Treating Cystic Fibrosis with Gene Therapy

Treating cystic Fibrosis with cistron TherapyIn this dissertation we sh on the whole consider the field of cistron therapy in circumstantial relation to cystic fibrosis.We examine the different speech communication sender apparatuss that micturate already been explored and concentrate primarily on the adeno-associated vectors. We examine the real state of re seem and consider the advantages and drawbacks of the assorted regularitys considered.We conclude with a raillery and compend of our conclusions and make a depend of assumptions relating to the future conduction of the researchin the field.The rate of progress in the field of element therapy has been enormous. We must remind ourselves that the first clinical constituent communicate experiment took space in 1989 when a patient with malignant melanoma true transmitt adaptedally modified auto logous T carrells. (Rosenberg SA et al1990)Gene therapy encompasses two major atomic number 18as. The in vivo field , where agents atomic number 18 incorporated into the target cells of the breathing body and the ex-vivo field where the target cells themselves ar genetically modified orthogonal the body and then re-implanted.Medical science has been employ the basic techniques of gene transfer for a foresighted time. The technique has been exploited when viral genes ar introduced to military personnel cells when a viral vaccine is administered. The key technologies that al down in the m byhed 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 good 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 m odified genetic corporeal is fitting to call down new proteins which provide restore deficient or unnatural functions of genetically diseased tissues, to generate tissues that engender 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 utilise of modified retroviral vectors (eg.MMLV) to return gene transfer into the chromosomes of a target cell and the adenovirus vectors when such integrating was non needed. Neither vector was itemly succeederful as the entire 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) decl ar the same ability to transfer genetic tag into the cellular genome and could do this in the non-dividing or dormant phase cells. (Amado R G et al 1999)In vitro, lentivir uses receive been shown to change the target cells tone of proteins for up to six months. alphaly, they dissolve be utilise for storely differentiated cells such as respiratory epithelium. The only cells that the lentivirus cannot penetrate the inwardness are those in the quiescent (G0) state as this blocks the reverse placement stages of protein synthesis. (Amado R G et al1999) cystic fibrosisCystic fibrosis is the most commonly lethal inherited recessive inthe caucasian population. It affects about 1 per 2,500 livebirths. Thetreatment of cystic fibrosis has alter enormously in the last fiftyyears with the life expectancy increasing from an fairish 10 years to30-40 years now.The ancient accept of death in affected individuals is the repeatedcycle of infection, inflammation and fibrosis of the respiratory tractwhich eventually culminates in respiratory bankruptcy and death.The disease itself is caused by mutations in the individual(a) gene forthe cystic fibrosis trans membrane conductance governor (CFTR) whichproduces a protein ready in sweat and pancreatic ducts, gut,seminiferous tubules, lungs and some(prenominal) otherwise tissues. The mutationsresult in an abnormal protein which, when expressed in the lungs,produces thick viscous and desiccated 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 genus Pseudomonas aeruginosa)(Porteous DJ et al 1997).An individual must receive a speculative copy of the cystic fibrosisgene from each parent in order to expand the clinical picture ofcystic fibrosis. Following normal genetic principles, if two carriersconceive a child, at that place 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 vectorsViruses have an ability to enter a soldier y cell and combine their owngenetic material with that of the soldiers cell. This is the basicrationale behind the science of gene transfer therapy. As we shalldiscuss in some detail in this dissertation, at that place 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 placing deoxyribonucleic acid 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 deoxyribonucleic acid withvarious plasmid or lipid complexes(Zabner et al 1997)The advantages of lipid or plasmid aided transfer weapons arethat they do not take care to generate the immunological responses thatare seen with the viral vectors. They can also be used to facilitatethe transport of much badr pieces of DNA which would otherwise belimited by the package 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 capers to theattention of the media. A retroviral manipulation of a case of X-SCID(X linked severe combine immunodeficiency) was set by gene therapywith an apparent degree of success (BBC 2002). This token 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 dischargely go away in the delimitateA multinational team used a retroviral vector to insert a functionalcopy of the gene into bone marrow fundament 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 stage of time. This was hailed in twain 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 influence 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 substantial a leukaemia-like illness. Genetic digest ofthe malignant cells suggested that the retroviral vector used in thetransfer had also activated an transforming gene 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 runs. (Lehrman S 1999)The prime reason for presenting these events is to demonstrate thefac t that there is both(prenominal) a metaphysical and practical risk of insertionalmutagenesis. Reduction of the risk requires greater specificity of thetargeting of the genetic deficit perhaps by directing the contemplationof 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 choose of theuse of retroviral vectors, one can cite the fact that they have ahighly efficient weapon of gene transfer together with lowimmunogenicity. It is a well researched and well canvass 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 receive high titres in theclinical situation (after Olsen, J. C. 1998).In broad terms, the principles behind the use of retroviral vec torsare that they must be modified in order not to be able to transmit anyovertly ghoulish 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 openhanded in mouse fibroblast cellsThere 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 erectively demo 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 h igher titre levels. (Rolls M et al 1999)At about the same time that the scientific press was knowledge aboutthe problems with retroviral transfer (see above) other investigatorswere working with adeno-associated