Treating Cystic Fibrosis with Gene Therapy

Treating Cystic Fibrosis with Gene Therapy 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. 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 SJ  1997) 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 cell’s 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 â€Å"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 â€Å"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 â€Å"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 Duan’s original paper (et al 2000) and theauthors describe what they call a â€Å"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 Yan’s 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 the  CFTR 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 IB3–1 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 â€Å"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 â€Å"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

Century In Canada

Nations are born out of conflict, and grow and thrive by learning from their mistakes. The 20th century in Canada was responsible for an abundance of great aspects that now exist in our country. Within that era the rights of women were recognized and altered, resulting in them being considered equals to men. Our army became recognized as an elite fighting force. Japanese Canadian internment camps were put in place as a result of the bombing of Pearl Harbor displayed nothing less then an unjust act.All leading up to the passing of the Canadian Charter Of Rights And Freedoms making certain that inhumane acts that have happened in our past will not happen again. After all, those who do not learn from their mistakes are doomed to repeat it. The early years of the 21st century were important to shaping Canada as a nation. 2 major events occurred during this time that helped to create our identity; the feminist movement began to take hold within and beyond our borders, as well as beginning to be recognized as a world-class military force.Women (prior to the famous five) were tremendously discriminated against and viewed as incapable of doing many acts. Emily Murphy, a self-taught legal expert, who championed in women and children's rights felt strongly in fighting for gender equality. In 1903 she began a campaign focusing on property rights of married women. With her hard work and dedication in 1911 the Dower Act was passed. The act stated that women had the right to one third of their husband's property and allowed for the surviving spouse to become the legal owner of the home.This signified a huge step for women because it proved there rights were beginning to be recognized and there was hope for one day being considered equals to men. Her career continued to progress when she along with other concerned women attended a trial for Edmonton prostitutes on October 17, 1933. The women were ordered to leave the court because the case was not to be viewed by â€Å"mixed company'. Murphy was furious and proposed that if women weren't allowed to view the case there should be a separate court for women, run by women.Emily later went on to become a Judge, Just like the generations of male lawyers/Judges in her family. Emily had to cope with the hurtful remarks from male lawyers questioning how she can be a Judge, and therefore be granted powers o make important/ valuable decisions, if she is not even considered ‘a person'. The law essentially categorized women as ‘crazy, unstable lunatics' and not considered a person. This got her fuming, fighting until there was Justice. Murphy gathered up 4 other women with the same political views as her, and together they made up the famous five.Emily, Henrietta Edwards, Irene Parlay, Louise McKinney and Nellie McClure were all strong willed women coming from well educated backgrounds and were devoted to social change and women suffrage. With the support of the female citizens they produced a petition and brought it to the Supreme Court. After a nail biting 5 weeks of debates the petition was denied. The women however were not discouraged and delivered the petition to Britain Privy Council, the highest role of government in Canada.On October 18, 1929 the Privy Council announces the Persons Case, explaining that women were legally considered persons and therefore could become members of the Senate of Canada. There is no question about it this milestone was the first of many for Canadian women, and because of the efforts of the Famous Five's women would now be considered equals to their male counterparts. The women of today owe a tremendous amount of gratitude for their efforts. World War I lasted from July of 1914 to November of 1918. During this time Canadians began to build an identity for themselves.This is evident during the battle of Vim Ridge. Canadian troops were ordered by Britain to conquer Vim Ridge, a prime piece of land that would be critical to the allies' efforts. Un fortunately the Germans had control over it, but Canadian troops lead by General Bang and General Currie were going to set out to conquer the ridge. Prior to the planned invasion the Canadian troops had to undergo weeks of exhausting practice drills, as it was crucial o stay undetected and surprise the enemy and that everything was executed perfectly, or the whole mission