which leaves very littlespace for other enhancing material. Because of this, the actual CFTRvector that has been used in the clinical trials to date uses only theminimal promoter activity of the AAVs-ITR itself to actually activateand drive the CFTR expression (Flotte TR et al. 1993). To look at this potentially important development in a little moredetail we can consider Duans original paper (et al 2000) and theauthors describe what they call a superenhancer. They describe acombination of a potent simian virus (SV40) and CMV immediate earlyenhancer elements as being packaged in one rAAV vector and a luciferasegene assisted by a small minima;l promoter in another rAAV vector. Invitro experiments suggested that either the SV40 or the intrinsicpromoter activity of the AAV-ITR was sufficient for this purpose. Theintermolecular recombination described above, was found to occur inboth vitro and in vivo experiments and was found to be sufficient tohave a greater than additive effect. Initial results from these varying methods are encouraging insofaras they are producing results of transgene expression which are 100-600times greater than with the unenhanced vector alone. (Yan, Z et al2000) Although not directly referable to our considerations of cysticfibrosis, we should note that Yans group and other workers have doneexperimental work which has culminated in the long term expression offunctional levels of erythropoetin with this two vector method in micein vivo. (Naffakh N et al 1995), This basic principle has been further enhanced by Sun (Sun L et al2000) with an ingenious manipulation of the system. They triedinserting the promoter and the first half of the coding sequence in onerAAV vector, immediately followed by a splice donor and then theupstream half of an intron. In the other rAAV vector was the downstreamhalf of the intron, the splice acceptor, the second half of the geneand the polyadenylation signal. To quote the author verbatim: This strategy is efficient enough to mediate high-level expressionand the intermolecular junctions are apparently stable enough tomediate expression for several months in vivo. Although this is clearly an ingenious augmentation of the sameprinciple , we should note that there are both advantages anddisadvantages to both pathways. The strategy that adopts the superenhancer takes its strengths fromthe fact that the recombination mechanisms optimise theposition-independent and orientation-independent functions of theenhancers. Consideration of the options would suggest that there arefour potential recombination outcomes from the process described.Either of the two vectors could be on the 5 end of the heterodimericmolecule and clearly either molecule could be in either orientation. With the superenhancer option, all four of these possibleintermolecular recombination outcomes should be functional fortransgene expression whereas if compared to the split intron strategy,by using the same reasoning, it is clear that only one out of the fourcould work. On the other side of the argument, the superenhancer option has thedisadvantage that the actual coding sequence of the gene to betransferred must still fall within the packaging capacity of the vectoritself whereas the split intron allows for a greater functionalexpansion of the packaging capacity. (after Flotte TR et al 2000) In either event it can be seen that these ingenious modificationseffectively eliminate the main size limitation of the rAAV deliverysystem. Although initial pre-clinical work is encouraging it appearsthat there is still some potential for a degree of immune responseparticularly if the host organism has not experienced the newlyproduced protein before. A number of studies have been done on animal (vertebrate andprimate) with only minimal success. Different administration methodshave been studied including direct administration into the lung (WagnerJ et al 1999), IM injection (Song, S et al 2001 B) and hepatic portalvein infusion (Song, S et al 2001 A) Human clinical trials have taken place with these vectors (Flotte T etal 1996)(Wagner J et al 1998) (Virella-Lowell, I et al 2000). Thestudies were done on adult male and female patients (18-47 yrs) whowere pseudomonas free and had recently been hospitalised for IVantibiotic infusions The disappointing results were probably a reflection of the factthat the CFTR defect is also interconnected in some way with aproinflammatory phenotype which appears to be triggered by the abnormalprotein via an unfolded protein response. The authors were able to showevidence that the rAAV-CFTR mechanism was able to correct the proteinproduction defect, they found it clinically difficult to transduce asufficient number of cells in the airway to reverse the inflammatoryresponse. It is proposed to run further experimental work which combines theCFTR expression with an anti inflammatory gene such as the IL-10.There is some in vitro work to suggest that this may be a possibleworkable approach (Teramoto, S et al 1998). Other work on ways ofenhancing the phenotypic expression of the modified genotype hassuggested that the use of various promoters and the rAAV-CMV/beta-actinhybrid promoter (CB-AAT) was found to be tone of the most efficient, atleast when it was compared to the other tested options such as the CMV,E1, U1a and U1b