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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 a
number of assumptions relating to the future direction of the research
in the field.
Introduction
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 autologous T cells. (Rosenberg SA et al
1990)
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 reimplanted.
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 nucleii 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 the
non-dividing state) was a major barrier for chromosomal gene
integration. (Sikorski R et al 1998). A breakthrough came with the
realisation that lentiviruses (eg 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 al
1999)
Cystic fibrosis
Cystic fibrosis is the most commonly lethal inherited recessive in
the caucasian population. It affects about 1 per 2,500 livebirths. The
treatment 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 are
commonly 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 that
it 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).
Retroviral vectors
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 of
the malignant cells suggested that the retroviral vector used in the
transfer had also activated an oncogene 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 oncogenes, 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 cell
line. (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)
Adeno vectors
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 oncogene
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-SCID
patients.
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 adeno
virus 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 al
1993). 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 cystic
fibrosis. (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.
Adeno Associated vectors
The adeno-associated vectors appear to have (at least on a
theoretical 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 et
al 1989). The adeno-associated vectors are particularly useful in
dealing 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, TR
et 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 I
diabetes 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 et
al 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 pool
may be less likely to produce effects that could be either potentially
disruptive to the host cell and less likely to activate oncogenes.
Phase I trials have demonstrated significant rises of CFTR levels in
both sinus and lung tissue with no evidence of vector-related toxicity.
(Wagner, JA et al 1999)
The adeno-associated vectors are constructed from proviral
adeno-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 et
al 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, S
et al 1999).
The vectors are thereby amenable to being separated by both their
physical characteristics and also their biological characteristics
(infectious units). They are carefully screened to ensure the absence
of any possible contamination from non-modified (replication competent
AAVs) 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 TR
et 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 al
2000). 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 from
a “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 al
1996) 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. In
vitro 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 100-600
times greater than with the unenhanced vector alone. (Yan, Z et al
2000)
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 mice
in vivo. (Naffakh N et al 1995),
This basic principle has been further enhanced by Sun (Sun L et al
2000) with an ingenious manipulation of the system. They tried
inserting the promoter and the first half of the coding sequence in one
rAAV 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 intron 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 and
primate) with only minimal success. Different administration methods
have been studied including direct administration into the lung (Wagner
J et al 1999), IM injection (Song, S et al 2001 B) and hepatic portal
vein infusion (Song, S et al 2001 A)
Human clinical trials have taken place with these vectors (Flotte T et
al 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 a
proinflammatory 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 adenovirus
vectors there is a marked lack of inflammatory toxicity with the rAAV
vectors.
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 (HNP1
and 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 serpin
enzyme 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 al
2004)
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 “cystic
fibrosis 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 in
the 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 function
in 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 al
1997) 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 vectors
for 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.
Analysis and Discussion
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 immunosuppression or modifications of various immunogenic
epitopes.
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. (Flotte
TR 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 including 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 long
term safety considerations, particularly those relating to mutagenesis
of ongogenes. On this point the Flotte group are optimistic and feel
able to make the comment:
The data from our laboratory strongly indicate that the bulk of rAAV
DNA 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 inadvertent 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)
References
Afione, SA, Conrad, CK, Kearns, WG, et al (1996)
In vivo model of adeno-associated virus vector persistence and rescue.
J Virol 70,3235-3241
Alton,EW et al. 1993
Nature Genetics. vol 5. 1993
Amado R G & Chen YJS 1999
Lentiviral Vectors—the Promise of Gene Therapy Within Reach?
Science. 285 (5428): 674-76.
BBC Online News.
”Bubble boy" saved by gene therapy.
3 April 2002,
Berns, KI, Linden, RM (1995)
The cryptic life style of adeno-associated virus.
Bioessays 17,237-245
Blacklow NR. 1988
Adeno-associated viruses of humans. In: Pattison JR, ed. Parvoviruses and human disease.