would be Jeopardized.Canadian aircrafts flew overhead the ridge and photographed what the ridge looked like. Returning back to Canadian grounds with information in hand this allowed for Canadian forces to set up an exact replica of what to expect during the attack in terms of characteristics of the ridge, and where the Germans were standing guard. Troops trained for weeks on the recreated Vim Ridge set, until they knew it so well they could perform their duties blindfolded. The Canadian forces had also set in place 2 techniques that would help them take over the ridge after they emerged from the tunnels being built to get as close as they can.They then would execute the creeping barrage and vim glide. The strategy consisted of making a smoke screen in front of them (produced by bombs) and crawling low to the ground, placing them beyond enemy lines. This allowed for the forces, when they were ready to attack, to take the enemy by surprise and hopefully conquer. On April 9, 1917 all 4 divisions of Canada's troops attacked, he plan was executed exactly as planned and the Canadians conquered the ridge. However, this battle was a tragic one as Canada was faced with heavy casualties, resulting in 3600 soldiers killed.The victory for Canada resulted in a nation and its armed forces being recognized for their brave men as well as smart planning and execution. Because of Vim Ridge we began to be a nation worth fearing. After the horror of the First World War and the tremendous achievement for women's rights, Canada was a nation well on it's way to great success. Though the war was devastating, we ulti mately came out of it stronger. Moving into the ass's women roles were continuing to evolve. The flapper girl age was beginning.What brought upon this revolution was the fact that during the war women had to disobey societies views on what a women should and should not do by stepping up and performing the boys jobs while they were off serving in the war. They had to remove their aprons, and leave the stove. This was a time they needed to step up and take over their husband's jobs in order to still provide for their family and be able to put dinner on the table. Once the war had ended women were not going to return back to their old roles. Women didn't want, and didn't feel like, they needed to succumb to society.They were comfortable in their new lives, though it was only intended to be temporary. This new wave of young women wanted to stand out and be different. All of these factors birthed the flapper girl. She appeared as a boy, dressed with short hair, higher skirts, and flatten ed chests. They had a strong appearance and attitude about them they felt liberated. The younger generation saw the time frame as a perfect opportunity to introduce this new style, given that women's rights were evolving, this would bring more attention onto them. It was overall a rebellious movement of expectations of women. (http://history sass. About. Mom/odd/sass/a/flappers. HTML) After the Japanese attacked Pearl Harbor, Hawaii on December 7, 1941 Canada took a step backwards on human rights and discrimination against the Japanese. At the time there were 22,000 Japanese Canadians living in British Columbia, some of wham's ancestors were the first immigrants coming for work to Canada in the sass. Though they had always been discriminated against by the largely white Canadian society, it was nothing compared to what was about to come. Days after the Pearl Harbor attack, Canadian companies began to fire all Japanese workers beginning with the Canadian Pacific Railways.Matters got worse when Japanese forces attacked Hong Kong and killed over 2000 Canadian soldiers stationed there for training. The Japanese began to be referred to as SAPS, and signs were being posted around the province harshly stating, â€Å"Keep out†. It was then the Federal Government designated a 100-mile wide strip, as a protected area to keep all Japanese until they would be further placed inland. They were finally ordered to pack a small suitcase and live in inverted over animal stalls awaiting their train to arrive. (http://www. CB. Ca/history/ OPPOSITENESS EPOCH APPEAL. HTML). Husbands, wives and children were all separated. The men were sent to work on road gangs, whereas the women and children were sent to shantytowns in the B. C wilderness. In January of 1943 the government forced the sale of all property/ belonging to the Japanese Canadians that includes their homes, cars and other valuables. The reasoning behind this was to erase any memories the Japanese built in Canada a nd to convince them not to assume living here when the war over. The writing was essentially on the walls, the Japanese were no longer welcome.Once the war ended the B. C federal government decided to release all Japanese from the camps. The Japanese were then faced with choosing between deportations back to Japan, specifically parts that were destroyed during the war, or moving east into the Rocky Mountains. The majority of them chose to move to Ontario, Quebec and the Prairies. Many families did move back to Japan as well. It was a long time coming, but finally on April 1st, 1949 after much protest, Japanese Canadians were finally allowed the freedom to live anywhere in Canada.