promoter constructs (Teramoto, S et al 1998) Overall, the initial results appear to be encouraging. A singleinjection of an rAAV-CB-AAT vector in animal studies has resulted inhigh level, stable transgene expression which has persisted over thelife span of the experimental animals and that there was no detectableinflammatory response in the animals who had received this form oftreatment (Flotte TR 2002) Flotte (et al 2002) reports that four human clinical trials at bothPhase I and Phase II level are currently underway examining the effectsof the rAAV-CFTR vector. They had an entry cohort of seven patientswith the vector being applied to the nasal lining, the maxillary sinusand the bronchus. The authors report no adverse effects being found andthat they have observed transgene expression at doses of 6 x 108 drp inthe sinus or 1 x 1013 drp in the lung. There are no reported interimfindings from the Phase II trials as yet. There is clearly a potential for clinical benefit on the basis of theresults found to date if one can extrapolate from in vitro and animalexperiments. The authors comment that, in contrast to the adenovirusvectors there is a marked lack of inflammatory toxicity with the rAAVvectors. Despite these positive comments, we should not, however, overlook thepotential limitations of this particular delivery system. These havebeen identified by various authors as: The inhibitory effect of preexisting airway inflammation on rAAV transduction in the lungs (Virella-Lowell, I et al 2000) A relative paucity of receptors on the apical surface of airway epithelial cells (Summerford, C et al 1998), The relatively weak nature of the minimal promoters used in the first-generation rAAV-CFTR vectors(Flotte, TR et al 1993), The potential for adverse long-term effects from rAAV vector DNA persistence. (Wu, P et al 2000) The Flotte group are currently investigating this problem by examiningthe hypothesis that the barriers in the airways of the cystic fibrosissufferer are primarily due to the neutrophil-derived -defensins (HNP1and HNP2) and are actually reversible by the mechanism of AAT proteindelivery (Virella-Lowell, I 2000) Wu and his co-workers have been looking at ways of manipulating thegenetic make up of the rAAV2 capsid and thereby trying to enhance thetargeting ability so that the vector specifically targets the serpinenzyme complex receptor on IB31 cells which is virtually specificfor the Cystic fibrosis bronchial cells Zabner, J (et al 2000), have considered alternative rAAV serotypesin the hope of finding one that will bind more specifically to thebronchial cells Other peripheral adjuncts have also been explored includingpromoters to enhance the effects of complementation and superenhancerswhich have been shown to improve the ability of the rAAV toconcatermerise with the help of smaller amounts of promoter agents(Duan, D et al 2000). Perhaps it is appropriate to conclude this section on considerationof adeno-associated vectors with a critical analysis of a very recentmulticentre, double-blind, placebo-controlled trial (Moss RB et al2004) This was a well constructed, fully statistically significant anddouble blinded trial which considered both the safety and thetolerability of repeated doses of adeno-associated serotype 2 vectorrepeatedly given by aerosol inhalation. The vector contained cysticfibrosis transmembrane conductance regulator (CFTR) complementary DNA(cDNA) [tgAAVCF], an adeno-associated virus (AAV) vector encoding thecomplete human CFTR cDNA. The entry cohort was comparatively small with 42 patients, of whom20 received the active agent. A number of indices of airway functionwere measured. Of particular interest to our considerations in thisdissertation was the fact that vector shedding was found in alltreated subjects up to 90 days after inoculation. And that all subjectswho received the active agent exhibited at least a fourfold increase inthe serum AAV2 neutralising antibody levels. Of the 20 treated patients, six subsequently underwent bronchoscopy.Of those six, gene transfer but not gene expression was demonstrated inall of them. On this basis, it would appear that the actual transfermechanism is effective, but there are other factors present whichappear to interfere with the subsequent expression of the gene in termsof protein production. The study did not comment on the possiblereasons for this. The authors were able to conclude that the delivery system workedwell with no evidence of adverse effects and that treated patientsdemonstrated an encouraging trend in improvement in pulmonary functionin patients with CF and mild lung disease. Lipid 67 We have discussed the various shortcomings of the virus-associatedvectors and this has prompted researchers to explore and consider otheroptimising options for facilitating gene transfer. Zabner (J et al1997) considered the use of cationic lipids in this process and foundone GL-67:DOPE (colloquially known as lipid 67) which appeared to beparticularly helpful in the process. Cationic lipids appear to show a degree of promise as possible vectorsfor CFTR cDNA transfer into respiratory epithelial cells of cysticfibrosis patients. Zabners group developed a preparation of plasmidencoding CFTR (pCF1-CFTR) and cationic lipid (GL-67:DOPE) whichappeared to facilitate the gene transfer to a