Boca Raton, FL: CRC Press; 1988; 165–174
Blacklow, NR, Hoggan, MD, Kapikian, AZ, et al (1989)
Epidemiology of adenovirus-associated virus infection in a nursery population. Am J Epidemiol 88,368-378
Caplen NJ, Alton EW, Middleton PG, Dorin JR, Stevenson BJ, Gao X, Durham SR, Jeffery PK, Hodson ME, Coutelle C, et al.1995
Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis.
Nat Med 1995 Jan;1(1):39-46..
Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, et al.2000
Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.
Science 2000;288: 669-72:
Conrad, CK, Allen, SS, Afione, SA, et al (1996)
Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung.
Gene Ther 3,658-668
Crystal RG. 1992
Gene therapy strategies for pulmonary disease.
Am J Med 1992; 92:44S-52S
Dark, J., et al. 1996
Transplantation. Shale, D. J., ed. Cystic Fibrosis.
BMJ Publishing Group. 1996. pp120-133.
Dong JY, Fan PD, Frizzell RA: 1996
Quantitative analysis of the packaging capacity of recombinant adeno- associated virus.
Hum Gene Ther 1996, 7:2101-2112.
Duan D, Sharma P, Yang J, Yue Y, Dudus L, Zhang Y, Fisher KJ, Engelhardt JF: 1998
Circular
intermediates of recombinant adeno-associated virus have defined
structural characteristics responsible for long-term episomal
persistence in muscle tissue.
J Virol 1998, 72:8568-8577.
Duan D, Yan Z, Yue Y, Engelhardt JF: 1999
Structural analysis of adeno-associated virus transduction circular intermediates.
Virology 1999, 261:8-14
Duan, D, Yue, Y, Yan, Z, et al (2000)
A new dual-vector approach to enhance recombinant adeno-associated
virus-mediated gene expression through intermolecular cis activation.
Nat Med 6,595-598
Egan, M, Flotte, T, Afione, S, et al (1992)
Defective regulation of outwardly rectifying Cl-channels by protein kinase A corrected by insertion of CFTR.
Nature 358,581-584
Erickson,R 1993
New Scientist. 2 Sep 1993.
Felgner P. 1997
Nonviral strategies for gene therapy.
Sci Am 1997;276:86-91.
Flotte TR, Afione SA, Solow R, Drumm ML, Markakis D, Guggino WB, Zeitlin PL, Carter BJ:1993
Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter.
J Biol Chem 1993, 268:3781-3790.
Flotte, TR, Afione, SA, Zeitlin, PL (1994)
Adeno-associated virus vector gene expression occurs in nondividing cells in the absence of vector DNA integration.
Am J Respir Cell Mol Biol 11,517-521
Flotte, T, Carter, B, Conrad, C, et al (1996)
A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with mild lung disease.
Hum Gene Ther 7,1145-1159
Flotte, TR, Carter, BJ (1998)
Adeno-associated virus vectors for gene therapy of cystic fibrosis.
Methods Enzymol 292,717-732
Flotte TR 2000
Size does matter: overcoming the adeno-associated virus packaging limit
Respiratory Research 2000, 1:16-18
Flotte TR Laube BL 2001
Gene Therapy in Cystic Fibrosis*
Chest. 2001;120:124S-131S.
Flotte TR 2002
Recombinant Adeno-Associated Virus Gene Therapy for Cystic Fibrosis and 1-Antitrypsin Deficiency
Chest. 2002;121:98S-102S.
Freeman SM, Whartenby KA, Freeman JL, Abboud CN, Marrogi AJ. 1996
In situ use of suicide genes for cancer therapy. Semin Oncol
1996;23:31-45. Zhang J, Russell SJ. Vectors for cancer gene therapy.
Cancer Metastasis Rev 1996;15:385-401.
Griesenbach U, Ferrari S, Geddes D Alton EW 2002
Gene therapy progress and prospects: cystic fibrosis.
Gene. Ther. 2002 Oct;9(20):1344-50.