Analysis of the Fashion Industry Essay

Fashion is one of the world’s most important creative industries. It has provided economic thought with a canonical example in theorizing about consumption and conformity. Social thinkers have long treated fashion as a window upon social class and social change. Cultural theorists have focused on fashion to reflect on symbolic meaning and social ideals. Fashion has also been seen to embody representative characteristics of modernity, and even of culture itself. Everyone wears clothing and inevitably participates in fashion to some degree. However, it would be an understatement to say that fashion influences just clothing; in fact, to be very precise fashion influences almost every aspect of our daily life. More often than not, Fashion trends are a reflection of the political, social and economic changes and developments around us. Fashion is an important part of not only arts but maybe even more of economy. At first glance only the aesthetic aspect of it is visible with the beauty, amazing creations and materials. But if one looks more carefully, the whole highly developed and profit producing industry lies behind this glitter. The fashion industry consists of four levels: the production of raw materials, principally textiles , leather and fur; the production of fashion goods by designers, manufacturers, contractors, and others; retail sales; and various forms of advertising and promotion. These levels consist of many separate but interdependent sectors, all of which are devoted to the goal of satisfying consumer demand for apparel under conditions that enable participants in the industry to operate at a profit. Not only The global fashion apparel industry is one of the most important sectors of the economy in terms of investment, revenue, trade and employment generation all over the world, but also The Business of Fashion is an essential daily resource for fashion creatives, executives and entrepreneurs. Selling is the final activity of business .In order to survive , the ability to sell has a great portion of importance among other business abilities. The fashion industry is rather a very sensitive industry toward changes in the cultural , social and economic factors . It is relatively harder or more complicated to understand why a product of a fashion industry sells while others are not. Companies perform various efforts to enhance their selling capabilities. Conducting a marketing analysis, that is, reviewing the strengths of an organization, its weaknesses, opportunities it can capitalize on for maximum profits, and the threats to achieving its full potential provides very invaluable information to the organization about the market and understanding the industry, as well. The analysis is used to define both the unfavorable and favorable factors and their impacts on goals of the business. The fashion industry, which is very volatile, is not an exception, it too has its own share of strengths and opportunities which once utilized by an organization can help it grow substantially and weaknesses and threats which the organization must strive to minimize to the lowest possible levels. Fashion marketing is based on the identification of market trends which are used to analyze, develop and configure related marketing strategies and promotional activities for fashion products. Fashion marketing is the application of a range of techniques and a business philosophy that centers upon the customer and potential customer of clothing and related products and services in order to meet the long term goals of the organization.The very nature of fashion, where change is intrinsic, gives emphasis to marketing activities . Essentially fashion marketing is composed of elements of fashion designing and marketing management which are combined together to develop a comprehensive marketing plan for fashion products. This marketing strategy and the related plan is composed of different tasks relative to the elements of promotions, advertising, retailing, branding, affiliate marketing, and distribution. In order to be successful fashion marketers have to be future oriented with forecast information about the market. The fashion industry demands that in order to have an effective fashion marketing strategy, companies need to understand and identify their customers, the trends in the fashion industry as well as how the branding and the marketing of their products effects the purchase behavior of the consumers. This knowledge is the main resource available to fashion marketers to develop a marketing strategy for their fashion based products . Marketing in the fashion industry is often difficult as the industry is very volatile with constant changes taking place in its external as well as internal environments. As a result the approaches taken towards fashion marketing have to be adjusted and revamped in order to effectively target the latest and future trends. A myriad of factors define the fashion retail market, namely the interactions among fashion companies and the interaction between fashion companies and the consumer. The retailers seem to have the most power in defining the market as they possess the power to market goods at prices desirable to them. However, the market price is in fact also determined by consumers. According to basic economic theory, the price of a good is determined by the demand by consumers and supply of the good by the producers in the economy. Especially for the fashion retail market, fashion trends are forecasted by analysing results of consumer’s emotions towards the previous trend. Hence, consumers do play a vital role in forming the fashion retail market as well. Consumer culture or consumerism, is â€Å"the theory that a progressively greater consumption of goods is economically beneficial†. Fashion creates a desire for ownership. Coupled with mass media and advertising, market makers sell lifestyles and consumers consume such products and lifestyles in hope for upward mobility in the social hierarchy. This creates a culture of hedonism through the impression that one can purchase a status and in turn, ‘happiness’. As such, this new consumer culture lays the foundations for consumption, and demand and supply, of fashion in a society. However, fashion comes and goes and is never constant. But with the ever changing trends and fashion, we see that people have to constantly buy and consume new goods and services to stay in fashion. The frequent renewal of fashion in our capitalistic society makes it an effective marketing strategy as the constant updating of ‘trends’ and the human desire to fit in keeps the fashion industry alive. The fashion industry is a product of the modern age. Prior to the mid-19th century, most clothing was custom made. It was handmade for individuals, either as home production or on order from dressmakers and tailors. By the beginning of the 20th century—with the rise of new technologies such as the sewing machine, the rise of global capitalism and the development of the factory system of production, and the proliferation of retail outlets such as department stores—clothing had increasingly come to be mass-produced in standard sizes and sold at fixed prices. Although the fashion industry developed first in Europe and America, today it is an international and highly globalized industry, with clothing often designed in one country, manufactured in another, and sold world-wide. For example, an American fashion company might source fabric in China and have the clothes manufactured in Vietnam, finished in Italy, and shipped to a warehouse in the United States for distribution to retail outlets internationally. The fashion industry has long been one of the largest employers in the United States, and it remains so in the 21st century.By any measure, the industry accounts for a significant share of world economic output. There is no doubt in the importance of fashion as an industry branch. The amount of money it produces and the number of people it employs makes it a very significant area in the world of economics. On the other side economic situation and changes within it also leave trace on fashion industry. Being so closely entwined any detailed analysis cannot be done without observing both