significantly greaterextent than previously tested lipid complexes. They performed in vivostudies which compared the gene transfer rate to the epithelial cellsof the nasal mucosa of DNA-lipid complex and DNA alone. In generalterms, their findings indicated that the DNA-lipid complex was far moreeffective in achieving gene transfer than was simply giving DNA. Theauthors felt able to conclude that: These results indicate that nonviral vectors can transfer CFTR cDNAto airway epithelia and at least partially restore the Cl- transportdefect characteristic of CF. However, improvements in the overallefficacy of gene transfer are required to develop a treatment for CF. In this dissertation we are primarily considering the issues ofgene therapy in direct relation to cystic fibrosis. Inevitably, thishas meant considering the issues on a wider front, as many areasoverlap on a theoretical or practical basis. The prime biochemical cellular defect in cystic fibrosis is anabnormality in the cystic fibrosis transmembrane conductance regulator(CFTR). From a theoretical perspective it should be obvious thatreplacement of the defective gene with a working alternative would bebest achieved in the neonatal period before the body had time todevelop substantial fibrotic changes in the lungs that were secondaryto repeated episodes of infection (Dark J et al 1996). If successful, this could be expected to reduce both morbidity andmortality for cystic fibrosis. We have been able to cite evidence thatgene transfer has been accomplished both in vitro and in vivo. We havediscussed the results of a number of research groups who haveinvestigated various delivery systems which, to varying extents, haveproved able to deliver at least a small quantity of functional respiteto the cystic fibrosis sufferer. It is also important to be fully aware of the possibility ofinadvertent side effects in the field of gene manipulation. We havehighlighted the problems with oncogene activation. But this appears tobe associated with some vectors more than others. In short, it wouldappear that the problems and limitations that appear with this type ofprocedure are a function of the parent virus. The initial work with adenoviruses appeared promising as genetransfer could be accomplished but the major drawback was the doselimiting inflammatory effects which arose primarily as a result of thelarge amount of viral protein which was required to achieve atherapeutic dose. The subsequent modifications which had a greaternumber of coding sequence deletions appeared to be more effective inanimal experiments as they generated a lesser response from the cellmediated immunity mechanisms and therefore had a greater duration ofaction. (Caplen NJ et al 1995). It seemed a logical step from there to produce vectors that had noviral genes at all. This did not produce any significant benefits orimprovements from the previous agents. A number of research teamsacross the world tried different subsidiary strategies including druginduced immunosuppression or modifications of various immunogenicepitopes. The plasmid-lipid complexes appeared to have a number of clinicallyimportant advantages insofar as they did not appear to generate anyimmunological response which is in distinct contrast to many viralvectors. Initial optimism did not appear to be translated intopractical application as the delivery systems explored appeared to beunable to deliver sufficiently large quantities through thepathological mucous layer that is the main feature of the cysticfibrosis patient. (Crystal RG 1992). The adeno-associated vectors have received a large amount of attentionwhen it became clear that alternative vectors were needed to optimisethe therapeutic effect. They have now reached the stage where animaltesting has lead to human Phase I and II clinical trials. As a group,they appear to have the advantage that they dont trigger theinflammatory reaction in the same way, or to the same extent as theadenovirus group. The major practical difficulty with this grouphowever, is the fact that because they are so small compared to thecomparatively large size of the CFTR gene it leaves no space forvector-specific sequences on which to base assays to help todistinguish the endogenous RNA from the vector-expressed RNA. (FlotteTR et al 2001) All the evidence that we have seen appears to suggest thatadeno-associated vectors have a satisfactory safety profile andcertainly appear to produce a longer duration of clinical effect thanthe other modalities. Another variable, and indeed challenge, in the field of genetherapy, is to find the optimum delivery vehicle. We have cited studiesthat have tried direct insufflation to the bronchial epithelium. Thisappears to be a superior mode of delivery to the aerosol which appearsto have the ability to cause agent specific reactions in the alveolarmembranes. There is continuing work which is currently looking at therelative merits of nebuliser delivery mechanisms as compared toconventional aerosol delivery systems. Others that have tried avoidingthe bronchial tree and utilising the respiratory epithelium byintroduction to the maxillary sinuses through intranasal antrostomies. Conclusion including future of gene therapy. In this dissertation we have presented evidence of from a number ofdifferent approaches to the