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Treating Cystic Fibrosis with Gene Therapy

Info: 8828 words (35 pages) Dissertation
Published: 2nd Nov 2021

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Tagged: MedicineGenomicsBiomedical Science

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 fifty years with the life expectancy increasing from an average 10 years to 30-40 years now.

The prime cause of death in affected individuals is the repeated cycle of infection, inflammation and fibrosis of the respiratory tract which eventually culminates in respiratory failure and death.

The disease itself is caused  by mutations in the single gene for the cystic fibrosis transmembrane conductance regulator (CFTR) which produces a protein found in sweat and pancreatic ducts, gut, seminiferous tubules, lungs and many other tissues. The mutations result 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 pathogens from 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 fibrosis gene from each parent in order to develop the clinical picture of cystic fibrosis. Following normal genetic principles, if two carriers conceive a child, there is a 25% chance that it will have cystic fibrosis, 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 own genetic material with that of the host cell. This is the basic rationale behind the science of gene transfer therapy.  As we shall discuss in some detail in this dissertation, there are a number of potential viral vectors that have been explored, evaluated and exploited in the search for an efficient and safe form of therapy. Viruses are not the only vector that can be utilised . Simply placing DNA in the nasal mucosa will produce some incorporation into the epithelial cells (Knowles MR et al 1998). This “absorption” can be demonstrably enhanced further by the combination of the DNA with various plasmid or lipid complexes (Zabner et al 1997)

The advantages of lipid or plasmid assisted transfer mechanisms are that they do not appear to generate the immunological responses that are seen with the viral vectors. They can also be used to facilitate the transport of much larger pieces of DNA which would otherwise be limited by the packaging consideration incumbent on the viral vectors.(Felgner P 1997).

The use of retroviral vectors is far from straightforward. The heavily publicised case in April 2000 brought some of the problems to the attention of the media. A retroviral manipulation of  a case of X-SCID (X linked severe combined immunodeficiency) was treated by gene therapy with an apparent degree of success (BBC 2002). This particular disease process is caused by a mutation on the gene which codes for the C chain of the cytokine receptors which is situated on the X chromosome and vital for the functional development of T Killer lymphocytes which are therefore completely absent in the condition

A multinational team used a retroviral vector to insert a functional copy of the gene into bone marrow stem cells which were then re-transfused back into the patient. (Cavazzana-Calvo M et al 2000).This particular case resulted in a return to normal levels of T cells in a comparatively short period of time. This was hailed in both the popular media and the peer reviewed journals as a major success and it can indeed be considered a landmark as it pioneered the successful use of an ex-vivo procedure that avoided direct in vivo transfer of the vector.

The reason for specifically highlighting this particular case is that following the initial optimism of the clinical team, two of the first ten patients with this condition who were treated in the same way subsequently developed a leukaemia-like illness. Genetic analysis ofthe malignant cells suggested that the retroviral vector used in the transfer had also activated an onco gene LIM-only2 (LMO2) which is known to be associated with some forms of leukaemia. The clinicians reviewing the situation felt that, although it was not the only cause of the malignancy it was one of the events that triggered it. Similar concerns have been raised in respect of other clinical trials. (Lehrman S 1999)

The prime reason for presenting these events is to demonstrate the fact that there is both a theoretical and practical risk of insertional mutagenesis. Reduction of the risk requires greater specificity of the targeting of the genetic deficit  perhaps by directing the expression of the therapeutic genes to various specific tissues utilising both transductional and transcriptional targeting. (Relph K et al 2004),

In terms of specific considerations of the arguments in favour of the use of retroviral  vectors, one can cite the fact that they have a highly efficient mechanism of gene transfer together with low immunogenicity. It is a well researched and well studied system and is known to selectively infect actively dividing cells. The converse arguments reflect their disadvantages including their ability to disturb or activate onco genes, the fact that they are difficulty to specifically target and it is difficult to obtain high titres in the clinical situation (after Olsen, J. C. 1998).

In broad terms, the principles behind the use of retroviral vectors are that they must be modified in order not to be able to transmit any overtly pathological coding. This involves the deletion of viral helper genes such as gag, pol and env to render the replication process invalid. 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 is the MoMuLV. It is an engineered vector which can store 8 kb of RNA without compromising packaging efficiency. It is a hybrid cell line easily grown in mouse fibroblast cells.

