key: cord-328899-kog99kk5
authors: Ferrari, Stefano; Geddes, Duncan M; Alton, Eric W.F.W
title: Barriers to and new approaches for gene therapy and gene delivery in cystic fibrosis
date: 2002-12-05
journal: Adv Drug Deliv Rev
DOI: 10.1016/s0169-409x(02)00145-x
sha: 
doc_id: 328899
cord_uid: kog99kk5

Clinical trials of gene therapy for cystic fibrosis suggest that current levels of gene transfer efficiency are probably too low to result in clinical benefit, largely as a result of the barriers faced by gene transfer vectors within the airways. The respiratory epithelium has evolved a complex series of extracellular barriers (mucus, lack of receptors, immune surveillance, etc.) aimed at preventing penetration of lumenally delivered materials, including gene therapy vectors. In addition, once in the cell, further hurdles have to be overcome, including DNA degradation, nuclear import and the ability to maintain long-term transgene expression. Strategies to overcome these barriers will be addressed in this review and include the use of: (i) clinically relevant adjuncts to overcome the extra- and intracellular barriers; (ii) less-conventional delivery routes, such as intravenous or in utero administration; (iii) more efficient non-viral vectors and ‘stealth’ viruses which can be re-administered; and (iv) new approaches to prolong transgene expression by means of alternative promoters or integrating vectors. These advances have the potential to improve the efficiency of gene delivery to the airway epithelium, thus making gene therapy a more realistic option for cystic fibrosis.

clearly an urgent need for a novel therapeutic approach. Cystic fibrosis (CF) is the most common reces-Preclinical studies carried out soon after the sively inherited lethal disease among Caucasian isolation of the gene showed that both viral and population, affecting approximately one in 2500 non-viral gene transfer agents (GTAs) were able to newborns. CF is caused by mutations in the cystic correct the chloride ion transport defect in CF fibrosis transmembrane conductance regulator transgenic mice. The success of these studies led (CFTR) gene, and to date around 1000 different several groups to initiate clinical trials of gene mutations have been identified. The underlying gene transfer in CF patients and three classes of GTAs mutation leads to defective production of the CFTR have been used so far: adenovirus, adeno-associated 2 protein, a cAMP-regulated chloride (Cl ) channel virus (AAV) and cationic lipids. Although the lung located in the apical membrane of epithelial cells.

remains the most medically relevant target, many Although the organs affected in CF also include the investigators chose to start their clinical studies by pancreas, gut, liver and reproductive tract, the clini-looking at gene transfer to the nasal mucosa. Nasal cal picture is dominated by pulmonary disease, with airway epithelia have a similar histology and the recurrent cycles of infection leading to inflammation, same CF-associated abnormalities in ion transport as bronchiectasis and, in greater than 90% of patients, pulmonary epithelia, but compared to lung, the nasal death from respiratory failure. The isolation of the cavity has an easier access for both gene transfer and gene responsible for CF in 1989 suggested the safety measurements and represents a reduced risk to feasibility of new therapeutic treatments based on the patient in case of the occurrence of side effects. CFTR gene transfer to patients with CF. The

Nine clinical trials using recombinant adenovirus rationale for the development of gene therapy proto-as a GTA have been published so far [1] [2] [3] [4] [5] [6] [7] [8] [9] , with five cols relies on the fact that heterozygotes appear to be involving administration of the virus to the lung phenotypically normal, expression of CFTR is low epithelium [2, [6] [7] [8] [9] . The results reported are not and the dysfunctional epithelial lining cells in the always consistent and of the 30 noses assessed most affected organ, the lung, are accessible through following a single application, approximately onenon-invasive techniques. Furthermore, although con-third showed some changes in chloride transport. No ventional treatments have increased life expectancy functional measurements were assessed in the lung of CF patients to approximately 30 years, there is studies, although some evidence of vector-derived CFTR mRNA was found. Common findings in all be different in CF subjects. More importantly, it is the studies published include (i) a dose-dependent now generally accepted that the efficiency of in vivo mild local inflammation and (ii) the progressive lack gene transfer with currently available vectors needs of expression following repeated administration.

to be increased. We will here review the progress Two phase I, single administration, dose escalation made in improving GTAs and the hurdles to efficient trials using AAV as a GTA have been reported so far.

transgene expression, which any vector has to over-AAV-CFTR was delivered to the maxillary sinus [10] come before entering the airway epithelial cells. or nebulised to the lungs of CF subjects [11] . In both cases AAV delivery was shown to be safe and vector DNA was found by PCR, up to 70 days in the 2 . Extracellular barriers maxillary sinuses [10] and 14-30 days in the airways [11] . When delivered to the maxillary sinus, some