problem of gene therapy in tacklingmonogenic conditions such as cystic fibrosis. As with most areas ofscientific exploration many blind alleys have to be explored beforean appropriate avenue of research becomes apparent. The initial enthusiasm the greeted the exploration of theplasmidDNA vector did not appear to be well founded although it isclear that further exploratory work is continuing in the field. The area of adeno-associated vectors appears to be currently themost promising with, at least in vitro, suggestions that many of thecurrent limiting problems may be on the verge of being solved. The major stumbling blocks at the moment are the difficulties ofproducing a high vector titre in the clinical situation and the longterm safety considerations, particularly those relating to mutagenesisof ongogenes. On this point the Flotte group are optimistic and feelable to make the comment: The data from our laboratory strongly indicate that the bulk of rAAVDNA in the lung, muscle, and liver is episomal and that rAAV genomesinteract with host cell proteins such as the DNA-dependent proteinkinase in the formation of stable high-molecular weight concatemers. It is the episomal situation of the gene that is currently thoughtto be the best insurance against inadvertent iatrogenic oncogenesis(Flotte et al 2002) but this is clearly no substitute for long termcareful and rigorous safety studies. It is often assumed, quite incorrectly, that the field of genetherapy is a discrete and academically isolated field. Progress in thisarea, as in so many other areas of research, is completely dependent ofdiscoveries and improvements in other areas of science. The future direction of research will be determined, to a degree,by improvements in our ability to manipulate cell types and cell linesoutside of the body as this will inevitably aid our ability to implantgenetically engineered agents. Reflection over the advances inknowledge from just the last decade indicates that new and innovativedelivery mechanisms will be developed, explored and evaluated. It islikely that the known short term problems of immunogenicity, low titredelivery and inefficient packaging will be addressed, very possiblywith new delivery vectors. It goes without saying that these investigations are hugelyexpensive in terms, not only of money, but of time, expertise andinvestment generally and therefore it is likely that the limitingfactor in terms of development will be the availability of resources(Russell S 1997) References Afione, SA, Conrad, CK, Kearns, WG, et al (1996) In vivo model of adeno-associated virus vector persistence and rescue. J Virol 70,3235-3241 Alton,EW et al. 1993 Nature Genetics. vol 5. 1993 Amado R G Chen YJS 1999 Lentiviral Vectorsthe Promise of Gene Therapy Within Reach? Science. 285 (5428): 674-76. BBC Online News. Bubble boy saved by gene therapy. 3 April 2002, Berns, KI, Linden, RM (1995) The cryptic life style of adeno-associated virus. Bioessays 17,237-245 Blacklow NR. 1988 Adeno-associated viruses of humans. In: Pattison JR, ed. Parvoviruses and human disease. Boca Raton, FL: CRC Press; 1988; 165174 Blacklow, NR, Hoggan, MD, Kapikian, AZ, et al (1989) Epidemiology of adenovirus-associated virus infection in a nursery population. Am J Epidemiol 88,368-378 Caplen NJ, Alton EW, Middleton PG, Dorin JR, Stevenson BJ, Gao X, Durham SR, Jeffery PK, Hodson ME, Coutelle C, et al.1995 Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Nat Med 1995 Jan;1(1):39-46.. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, et al.2000 Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000;288: 669-72: Conrad, CK, Allen, SS, Afione, SA, et al (1996) Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung. Gene Ther 3,658-668 Crystal RG. 1992 Gene therapy strategies for pulmonary disease. Am J Med 1992; 92:44S-52S Dark, J., et al. 1996 Transplantation. Shale, D. J., ed. Cystic Fibrosis. BMJ Publishing Group. 1996. pp120-133. Dong JY, Fan PD, Frizzell RA: 1996 Quantitative analysis of the packaging capacity of recombinant adeno- associated virus. Hum Gene Ther 1996, 7:2101-2112. Duan D, Sharma P, Yang J, Yue Y, Dudus L, Zhang Y, Fisher KJ, Engelhardt JF: 1998 Circularintermediates of recombinant adeno-associated virus have definedstructural characteristics responsible for long-term episomalpersistence in muscle tissue. J Virol 1998, 72:8568-8577. Duan D, Yan Z, Yue Y, Engelhardt JF: 1999 Structural analysis of adeno-associated virus transduction circular intermediates. Virology 1999, 261:8-14 Duan, D, Yue, Y, Yan, Z, et al (2000) A new dual-vector approach to enhance recombinant adeno-associatedvirus-mediated gene expression through intermolecular cis activation. Nat Med 6,595-598 Egan, M, Flotte, T, Afione, S, et al (1992) Defective regulation of outwardly rectifying Cl-channels by protein kinase A corrected by insertion of CFTR. Nature 358,581-584 Erickson,R 1993 New Scientist. 