Grimm, D, Kern, A, Rittner, K, et al (1998)
Novel tools for production and purification of recombinant adeno-associated virus vectors.
Hum Gene Ther 9,2745-2760
Hyde,SC et al. 1993
Nature. vol 362. 1993.
Kay MA, Manno CS, Ragni MV, Larson PJ, Couto LB, McClelland A, et al. 2000
Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.
Nature Genet 2000;24: 257-61.
Kay MA, Nakai H. 2003
Looking into the safety of AAV vectors.
Nature 2003;424: 251.
Kearns WG, Afione SA, Fulmer SB, Pang MC, Erikson D, Egan M, Landrum MJ, Flotte TR, Cutting GR:1996
Recombinant adeno-associated virus (AAV-CFTR) vectors do not integrate
in a site-specific fashion in an immortalized epithelial cell line.
Gene Ther 1996, 3:748-755.
Knowles MR, Noone PG, Hohneker K, Johnstone LG, Boucher RC, Efthimoiou J, Crawford C, Brown R, Schwartzbach C, pearlman R 1998
A double-blind, placebo controlled, dose ranging study to evaluate the
safety and biological efficacy of the lipid-DNA complex GR213487B in
the nasal epithelium of adult patients with cystic fibrosis.
Hum Gene Ther. 1998 Jan 20;9(2):249-69.
Lehrman S. 1999
Virus treatment questioned after gene therapy death.
Nature 1999;401: 517-8
Moss RB, Rodman D, Spencer LT, Aitken M, Zeitlin P, Waltz D, Milla C, Brody AS, Clancy JP, Ramsay B, Hamblett N, Heald A, 2004
Repeated adeno-associated virus serotype 2 aerosol-mediated cystic
fibrosis transmembrane regulator gene transfer to the lungs of patients
with cystic fibrosis : a multicenter, double-blind, placebo-controlled
trial - clinical investigations
Chest Feb 2004
Muzyczka N: 1994
Adeno-associated virus (AAV) vectors: will they work?
J Clin Invest 1994, 94:1351.
Naffakh N, Henri A, Villeval JL, Rouyer-Fessard P, Moullier P, Blumenfeld N, et al. 1995
Sustained delivery of erythropoietin in mice by genetically modified skin fibroblasts.
Proc Natl Acad Sci U S A 1995;92:3194-8.
Naldini, L., et al. 1996
In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.
Science. 1996. 272: 263-267.
Nichols, E. K. 1998
Human Gene Therapy. Cambridge,
Massachusettes: Harvard University Press, 1998.
Olsen, J. C. 1998.
Gene Transfer Vectors Derived From Equine Infectious Anemia Virus.
Gene Therapy. 5: 1481-1487.
Porteous DJ, Dorin JR, McLachlan G, Davidson-Smith H, Davidson H, Stevenson BJ, et al. 1997
Evidence for safety and efficacy of DOTAP cationic liposome mediated
CFTR gene transfer to the nasal epithelium of patients with cystic
fibrosis.
Gene Ther 1997;4:210-18.
Relph K, Kevin Harrington, and Hardev Pandha 2004
Recent developments and current status of gene therapy using viral vectors in the United Kingdom
BMJ, Oct 2004; 329: 839 - 842 ;
Rolls, M. M., et al. 1999
Novel infectious particles generated by expression of the vesicular stomatitis virus glycoprotein from a self replication RNA.
Cell. 1999. 79: 497-506.
Rosenberg SA, Aebersold P, Cornetta K, Kasid A, Morgan AA, Moen R, et al. 1990
Gene transfer into humans: immunotherapy of patients with advanced
melanoma using tumor infiltrating lymphocytes modified by retroviral
gene transduction.
N Engl J Med 1990;323:570-8.
Russell SJ 1997
Science, medicine, and the future : Gene therapy
BMJ, Nov 1997; 315: 1289 – 1292
Santis,G. Geddes,D. 1994
Post Grad Med J. vol 70. 1994.