The respiratory epithelium presents a particular degree of dose-dependent chloride transport correc-challenge for GTAs, since one function of the upper tion was observed. However, in both studies no respiratory tract is to keep foreign particles out of the vector-derived mRNA was detected.

lung. Airway epithelia have evolved a complex Cationic lipids have been used in eight clinical series of barriers to prevent penetration of lumenally trials [12] [13] [14] [15] [16] [17] [18] [19] , with two of them involving nebulisa-delivered materials (including both viral and nontion of the lipoplexes to the lower airways [16, 19] . viral GTAs) into the cell or interstitial compartment. Different lipoplexes were used including DC-Chol-These barriers consist of: (i) a well-defined mucus DOPE [12, 14, 17] , DOTAP [15] , GL-67 [13, 16, 19] layer that may bind inhaled vectors and remove them and EDMPC-cholesterol [18] , with DNA doses via mucus clearance mechanisms, (ii) a glycocalyx ranging from 10 mg to 1.25 mg when administered to that may bind vectors and prevent binding to cell the nose and up to 42.2 mg when nebulised to the surface receptors and perhaps most importantly, and lungs [16] . Results were similar to those reported (iii) an apical cell membrane that is relatively devoid with adenoviral vectors and a correction towards of viral receptors and growth / tropic receptors (Fig. normal values of the chloride defect was observed 1). This series of barriers is complemented by both in the nose (about 20%) [12, [14] [15] [16] and in the epithelial tight junctions that are 'moderately leaky' lung (25%) [16] , lasting between 7 days and 3 weeks to ions but quite 'tight' for larger solutes, thereby [16] . Interestingly, in one study naked DNA was preventing penetration by current vectors from the reported to be as efficient as lipoplexes [13] . Unlike lumenal surfaces into the interstitium. In addition, viruses, it has been reported that lipoplexes can be CF lungs are characterised by the presence of a successfully re-administered without apparent loss of discontinuous barrier of purulent secretions, which efficacy [17] . However, mild flu-like symptoms were contain exogenous actin, DNA and inflammatory noted in both lung trials following aerosolisation of products that can modify the integrity of a lipoplex liposome-DNA complexes and are probably related and limit gene delivery to the airways. CF sputum to the presence of unmethylated CpG motifs in has been shown to retard the movement of particles bacterially derived plasmid DNA [16, 19] .

having a size comparable with lipoplexes and to In summary, within 13 years of the cloning of the almost completely block 560-nm particles [20] . CFTR gene tremendous progress has been made and Furthermore, binding of negatively charged CF proof-of-principle for correction of the basic chloride mucus components to the gene complexes may defect has been established within the target organ in change their surface charge and size, resulting in a vivo in CF subjects. However, in none of the clinical decreased transport of the lipoplex through the trials cited, has sodium hyperabsorption been altered. mucus and therefore in a decreased cellular uptake Each of the three GTAs used has achieved limited [21] . success, with none outshining the others. Interesting-Using an ex vivo sheep trachea model, we demonly, despite non-viral vectors being arguably less strated that cationic lipid-mediated gene transfer, but efficient than viruses in animal models and labora-not adenoviral vectors or recombinant Sendai virus tory studies, clinical trials have shown that this might [22] , was inhibited by normal mucus, with removal Extracellular barriers include the presence of infected mucus and sputum, mucociliary clearance and tight junctions between the cells, which limit the access of viral vectors to receptors localised on the basolateral membrane. Alternatively, gene transfer agents could be delivered intravenously (I / V route), even if it is unlikely that airway epithelial cells will be targeted. Inflammation, CTL-mediated degradation of transduced cells and neutralizing antibodies can further limit transgene expression or re-administration of the gene transfer vectors. Hidden stem or progenitor cells could be targeted by lentiviral vectors. Once inside the cell, the genetic material has to overcome endosome and cytoplasmic degradation and to get into the nucleus. CE, ciliated cell; B, basal cell; G, goblet cell; TJ, tight junctions; C, capillary; SMG, submucosal gland.

of this layer significantly increasing transgene ex-nant human DNase has been shown to reduce sputum pression [23] . Similar results were obtained when viscoelasticity, facilitate the transport of nanospheres cationic polymers, such as PEI, were used. However, though CF sputum [20] and, when mixed with we have recently shown that the mucus barrier can, lipoplexes or polyplexes, not to alter their ability to in part, be overcome by treatment with mucolytic mediate gene delivery [28] . agents, such as Nacystelyn, when either a cationic Recent findings have suggested that the glycocalyx lipid (GL-67 or EDMPC:Chol) or a cationic polymer may also represent another major obstacle to gene (PEI) are used to deliver genes to airway epithelium transfer to the airway epithelium. The glycocalyx is [24] .