2 Sep 1993. Felgner P. 1997 Nonviral strategies for gene therapy. Sci Am 1997;276:86-91. Flotte TR, Afione SA, Solow R, Drumm ML, Markakis D, Guggino WB, Zeitlin PL, Carter BJ:1993 Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter. J Biol Chem 1993, 268:3781-3790. Flotte, TR, Afione, SA, Zeitlin, PL (1994) Adeno-associated virus vector gene expression occurs in nondividing cells in the absence of vector DNA integration. Am J Respir Cell Mol Biol 11,517-521 Flotte, T, Carter, B, Conrad, C, et al (1996) A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with mild lung disease. Hum Gene Ther 7,1145-1159 Flotte, TR, Carter, BJ (1998) Adeno-associated virus vectors for gene therapy of cystic fibrosis. Methods Enzymol 292,717-732 Flotte TR 2000 Size does matter: overcoming the adeno-associated virus packaging limit Respiratory Research 2000, 1:16-18 Flotte TR Laube BL 2001 Gene Therapy in Cystic Fibrosis* Chest. 2001;120:124S-131S. Flotte TR 2002 Recombinant Adeno-Associated Virus Gene Therapy for Cystic Fibrosis and 1-Antitrypsin Deficiency Chest. 2002;121:98S-102S. Freeman SM, Whartenby KA, Freeman JL, Abboud CN, Marrogi AJ. 1996 In situ use of suicide genes for cancer therapy. Semin Oncol1996;23:31-45. Zhang J, Russell SJ. Vectors for cancer gene therapy. Cancer Metastasis Rev 1996;15:385-401. Griesenbach U, Ferrari S, Geddes D Alton EW 2002 Gene therapy progress and prospects: cystic fibrosis. Gene. Ther. 2002 Oct;9(20):1344-50. Grimm, D, Kern, A, Rittner, K, et al (1998) Novel tools for production and purification of recombinant adeno-associated virus vectors. Hum Gene Ther 9,2745-2760 Hyde,SC et al. 1993 Nature. vol 362. 1993. Kay MA, Manno CS, Ragni MV, Larson PJ, Couto LB, McClelland A, et al. 2000 Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nature Genet 2000;24: 257-61. Kay MA, Nakai H. 2003 Looking into the safety of AAV vectors. Nature 2003;424: 251. Kearns WG, Afione SA, Fulmer SB, Pang MC, Erikson D, Egan M, Landrum MJ, Flotte TR, Cutting GR:1996 Recombinant adeno-associated virus (AAV-CFTR) vectors do not integratein a site-specific fashion in an immortalized epithelial cell line. Gene Ther 1996, 3:748-755. Knowles MR, Noone PG, Hohneker K, Johnstone LG, Boucher RC, Efthimoiou J, Crawford C, Brown R, Schwartzbach C, pearlman R 1998 A double-blind, placebo controlled, dose ranging study to evaluate thesafety and biological efficacy of the lipid-DNA complex GR213487B inthe nasal epithelium of adult patients with cystic fibrosis. Hum Gene Ther. 1998 Jan 20;9(2):249-69. Lehrman S. 1999 Virus treatment questioned after gene therapy death. Nature 1999;401: 517-8 Moss RB, Rodman D, Spencer LT, Aitken M, Zeitlin P, Waltz D, Milla C, Brody AS, Clancy JP, Ramsay B, Hamblett N, Heald A, 2004 Repeated adeno-associated virus serotype 2 aerosol-mediated cysticfibrosis transmembrane regulator gene transfer to the lungs of patientswith cystic fibrosis : a multicenter, double-blind, placebo-controlledtrial clinical investigations Chest Feb 2004 Muzyczka N: 1994 Adeno-associated virus (AAV) vectors: will they work? J Clin Invest 1994, 94:1351. Naffakh N, Henri A, Villeval JL, Rouyer-Fessard P, Moullier P, Blumenfeld N, et al. 1995 Sustained delivery of erythropoietin in mice by genetically modified skin fibroblasts. Proc Natl Acad Sci U S A 1995;92:3194-8. Naldini, L., et al. 1996 In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996. 272: 263-267. Nichols, E. K. 1998 Human Gene Therapy. Cambridge, Massachusettes: Harvard University Press, 1998. Olsen, J. C. 1998. Gene Transfer Vectors Derived From Equine Infectious Anemia Virus. Gene Therapy. 5: 1481-1487. Porteous DJ, Dorin JR, McLachlan G, Davidson-Smith H, Davidson H, Stevenson BJ, et al. 1997 Evidence for safety and efficacy of DOTAP cationic liposome mediatedCFTR gene transfer to the nasal epithelium of patients with cysticfibrosis. Gene Ther 1997;4:210-18. Relph K, Kevin Harrington, and Hardev Pandha 2004 Recent developments and current status of gene therapy using viral vectors in the United Kingdom BMJ, Oct 2004; 329: 839 842 ; Rolls, M. M., et al. 1999 Novel infectious particles generated by expression of the vesicular stomatitis virus glycoprotein from a self replication RNA. Cell. 1999. 79: 497-506. Rosenberg SA, Aebersold P, Cornetta K, Kasid A, Morgan AA, Moen R, et al. 1990 Gene transfer into humans: immunotherapy of patients with advancedmelanoma using tumor infiltrating lymphocytes modified by retroviralgene transduction. N Engl J Med 1990;323:570-8. Russell SJ 1997 Science, medicine, and the future : Gene therapy BMJ, Nov 1997; 315: 1289 1292 Santis,G. Geddes,D. 1994 Post Grad Med J. vol 70. 1994. Sikorski R, Peters R 1998 Gene Therapy: Treating with HIV. Science. 282 (5393): 1438a. Song, S, Morgan, M, Ellis, T, et al (1998) Sustained secretion of human alpha-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors. Proc Natl Acad Sci U S A 95,14384-14388 Song, S, Embury, J, Laipis, P, et al (2001 A) Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT)after portal vein injection of recombinant adeno-associated virus(rAAV) vectors. Gene Ther 8,1299-1306 Song, S, Laipis, PJ, Berns, KI, et al (2001 B) Effect of DNA-dependent protein kinase on the molecular fate of the rAAV2 genome in skeletal muscle. Proc Natl Acad Sci U S A 98,4084-4088 Stern M, K Ulrich, D M Geddes and E W F W Alton 2003 Poly (D,L-lactide-co-glycolide)/DNA microspheres to facilitate prolongedtransgene expression in airway epithelium in vitro, ex vivo and in vivo Gene Therapy (2003) 10, 1282-1288. Stribling,R et al. 1992 Proc Nat Acad Sci. vol 89. 