There is a subdivision of the retroviral vectors known as the lentivirus, which is the only retroviral vector capable of integrating into the chromosomes of non-dividing cells. This has been effectively demonstrated in vitro (Naldini L et al 1996).

The biggest problem with the lentivirus vectors is that they appeared to only produce very low titres. Some recent research suggested that a modification to a amphotropic envelope protien was capable of allowing higher titre levels. (Rolls M et al 1999)

At about the same time that the scientific press was learning about the problems with retroviral transfer (see above) other investigators were working with adeno-associated viruses (AAVs). A similar process was invoked using adeno-associated viruses to correct a genetic defect involving coagulation factor IX. The adeno-associated viruses were used as they were considered to be amongst the safest candidates for gene transfer. They do not naturally cause disease processes in humans and have only rarely been found to incorporate in a random fashion into the human genome. Although it is noted that adenoviruses do cause onco gene activation 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 the trial author (in later research work) published a study which suggested that, in study mice, the vector used in the trials actually integrated itself into gene containing regions of DNA more frequently that it did into non-coding regions (Kay MA et al 2003). The findings were reported as the fact that new genetic material was randomly distributed amongst all of the chromosomes particularly at sites of gene activity. On this basis, there appears to be at least a theoretical basis for the possibility of similar cellular defects such as occurred in the X-SCIDpatients.

Adenoviruses are comparatively simple structures. They are categorised as double stranded DNA viruses. They have icosahedral capsids with twelve vertices and seven surface proteins. The virion itself is spherical and non-enveloped and in the region of 70-90 nm in size.

Their natural history is that they are spread easily in the natural state by the faeco-oral route and also by respiratory inhalation which clearly 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 code for specific proteins and they do not produce infection pathogenic viral offspring.

The early trials into this particular area were reviewed by Griesenbach (Griesenbach U et al 2002) who pointed out that the cystic fibrosis gene was first cloned in 1989 and in the subsequent  years, 18 different trials were carried out, all with rather low degrees of success. They collectively trialed three different vectors, namely adenoviruses, adeno-associated viruses 2 and cationic liposomes, and almost universally found that each vector had a very low rate of clinically significant gene transfer and none was sufficient to achieve clinical benefit

Plasmid Complexes

At its most basic level, a plasmid is a small accessory collection of DNA which is found in the cytoplasm outside of the nucleus. They are capable of independent replication and can be manipulated with rather more ease than nuclear DNA.

Early investigations into the field of gene transfer explored the possibility of plasmid vectors  and demonstrated the feasibility of the method to effect CFTR gene transfer in vitro (Alton EW 1993). Other teams had demonstrated the fact that, in clinical use the plasmid-liposome is both nontoxic and non-immunogenic (Hyde,SC et al1993).  This appeared to raise the possibility that many of the immunological problems encountered by teams working with viral mediated gene transfer mechanisms might be circumvented.

In vivo work (Yoshimura,K et al. 1992) had demonstrated that genes could be transferred into the cytoplasm by this method and Stribling, R(et al 1992) demonstrated that, once there, they would then replicate normally. Alton experimented with a CFTR-plasmid preparation in mice and demonstrated that it was capable of correcting the chloride levels in cystic fibrosis mice back to normal levels (Alton EW 1993)

Although the initial results were encouraging, clinical trials were disappointing as the plasmid complex could not easily penetrate the thick mucous residues in the diseased lungs of patients with cysticfibrosis. (Erickson,R 1993)

The plasmids typically have a positively charged head-group which is able to bind to the DNA strand and a hydrophobic tail group which facilitates the transfer of the complex across the cellular membranes. Initial studies suggest that  between 100-1000 times more DNA is required to effect successful gene transfer when this method is compared 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 that the respiratory epithelium is exposed to the DNA over a long period.Their solution was to encapsulate the CFTR-plasmid in a slow release biocompatible polymer. Clinical trials are underway but not yet reported.

The adeno-associated vectors appear to have (at least on atheoretical basis) a number of advantages over the vectors that we have already discussed. They are based on a virus vector that is already non-pathogenic (Berns, KI et al 1995) and has a mechanism that allows it 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 disorders such as cystic fibrosis and alpha 1 antitrypsin deficiency. (Flotte, TRet al 1998).