Application of Nursing Theory - Free Essay Example

Sample details Pages: 4 Words: 1123 Downloads: 7 Date added: 2019/10/10 Did you like this example? Introduction Nursing theories are important when it comes to nursing practice. They have been used in education, staffing, leadership, advanced practice and information. One of the main concerns in nursing is nursing care and practice whereby leadership is involved. Don’t waste time! Our writers will create an original "Application of Nursing Theory" essay for you Create order The nurses are required to establish a relationship with the patients in order to help them in the recovery process. One theory addressing this topic is the Hildegard Peplau’s Interpersonal Theory. Peplau defines that the relationship developed between the patient and the nurse as key to aid recovery process. The theory addresses the relationship from orientation, identification, exploitation and resolution. The nurses are seen as the leaders in the whole process and it’s their responsibility to ensure a favorable interpersonal relationship is developed. Failure to develop the relationship may affect the entire recovery process. Once the relationship has been developed, the nurse is free to engage the patient and the patient is also free to engage the nurse on issues affecting him. The patient feels free with the nurse and in turn nursing care is enhanced. The paper will address the issue of nursing care and how the theory can be applied to deal with it. Nursing care Nursing care has been a major concern when it comes to nursing practice. In the nursing profession, the aim is ensure that the patients are provided with the necessary care to aid their recovery process. Nursing care can be hospital based or home based. The nurses are required to take care of the patients and provide a regular check up. In case of an emergency, the nurse is the one to respond to it. Nursing care is important to patient recovery and it ensures that the patient is on the right track towards recovery. Failure to provide adequate care to the patient can have a negative impact. The issue has been addressed internationally as one of the major concerns in the health sector. Without proper nursing care, the entire healthcare system can be a failure. Nursing care provides a favorable environment for the patient to recover. It also helps the patients psychologically and emotionally. Psychological harm can hinder the recovery process and thus the nurses are required to help the patients overcome such issues. Nursing care is important to the nurses as it defines their role in the healthcare system. The issue has been listed as one of the major issues affecting healthcare delivery process. However, the main stakeholders affected by the issue are the patients and the nurses. It entirely involves the relationship between the patients and the nurses. Nurses are the leaders tasked to manage the entire relationship and ensure that it’s effective in aiding patient recovery. Failure of the nurses to address the issue may lead to failure of the entire healthcare delivery process. Application of Peplau’s interpersonal theory in nursing care This theory has four phases in which relations between a nurse and patient are involved. The first phase is referred to as orientation. It is characterized by the meeting of a patient and nurse as strangers during the time in which the patient is seeking for assistance. The nurse plays the role of helping the patient understand his/her problem and then determine the reason the patient is in need of help. The second phase is referred to as identification. In this stage, the patient takes part in the process of setting goals. He/she acquires a feeling of belonging which helps him/her to selectively respond to the ones in a position to address his/her demands. The second phase then transits to exploitation phase. In this stage, the patient makes attempts to gain full value from the benefits he/she gained from the relationship. The nurse then lays down new goals which will be achieved by a way of personal effort. At this stage, power is shifted to the patient as he/she works on a mechani sm of achieving the newly set goals. The last stage of the relationship between a nurse and a patient is referred to as resolution. This only happens in the case where the first three phases have been completed in a successful manner. It is characterized by a patient putting aside old goals gradually and then adopts new ones. It is at this phase where the patient frees himself/herself from the nurse and hence the relationship comes to completion. All through the process, the nurse works together with the patient in administering the following roles: The role of counseling- in the process of administering care, a nurse attains the role of a counselor by working to find a solution to the patient’s problems. Leadership role- as a care giver, a nurse plays the role of a leader by working with the patient in a democrat way. iii. [bookmark: _GoBack]Stranger- the nurse is viewed as a stranger in the way he accepts the patients objectively. Teaching role- as a teacher, the nurse offers new information to the patient and hence helps him/her in the process of learning. The phase of orientation is initiated during the time when a patient has a feeling that he/she is in need of care. It is from this point that assistance from a profession is sought. The main focus by the nurse is to gather more information about the patient. The patient’s mental status is one of the key areas in which the nurse is interested in. In the process of coming up with a plan, the educative requirements of the patient are put into consideration first. The power making the process a success lies in the hands of the patient and hence a good relationship between the nurse and a patient is very important. In the phase of working, a lot of attention is given to the efforts of the patient to attain and then employ knowledge on personal strengths, available resources, and the illness. In the process of administering nursing care, the relationship between a nurse and the patient is flexible in a way that the two can work in a interdependently, dependently or independently dep ending on patient’s level of anxiety, needs, self-awareness, and developmental capacity. Conclusion Nursing theories are important when it comes to nursing practice and delivery of effective healthcare. These theories have been developed to address issues arising in the nursing department. One of the issues of concern is the nursing care which involves the patient and the nurse. Concerning the issue, Hildegard Peplau developed the Interpersonal Theory which addressed the relationship between the patient and the nurse. The nurses are required to provide nursing care to the patients as well as ensuring there is a favorable relationship between the patient and the nurse.