1992. Summerford, C, Samulski, RJ (1998) Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J Virol 72,1438-1445 Sun L, Li J, Xiao X: 2000 Overcoming adeno-associated virus vector size limitation through viral DNA heterodimerization. Nat Med 2000, 6:599-602. Teramoto, S, Bartlett, JS, McCarty, D, et al (1998) Factors influencing adeno-associated virus-mediated gene transfer tohuman cystic fibrosis airway epithelial cells: comparison withadenovirus vectors. J Virol 72,8904-8912 Virella-Lowell, I, Poirier, A, Chesnut, KA, et al (2000) Inhibition of recombinant adeno-associated virus (rAAV) transduction by bronchial secretions from cystic fibrosis patients. Gene Ther 7,1783-1789 Wagner, JA, Moran, ML, Messner, AH, et al (1998) A phase I/II study of tgAAV-CF for the treatment of chronic sinusitis in patients with cystic fibrosis. Hum Gene Ther 9,889-909 Wagner, JA, Messner, AH, Moran, ML, et al (1999) Safety and biological efficacy of an adeno-associated virusvector-cystic fibrosis transmembrane conductance regulator (AAV-CFTR)in the cystic fibrosis maxillary sinus. Laryngoscope 109,266-274 Wagner JA, Nepomuceno IB, Messner AH, et al. 2002 A phase II, double-blind, randomized, placebo-controlled clinical trialof tgAAVCF using maxillary sinus delivery in CF patients withantrostomies. Hum Gene Ther 9,889-909 2002 Wu, P, Xiao, W, Conlon, T, et al (2000) Mutational analysis of the adeno-associated virus type2 (AAV2) capsidgene and construction of AAV2 vectors with altered tropism. J Virol 74,8635-8647 Yan, Z, Zhang, Y, Duan, D, et al (2000) From the cover: trans-splicing vectors expand the utility of adeno- associated virus for gene therapy. Proc Natl Acad Sci U S A 97,6716-6721 Yoshimura,K et al. 1992 Nuclei Acids Res. vol 20. 1992. Zabner J, Cheng SH, Meeker D, Launspach J, Balfour R, Perricone MA,Morris JE, marshall J, Fasbender A, Smith AE Department, Welsh MJ1997 Comparison of DNA-lipid complexes and DNA alone for gene transfer to cystic fibrosis airway epithelia in vivo. J,Clin. Invest. 1997 Sep 15;100(6):1529-37. Zabner, J, Seiler, M, Walters, R, et al (2000) Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apicalsurfaces of airway epithelia and facilitates gene transfer. J Virol 74,3852-3858 Zolotukhin, S, Byrne, BJ, Mason, E, et al (1999) Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 6,973-985

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Passport Program In The Success Of Hero Honda Commerce Essay Example For Students

Passport Program In The Success Of Hero Honda Commerce Essay Hero Honda Motors Limited, based in Delhi, India is a joint venture between the Hero Group of India and Honda of Japan. Verification. It has been referred to as the universe s biggest maker of 2-wheeled motorised vehicles since 2001, when it produced 1.3 million minibikes in a individual twelvemonth. During the financial twelvemonth 2008-09, the company has sold 3.28 million motorcycles and the net net income of the company stood at Rs. 1281.7 crore, up 32 % from the old financial twelvemonth. The company s most popular theoretical account is the Hero Honda s Splendor, which is the universe s largest-selling bike, selling more than one million units per twelvemonth. The company introduced new coevals bikes that set industry benchmarks for fuel thrift and low emanation. A legendary Fill it Shut it Forget it run captured the imaginativeness of commuters across India, and Hero Honda sold 1000000s of motorcycles strictly on the committedness of increased milage. Over 20 million Hero Honda two Wheelers tread Indian roads today. These are about every bit many as the figure of people in Finland, Ireland and Sweden set together! Hero Honda has systematically grown at dual figures since origin ; and today, every 2nd bike sold in the state is a Hero Honda. Every 30 seconds, person in India bargain Hero Honda s top -selling bike Luster. This gay season, the company sold half a million two Wheelers in a individual month-a effort unparalleled in planetary automotive history. Hero Honda motorcycles presently roll out from its three globally benchmarked fabrication installations. Two of these are based at Dharuhera and Gurgaon in Haryana and the 3rd provin ce of the art fabrication installation was inaugurated at Haridwar, Uttrakhand in April this twelvemonth. These workss together are capable of bring forthing out 4.4 million units per twelvemonth. Hero Honda s extended gross revenues and service web now spans over 3000 client touch points. These consist a mix of franchises, service and trim points, trim parts stockiest and authorised representatives of traders located across different geographicss. Hero Honda values its relationship with clients. Its alone CRM enterprise Hero Honda Passport Program, one of the largest plans of this sort in the universe, has over 3 million members on its roll. The plan has non merely helped Hero Honda understand its clients and present value at different monetary value points, but has besides created a loyal community of trade name embassadors. Having reached an impregnable pole place in the Indian two Wheeler market, Hero Honda is invariably working towards consolidating its place in the market top ographic point. The company believes that altering demographic profile of India, increasing urbanisation and the authorization of rural India will add 1000000s of new households to the economic mainstream. This would supply the growing ballast that would prolong Hero Honda