In addition, some workers have also developed vectors which are capable of  producing either inducible or constitutive expression of the cytokine, interlukin-10 (IL-10) which is an important anti-inflammatory protein which, on a theoretical basis, could be useful not only in cystic fibrosis but in other disease process which have 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 stage of early clinical trials (Wagner J et al 2002).

With specific reference to the implications of cystic fibrosis, we can point to trials which have resulted in the expression of cystic fibrosis 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), and again in early phase I clinical trials (Wagner, J et al 1998)

The rAAV-IL-10 model has been studied in bronchial cell cultures from cystic fibrosis patients, to determine the functional consequences of CFTR complementation. This has not yet been demonstrated in vivo with 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  it is possible to achieve long term gene transfer and functional expression 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 DNA in this form does not appear to be assimilated into the genetic material of the chromosome, but persists in log strings or concatemers that are episomal, which is in complete contrast to what happens when the naturally occurring agent infects the cell. There is some evidence to suggest that host cell intrinsic factors such as DNA-dependent protein kinase play some role in this process (Song, S 2001).

The significance of this finding could be that the exclusion of the functional, newly introduced DNA from the rest of the nuclear gene poolmay be less likely to produce effects that could be either potentially disruptive to the host cell and less likely to activate onco genes.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 proteins deleted and substituting the appropriate gene (CFTR or equivalent) between the rAAV2 inverted terminal repeats together with other signal sequences such as promoter and polyadenylation sequences (Flotte, TR etal 1994)

The packaging processes allows for about 5 kb of rAAV genomes to be carried  in the vectors which are prepared using a cotransfection technique utilising human embryonic kidney cells (HEK-293) where the vector plasmid is cotransfected into the cells with helper agents (plasmid pDG) being used to encode the rAAV2-rep and -cap genes together with the adenovirus helper functions (Grimm, D et al 1998).These are incubated for between 48 and 72 hrs. The cells are then lysed and the resultant agents are then separated by ultracentrifugation against 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 absence of any possible contamination from non-modified (replication competentAAVs) prior to clinical usage. (Muzyczka N 1994)

The comparatively small “payload” of the adeno-associated vectors is proving to be a significant problem. The vector itself is small when compared to the comparatively large size of the CFTR gene. (Flotte TRet al 1993) It does not leave any room to manoeuvre to manipulate the vector-specific sequences in the way that we have described with the retroviral and adenoviral groups. (Flotte TR et al 2001).

A number of authors have characterised the problem with the observation that the rAAV is typically about 20 nm across which allows packaging of about 4.7 kb (kilobases) of transferable modified gene (exogenous DNA). (Dong JY et al 1996), If  it is combined with other enhancers such as the promoter, the polyadenylation signal, this clearly reduces the capacity for the DNA component. (Duan D et al2000). The Yan paper (Yan Z et al 2000) has outlined a novel exploitation of the unique ability of the rAAV genomes to link together in strings which appears to have the ability to bypass this particular limitation.( Flotte TR 2000).

The mechanism itself is the capacity of two distinct rAAV genomes that happen to simultaneously infect the same target cell to undergo an intermolecular recombination insider the transduced nucleus of the target cell.

This was a chance finding which arose from work involving rAAV-derived episomes (Kearns WG et al 1996) in primate airways. It was found that some of these episomes were configured as circular head to tail 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 cellular penetrations which combined within the palindromic inverted terminal repeat sequences that are an intrinsic part of the AAV genome structure. The authors were of the opinion that it was likely to be the latter eventuality (Duan D et al 1998)

It was a logical progression to try to exploit this phenomenon and thereby bypass the limitations imposed by the relatively small packaging capacity of rAAV. The adeno-associated vectors capsid only has a capacity of about 5 kb. If we consider that the 145 nucleotide stretch of the AAV-ITR (inverted terminal repeat) sequence has to be in place at both ends of the single-strand DNA for the vector DNA to be both replicated and packaged, this only leaves in the region of 4.7 kb of 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 little space for other enhancing material. Because of this, the actual CFTR vector that has been used in the clinical trials to date uses only the minimal promoter activity of the AAVs-ITR itself to actually activate and drive the CFTR expression (Flotte TR et al. 1993).