Sikorski R, Peters R 1998
Gene Therapy: Treating with HIV.
Science. 282 (5393): 1438a.
Song, S, Morgan, M, Ellis, T, et al (1998)
Sustained secretion of human alpha-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors.
Proc Natl Acad Sci U S A 95,14384-14388
Song, S, Embury, J, Laipis, P, et al (2001 A)
Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT)
after portal vein injection of recombinant adeno-associated virus
(rAAV) vectors. Gene Ther 8,1299-1306
Song, S, Laipis, PJ, Berns, KI, et al (2001 B)
Effect of DNA-dependent protein kinase on the molecular fate of the rAAV2 genome in skeletal muscle.
Proc Natl Acad Sci U S A 98,4084-4088
Stern M, K Ulrich, D M Geddes and E W F W Alton 2003
Poly (D,
L-lactide-co-glycolide)/DNA microspheres to facilitate prolonged
transgene expression in airway epithelium in vitro, ex vivo and in vivo
Gene Therapy (2003) 10, 1282-1288.
Stribling,R et al. 1992
Proc Nat Acad Sci. vol 89. 1992.
Summerford, C, Samulski, RJ (1998)
Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions.
J Virol 72,1438-1445
Sun L, Li J, Xiao X: 2000
Overcoming adeno-associated virus vector size limitation through viral DNA heterodimerization.
Nat Med 2000, 6:599-602.
Teramoto, S, Bartlett, JS, McCarty, D, et al (1998)
Factors influencing adeno-associated virus-mediated gene transfer to
human cystic fibrosis airway epithelial cells: comparison with
adenovirus vectors.
J Virol 72,8904-8912
Virella-Lowell, I, Poirier, A, Chesnut, KA, et al (2000)
Inhibition of recombinant adeno-associated virus (rAAV) transduction by bronchial secretions from cystic fibrosis patients.
Gene Ther 7,1783-1789
Wagner, JA, Moran, ML, Messner, AH, et al (1998)
A phase I/II study of tgAAV-CF for the treatment of chronic sinusitis in patients with cystic fibrosis.
Hum Gene Ther 9,889-909
Wagner, JA, Messner, AH, Moran, ML, et al (1999)
Safety and biological efficacy of an adeno-associated virus
vector-cystic fibrosis transmembrane conductance regulator (AAV-CFTR)
in the cystic fibrosis maxillary sinus.
Laryngoscope 109,266-274
Wagner JA, Nepomuceno IB, Messner AH, et al. 2002
A phase II, double-blind, randomized, placebo-controlled clinical trial
of tgAAVCF using maxillary sinus delivery in CF patients with
antrostomies.
Hum Gene Ther 9,889-909 2002
Wu, P, Xiao, W, Conlon, T, et al (2000)
Mutational analysis of the adeno-associated virus type2 (AAV2) capsid
gene and construction of AAV2 vectors with altered tropism.
J Virol 74,8635-8647
Yan, Z, Zhang, Y, Duan, D, et al (2000)
From the cover: trans-splicing vectors expand the utility of adeno- associated virus for gene therapy.
Proc Natl Acad Sci U S A 97,6716-6721
Yoshimura,K et al. 1992
Nuclei Acids Res. vol 20. 1992.
Zabner J, Cheng SH, Meeker D, Launspach J, Balfour R, Perricone MA,
Morris JE, marshall J, Fasbender A, Smith A&E Department, Welsh MJ
1997
Comparison of DNA-lipid complexes and DNA alone for gene transfer to cystic fibrosis airway epithelia in vivo.
J,Clin. Invest. 1997 Sep 15;100(6):1529-37.
Zabner, J, Seiler, M, Walters, R, et al (2000)
Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical
surfaces of airway epithelia and facilitates gene transfer.
J Virol 74,3852-3858
Zolotukhin, S, Byrne, BJ, Mason, E, et al (1999)
Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield.
Gene Ther 6,973-985
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