in the old ages to come. As Brijmohan Lall Munjal, the Chairman, Hero Honda Motors compactly points out, We pioneered India s bike industry, and it s our duty now to take the industry to the following degree. We ll make all it takes to make at that place. Outline1 Company profile2 History3 List of theoretical accounts4 Hero Honda Passport programme.5 What are the benefits of having a hero Honda passport?6 For how long is the Hero Honda Passport valid?7 decision8 Reappraisal of literature Company profile Hero is the trade name name used by the Munjal brothers for their flagship company Hero Cycles Ltd. A joint venture between the Hero Group and Honda Motor Company was established in 1984 as the Hero Honda company, India. During the 1980s, the company introduced bikes that were popular in India for their fuel economic system and low cost. A popular advertisement run based on the motto Fill it Shut it Forget it that emphasized the bike s fuel efficiency helped the company grow at a double-digit gait since origin. Hero Honda has three fabrication installations based at Dharuhera and Gurgaon in Haryana and at Hardware in Uttarakhand. These workss together are capable of churning out 3.9 million motorcycles per twelvemonth. Hero Honda s has a big gross revenues and service web with over 3,000 franchises and service points across India. Hero Honda s client trueness plan, the Hero Honda Passport Program, claims to be one of the largest plans of its sort in the universe with over 3 million members. The 2006 Forbes 200 Most Respected companies list has Hero Honda Motors ranked at 108. History India became the 2nd largest two Wheeler maker in the universe and get downing in the fiftiess with the Automobile Products of India ( API ) that manufactured the Lambrettas and Bajaj Auto Ltd. with its association with Piaggio of Italy ( maker of Vespa scooters ) as the largest makers within the state commendation needed. The licence raj that existed between the 1940s to 1980s in India did non let foreign companies to come in the market and imports were tightly controlled. This regulative labyrinth, before the economic liberalisation, made concern easier for local participants to hold a marketer s market. Customers in India were forced to wait up to 12 old ages to purchase a scooter from Bajaj. The Chief executive officer of Bajaj commented that he did non necessitate a selling section, merely a despatch section. By the twelvemonth 1990, Bajaj had a waiting list that was 26 times its one-year end product for scooters. The bike section had the same long delay times with three makers: Royal Enfield, Ideal Jawa, and Escorts. Royal Enfield made a 350cc Bullet with the lone four-stroke engine at that clip and took the higher terminal of the market but there was small competition for their clients. Ideal Jawa and Escorts took the center and lower terminal of the market severally. In the mid-1980s, the Indian authorities ordinances changed and permitted foreign companies to come in the Indian market through minority joint ventures. The two-wheeled market changed with four Indo-Japanese joint ventures: Hero Honda, TVS SUZUKI, Bajaj Kawasaki and Kinetic Motor Company ( Kinetic Honda ) . The entry of these foreign companies changed the Indian market kineticss from the supply side to the demand side. With a larger choice of two-wheelers on the Indian market, consumers started to derive influence over the merchandises they bought and raised higher client outlooks. The industry produced more theoretical accounts, titling options, monetary values, and different fuel efficiencies. The foreign companies new engineerings helped do the merchandises more dependable and with better quality. Indian companies had to alter to maintain up with their planetary opposite numbers. List of theoretical accounts Achiever Ambition 133, Ambition 135 Prevent Computer Crime EssaySo wholly you have to make to gain points A ; wagess is merely maintain keeping your Hero Honda bike, purchase echt Hero Honda Spare Parts and accoutrements and we ll give you one point for every rupee you spend! A Star Club and Treasured Rewards This alone nine has been introduced for those members who get their motorcycles serviced on a regular basis from authorized Hero Honda Service Centers. The Star Club rank non merely identifies the member as particular for the HHPP household, but besides brings in some added benefits like: 30 % price reduction on labourA A 7.5 % price reduction on spares, till the cogency of the Passport Introducing Treasured RewardGood intelligence for all Passport Programme members who have crossed the 1 hundred thousand point grade in the Hero Honda Passport Programme.A A new wagess construction has been introduced for all such members The HHPP Treasured Rewards. The HHPP Treasured Rewards will be available at 8 prescribed mileposts get downing from 1.25 lakh points and stoping at 3 hundred thousand points, with each milepost at a spread of 25,000 points. In other words, the new mileposts are placed at 1.25 hundred thousand, 1.5 hundred thousand and so on boulder clay 3 lakhs Each clip a member reaches a milepost a Motorcycle Privilege Voucher of Rs. 1250/- will be given to him. This verifier may be used to avail a price reduction on the purchase of a new Hero Honda bike. This verifier is movable