To look at this potentially important development in a little more detail we can consider Duan’s original paper (et al 2000) and the authors describe what they call a “superenhancer”. They describe a combination of a potent simian virus (SV40) and CMV immediate early enhancer elements as being packaged in one rAAV vector and a luciferase gene assisted by a small minima;l promoter in another rAAV vector. Invitro experiments suggested that either the SV40 or the intrinsic promoter activity of the AAV-ITR was sufficient for this purpose. The intermolecular recombination described above, was found to occur in both vitro and in vivo experiments and was found to be sufficient to have a greater than additive effect.

Initial results from these varying methods are encouraging insofar as 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 cystic fibrosis, we should note that Yan’s group and other workers have done experimental work which has culminated in the long term expression of functional 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 tried inserting the promoter and the first half of the coding sequence in onerAAV vector, immediately followed by a splice donor and then the upstream half of an intron. In the other rAAV vector was the downstream half of the intron, the splice acceptor, the second half of the gene and the polyadenylation signal. To quote the author verbatim:

This strategy is efficient enough to mediate high-level expression and the intermolecular junctions are apparently stable enough to mediate expression for several months in vivo.

Although this is clearly an ingenious augmentation of the same principle, we should note that there are both advantages and disadvantages to both pathways.

The strategy that adopts the superenhancer takes its strengths from the fact that the recombination mechanisms optimise the position-independent and orientation-independent functions of the enhancers.  Consideration of the options would suggest that there are four potential recombination outcomes from the process described. Either of the two vectors could be on the 5’ end of the heterodimeric molecule and clearly either molecule could be in either orientation.

With the superenhancer option, all four of these possible intermolecular recombination outcomes should be functional for transgene expression whereas if compared to the split intron strategy, by using the same reasoning, it is clear that only one out of the four could work.

On the other side of the argument, the superenhancer option has the disadvantage that the actual coding sequence of the gene to be transferred must still fall within the packaging capacity of the vector itself whereas the split in tron allows for a greater functional expansion of the packaging capacity. (after Flotte TR et al 2000)

In either event it can be seen that these ingenious modifications effectively eliminate the main size limitation of the rAAV delivery system. Although initial pre-clinical work is encouraging it appears that there is still some potential for a degree of immune response particularly if the host organism has not experienced the newly produced protein before.

A number of studies have been done on animal (vertebrate andprimate) with only minimal success. Different administration methods have 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). The studies were done on adult male and female patients (18-47 yrs) who were pseudomonas free and had recently been hospitalised for IV antibiotic infusions

The disappointing results were probably a reflection of the fact that the CFTR defect is also interconnected in some way with  aproinflammatory phenotype which appears to be triggered by the abnormal protein via an unfolded protein response. The authors were able to show evidence that the rAAV-CFTR mechanism was able to  correct the protein production defect, they found it clinically difficult to transduce a sufficient number of cells in the airway to reverse the inflammatory response.

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 possible workable approach (Teramoto, S et al 1998). Other work on ways of enhancing the phenotypic expression of the modified genotype has suggested that the use of various promoters and the rAAV-CMV/beta-actin hybrid promoter (CB-AAT) was found to be tone of the most efficient, at least 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 single injection of an rAAV-CB-AAT vector in animal studies has resulted in high level, stable transgene expression which has persisted over the life span of the experimental animals and that there was no detectable inflammatory response in the animals who had received this form of treatment (Flotte TR 2002)

Flotte (et al 2002) reports that four human clinical trials at both Phase I and Phase II level are currently underway examining the effects of the rAAV-CFTR vector. They had an entry cohort of seven patients with the vector being applied to the nasal lining, the maxillary sinus and the bronchus. The authors report no adverse effects being found and that they have observed transgene expression at doses of 6 x 108 drp in the sinus or 1 x 1013 drp in the lung. There are no reported interim findings from the Phase II trials as yet.