viruses (AAVs). A similar processwas invoked using adeno-associated viruses to enlighten a genetic defectinvolving coagulation factor IX. The adeno-associated viruses were usedas they were considered to be amongst the safest panoramas for genetransfer. They do not naturally cause disease processes in humans andhave only seldom been found to incorporate in a random fashion into thehuman genome. Although it is illustrious 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), just thetrial indite (in later research work) published a pack which suggestedthat, in study mice, the vector used in the trials unquestionablely integrateditself into gene cont aining 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 distri furthered amongstall of the chromosomes particularly at sites of gene activeness. On thisbasis, there appears to be at least a theoretical basis for thepossibility of similar cellular defects such as go onred in the X-SCIDpatients.Adenoviruses are comparatively simple structures. They arecategorised as double stranded DNA viruses. They have icosahedralmirids with twelve vertices and cardinal fold 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 thera py 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 epochal gene transfer and none was sufficient to achieveclinical take in Plasmid ComplexesAt its most basic level, a plasmid is a small accessory collection ofDNA which is found in the cytoplasm out of doors of the karyon. They arecapable of independent replication and can be manipulated with rathermore ease than nuclear DNA. early(a) investigations into the field of gene transfer explored thepossibility of plasmi d vectors and demonstrated the feasibility of themethod to picture 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 were bilk 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- convention 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 time 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 (Bern s, 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 anti turn inpsin deficiency. (Flotte, TRet al 1998).In addition, some workers have also developed vectors which are capableof producing either inducible or constitutive flavor 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 refer ence 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 ( hierarchs) (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 localize the functional con rangesof 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 potential 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 mou se studies, the vector-introduced DNAin this form does not appear to be assimilated into the geneticmaterial of the chromosome, but persists in log thread or concatemersthat are episomal, which is in complete contrast to what happens whenthe naturally occurring agent infects the cell. There is some take the standto suggest that host cell intrinsic factors such as DNA-dependentprotein kinase play some share 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 little likely to produce make 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 anatropous terminal repeats together with other signalsequences such as relay link 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 set-apart 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 carefull screened to ensure the absenceof any possible contamination from non-modified (replication competentAAVs) prior to clinical usage. (Muzyczka N 1994)The comparatively small commitment 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 draw to manipulate thevector-specific sequences in the way that we have findd 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). ( peal 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) h as 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 searching 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 flight paths. 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 at heart the palindromic inverted terminalrepeat sequences that are an intrinsic part of the AAV genomestructure. The authors were of the opinion that i t 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 the negligible 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 minimal promoter in another(prenominal) 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 one-dimensional 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 distinguish that Yans group and other workers have done observational 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 sunniness (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 conferrer and then theupstream half of an intron. In the other rAAV vector was the downstreamhalf of the intron, the splice acceptor, the flake half of the geneand the polyadenylation signal. To quote the author verbatimThis strategy is efficient becoming to mediate high-level expressionand the intermolecular junctions are apparently stable decent 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. experimental condition 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 sever 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 stable 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 primary(prenominal) 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 beingness 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 extract (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 infusionsThe 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 airline business 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-A AT) 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 soon 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. The re are no reported interimfindings from the Phase II trials as yet. There is clearly a potential for clinical good on the basis of theresults found to date if one can generalise from in vitro and animalexperiments. The authors gossip that, in contrast to the adenovirusvectors there is a tag 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 curren tly 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 cellsZabner, J (et al 2000), have considered alternative rAAV serotypesin the hope of finding one that will bind more specifically to thebronchial cellsOther circumferential adjuncts have also been explored includingpromoters to enhance the effects of complementation and superenhancerswhich have been shown to amend the ability of the rAAV toconcatermerise with the help of smaller amounts of promoter agen ts(Duan, D et al 2000). maybe 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 maturation 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 throw in 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 rise in pulmonary functionin patients with CF and mild lung disease. Lipid 67We 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 t he use of cationic lipids in this process and foundone GL-67DOPE (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