and can be given to friends or relations who may be be aftering to purchase a new Hero Honda bike Under the new Rewards Programme, the manner of entering points will alter. The points will now be recorded in 2 books one would be the old Passport with Additional Treasured Rewards sheets for come ining points earned through service, spares and accoutrements and the other would be a Referral Book which will be used for come ining referral points. The R eferral Book will get down from 1 lakh points. On traversing 1 lakh points the members need to retain a photocopy of their old Pass with them and direct the passport to the HHPP Programme Centre instantly. On reception, the old Passport with Additional treasured wagess sheets and the new Referral Book will be sent across to the members enabling them to get down redeeming wagess beyond 1 lakh points. decision Hero Honda, the World No. 1 two-wheeled company, today announced that it has late achieved a landmark member base of more than 2 million clients for its Passport Programme . Extending across 1000 authorized Hero Honda franchises and SSPs, the plan has now become India s largest Customer Relationship Management ( CRM ) Program. The unprecedented success of the CRM plan is a testimony of the religion that the clients repose in the company and its merchandises. It was born out of a demand to maximise committedness and beef up the emotional value that binds our diverse set of clients to the Hero Honda trade name. With its disposed tagline Rishta Dil Ka , the Passport Programme is a strong platform for the company to construct long-run relationships with its clients, beyond the point of sale, and pass on its trade name image of trust and reliability . The Hero Honda Passport Programme rank is a alone inaugural unfastened to the proprietors of Hero Honda bikes. The plan offers man y touchable and intangible benefits to both the clients every bit good as to the traders. Members earn points for incurring outgo at the Hero Honda dealership/workshop, which can subsequently be redeemed against benefits. On the juncture of the memorialization of the 2 million-passport plan milepost, Mr. Pawan Munjal, Managing Director, Hero Honda Motors Ltd. said, At Hero Honda our enterprise is to offer the best in merchandise quality to our clients ensuing in highest degrees of client satisfaction. The Passport programme non merely builds digesting relationships with our clients but besides offer them some extra value , beyond merely the touchable benefits of our merchandises. This milepost merely reaffirms our clients religion in us, which has been polar in the company s growing . The Hero Honda Passport programme was rolled out nationally in the twelvemonth 2001, after a successful pilot in four provinces in the twelvemonth 2000. A alone characteristic of the programme is i ts reward theoretical account, harmonizing to which every rupee spent by the member translates into a wages point. These reward points can so be redeemed for exciting gifts. One of the most important benefits of this plan is a free personal accident policy worth Rs. 1 hundred thousand. Insurance claim of over Rs. 6 crore has already been disbursed to dependent households, representing Hero Honda s concern non merely for the safety of its clients, but besides their household members. With the aid of this plan, the company has already received over 900,000 referrals therefore besides giving a bonus to gross revenues through favorable word of oral cavity. Reappraisal of literature With a member base of more than 20 lakh clients widening across 1000 authorised traders and SSP s, Hero Honda Passport Program is today the state s largest Customer Retention Management ( CRM ) plan. Under the disposed tagline Rishta Dil Ka , the plan has become a strong platform for us to construct long-run relationship with our clients beyond the point of sale, and take frontward our trade name image of trust and reliability . It was born out of a demand to maximise committedness and beef up the emotional value that binds our diverse set of clients to the Hero Honda trade name. The plan represents a cardinal alteration in looking at the client as a long-run plus instead than a mere mark for gross revenues dealing. Marketing plan designed to heighten trade name trueness by cultivating an ongoing relationship between a seller and his client. Successful trueness plans encourage the consumer to purchase often, to increase the sum spent each clip, and to concentrate all or most of their related purchases on that trade name. Most loyalty plans offer fringe benefits for rank in a nine or plan and wages purchases. Wagess may be based on the dollar value of purchases made or on the frequence of purchases. The most well-known trueness plans are air hose frequent-flyer plans that offer price reductions against future travel called award stat mis. Most big supermarket ironss now have frequent-buyer nines that offer no-coupon price reductions every bit good as newssheets and affiliate price reductions. Loyalty plan tactics besides include regular communicating with clients such as reminder mailings, private recognition cards, cross-sell and up-sell offers, satisfaction and sentiment studies, and aggregatio n of information for member databases. See besides frequency price reduction.