There is clearly a potential for clinical benefit on the basis of the results found to date if one can extrapolate from in vitro and animal experiments. 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 the potential limitations of this particular delivery system. These have been 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 examining the hypothesis that the barriers in the airways of the cystic fibrosis sufferer are primarily  due to the neutrophil-derived -defensins (HNP1and HNP2) and are actually reversible by the mechanism of AAT protein delivery (Virella-Lowell, I 2000)

Wu and his co-workers have been looking at ways of manipulating the genetic make up of the rAAV2 capsid and thereby trying to enhance the targeting ability so that the vector specifically targets the serpinenzyme complex receptor on IB3–1 cells – which is virtually specific for the Cystic fibrosis bronchial cells

Zabner, J (et al 2000), have considered alternative rAAV serotypes in the hope of finding one that will bind more specifically to the bronchial cells

Other peripheral adjuncts have also been explored including promoters to enhance the effects of complementation and superenhancers which have been shown to improve the ability of the rAAV to concatermerise with the help of smaller amounts of promoter agents (Duan, D et al 2000).

Perhaps it is appropriate to conclude this section on consideration of adeno-associated vectors with a critical analysis of a very recent multicentre, double-blind, placebo-controlled trial (Moss RB et al2004)

This was a well constructed, fully statistically significant and double blinded trial which considered  both the safety and the tolerability of repeated doses of adeno-associated serotype 2 vector repeatedly given by aerosol inhalation. The vector contained “cysticfibrosis transmembrane conductance regulator (CFTR) complementary DNA(cDNA) [tgAAVCF], an adeno-associated virus (AAV) vector encoding the complete human CFTR cDNA.”

The entry cohort was comparatively small with 42 patients, of whom 20 received the active agent. A number of indices of airway function were measured. Of particular interest to our considerations in this dissertation was the fact that  vector shedding was found in all treated subjects up to 90 days after inoculation. And that all subjects who 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 in all of them. On this basis, it would appear that the actual transfer mechanism is effective, but there are other factors present which appear to interfere with the subsequent expression of the gene in terms of protein production. The study did not comment on the possible reasons for this.

The authors were able to conclude that the delivery system worked well with no evidence of adverse effects and that treated patients demonstrated 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-associated vectors and this has prompted researchers to explore and consider other optimising options for facilitating gene transfer. Zabner (J et al1997) considered the use of cationic lipids in this process and found one  – GL-67:DOPE (colloquially known as lipid 67) which appeared to be particularly helpful in the process.

Cationic lipids appear to show a degree of promise as possible vectorsfor CFTR cDNA transfer into respiratory epithelial cells of cystic fibrosis patients. Zabner’s group developed a preparation of plasmid encoding CFTR (pCF1-CFTR) and cationic lipid (GL-67:DOPE) which appeared to facilitate the gene transfer to a significantly greater extent than previously tested lipid complexes. They performed in vivo studies which compared the gene transfer rate to the epithelial cells of the nasal mucosa of DNA-lipid complex and DNA alone. In general terms, their findings indicated that the DNA-lipid complex was far more effective in achieving gene transfer than was simply giving DNA. The authors felt able to conclude that:

These results indicate that nonviral vectors can transfer CFTR cDNA to airway epithelia and at least partially restore the Cl- transport defect characteristic of CF. However, improvements in the overall efficacy of gene transfer are required to develop a treatment for CF.

In this dissertation we are primarily considering the issues of gene therapy in direct relation to cystic fibrosis. Inevitably, this has meant considering the issues on a wider front, as many areas overlap on a theoretical or practical basis.

The prime biochemical cellular defect in cystic fibrosis is an abnormality in the cystic fibrosis transmembrane conductance regulator (CFTR). From a theoretical perspective it should be obvious that replacement of the defective gene with a working alternative would be best achieved in the neonatal period before the body had time to develop substantial fibrotic changes in the lungs that were secondary to repeated episodes of infection (Dark J et al 1996).

If successful, this could be expected to reduce both morbidity and mortality for cystic fibrosis. We have been able to cite evidence that gene transfer has been accomplished both in vitro and in vivo. We have discussed the results of a number of research groups who have investigated various delivery systems  which, to varying extents, have proved able to deliver at least a small quantity of functional respite to the cystic fibrosis sufferer.

It is also important to be fully aware of the possibility of inadvertent side effects in the field of gene manipulation. We have highlighted the problems with oncogene activation. But this appears to be associated with some vectors more than others. In short, it would appear that the problems and limitations that appear with this type of procedure are a function of the parent virus.

The initial work with adenoviruses appeared promising as gene transfer could be accomplished but the major drawback was the dose limiting inflammatory effects which arose primarily as a result of the large amount of viral protein which was required to achieve a therapeutic dose. The subsequent modifications which had a greater number of coding sequence deletions appeared to be more effective in animal experiments as they generated a lesser response from the cell mediated immunity mechanisms and therefore had a greater duration of action. (Caplen NJ et al 1995).

It seemed a logical step from there to produce vectors that had no viral genes at all. This did not produce any significant benefits or improvements from the previous agents. A number of research teams across the world tried different subsidiary strategies including drug induced immuno suppression or modifications of various immuno genicepitopes.

The plasmid-lipid complexes appeared to have a number of clinically important advantages insofar as they did not appear to generate any immunological response which is in distinct contrast to many viral vectors. Initial optimism did not appear to be translated into practical application as the delivery systems explored appeared to be unable to deliver sufficiently large quantities through the pathological mucous layer that is the main feature of the cystic fibrosis patient. (Crystal RG 1992).

The adeno-associated vectors have received a large amount of attention when it became clear that alternative vectors were needed to optimise the therapeutic effect.  They have now reached the stage where animal testing has lead to human Phase I and II clinical trials. As a group,they appear to have the advantage that they don’t trigger the inflammatory reaction in the same way, or to the same extent as the adenovirus group. The major practical difficulty with this group however, is the fact that because they are so small – compared to the comparatively large size of the CFTR gene – it leaves no space for vector-specific sequences on which to base assays to help to distinguish the endogenous RNA from the vector-expressed RNA. (FlotteTR et al 2001)

All the evidence that we have seen appears to  suggest that adeno-associated vectors have a satisfactory safety profile and certainly appear to produce a longer duration of clinical effect than the other modalities.

Another variable, and indeed challenge, in the field of gene therapy, is to find the optimum delivery vehicle. We have cited studies that have tried direct insufflation to the bronchial epithelium. This appears to be a superior mode of delivery to the aerosol which appears to have the ability to cause agent specific reactions in the alveolar membranes. There is continuing work which is currently looking at the relative merits of nebuliser delivery mechanisms as compared to conventional aerosol delivery systems. Others that have tried avoiding the bronchial tree and utilising the respiratory epithelium by introduction to the maxillary sinuses through intranasal antrostomies.

Conclusion and future of gene therapy

In this dissertation we have presented evidence of from a number of different approaches to the problem of gene therapy in tackling monogenic conditions such as cystic fibrosis. As with most areas of scientific exploration many “blind alleys” have to be explored before an appropriate avenue of research becomes apparent.

The initial enthusiasm the greeted the exploration of the plasmid–DNA vector did not appear to be well founded although it is clear that further exploratory work is continuing in the field.

The area of adeno-associated vectors appears to be currently the most promising with, at least in vitro, suggestions that many of the current limiting problems may be on the verge of being solved.

The major stumbling blocks at the moment are the difficulties of producing a high vector titre in the clinical situation and the longterm safety considerations, particularly those relating to mutagenesis of 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 genomes interact with host cell proteins such as the DNA-dependent protein kinase in the formation of stable high-molecular weight concatemers.

It is the episomal situation of the gene that is currently thought to be the best insurance against in advertent iatrogenic oncogenesis (Flotte et al 2002) but this is clearly no substitute for long term careful and rigorous safety studies.

It is often assumed, quite incorrectly, that the field of gene therapy is a discrete and academically isolated field. Progress in this area, as in so many other areas of research, is completely dependent of discoveries 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 lines outside of the body as this will inevitably aid our ability to implant genetically engineered agents. Reflection over the advances in knowledge from just the last decade indicates that new and innovative delivery mechanisms will be developed, explored and evaluated. It is likely that the known short term problems of immunogenicity, low titre delivery and inefficient packaging will be addressed, very possibly with new delivery vectors.

It goes without saying that these investigations are hugely expensive in terms, not only of money, but of time, expertise and investment generally and therefore it is likely that the limiting factor in terms of development will be the availability of resources (Russell S 1997)


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