composed of many carbohydrate-bearing structures, Similarly, sputum and bronchoalveolar lavage including glycoproteins, glycolipids and proteoglyfluid (BALF) recovered from CF patients have been cans. Treatment with agents to remove components shown to inhibit liposome- [25] , adenovirus- [26] of the glycocalyx, such as neuroaminidase, has been and AAV-mediated gene transfer efficiency [27] .

shown to enhance the susceptibility of polarised cells However, when CF sputum was treated with recom-to transduction by Ad or AAV vectors [29] . binant human DNase, an increased liposome-me-Attempts have been made to circumvent barriers at diated gene delivery was observed [25] . Recombi-the mucosal surface by delivering transgenes via the bloodstream. The intravenous route may make it 3 . Cell surface barriers possible to access the basolateral membrane of airway epithelial cells, characterised by a higher rate

The early studies with model systems that emof endocytosis and an increased density of viral ployed poorly differentiated airway epithelial cells receptors. However, the difficulties in overcoming suggested that gene transfer efficiency for a variety the large number of barriers between the vascular of vectors would be high. However, with the advent compartment and airway epithelial cells (endothelial of well-differentiated culture systems it became clear cells, endothelial cell basement membrane, inter-that (i) the airway lumen-facing columnar cell, the stitium and epithelial basement membrane) make this predominant cell type that must be transduced in route challenging. A large number of studies have vivo, is relatively resistant to viral and nonviral gene been conducted to identify which cells in the lung transfer and (ii) the apical surfaces of well-differenare transduced after intravenous (i.v.) injection of tiated airway epithelial cells have a low basal and liposome-DNA complexes. Most of these have stimulated rate of endocytosis. In contrast, currently concluded that the majority of the cells transfected available cell cultures are composed of basal or are the pulmonary endothelial cells, with some poorly differentiated cells that are highly transducstudies reporting transfection of type I / II alveolar ible, but do not mirror the airway lumen in vivo [35] . epithelial cells [30] . Only a few studies have reported airway epithelial cell transfection after i.v.

3 .1. Both synthetic and viral vectors are unable to injection of cationic lipid-DNA complexes [31, 32] . decreased progressively as the cells became more This may have been due to properties of this specific polarised and differentiated [36] . Similar reports formulation (such as a longer circulation time) since demonstrated that much of the reduction in gene similar results were not obtained when other GTAs delivery was due to a decrease in binding of the such as DOTAP-DOPE, DC-Chol-DOPE. GL-67A cationic lipid to the surface of mature airway epiand 22 kDa PEI were administered [32] .

thelial cells [37] and also to a decline in the rate of Independently from the route of administration, internalisation of the bound complexes into these innate immune defence mechanisms and pre-existing cells [38] . The hypothesis is that reduction in binding immunity are likely to play a crucial role in defend-may be due to differences in surface charge between ing the lung from foreign particles, including gene poorly and well-differentiated cells. While poorly therapy materials. Airway macrophages are known to differentiated cells exhibited receptor-mediated endoreduce the amount of GTA able to reach the epitheli-cytosis, pinocytosis and phagocytosis, well-differenal cells either via a direct mechanism (phagocytosis) tiated cell were only capable of receptor-mediated or, since they are antigen presenting cells, through endocytosis [37] . Some reports have indicated that stimulation of the host immune system. Pre-existing cell proliferation may influence cationic lipid-meimmunity from antibodies to wild-type viruses, such diated transfection activity, either by making epias adenovirus and AAV, could hinder their ability to thelial cells themselves more susceptible to transfectarget epithelial cells efficiently. A survey of normal tion or by temporary disruption of the tight junctions and CF subjects has shown that virtually all subjects [38] . This would allow the lipoplexes to either access had antibodies to Ad5 and to AAV2 (the two most the basolateral surface of well-differentiated cells or used viral vectors so far), although only 55 and 32%, to access more immature, less differentiated cells. respectively, were neutralising antibodies [33] . It has With regard to viral vectors, it has been demonalso been observed that individuals with a higher strated that the receptors for adenovirus [39], AAV-2 baseline anti-Ad neutralising antibody titre mounted [40] and retrovirus are localised to the basolateral a higher neutralising antibody response after vector membrane, and are therefore not accessible when administration [34] .

these vectors are delivered topically.

first examples reported is the P2Y -purinoreceptor, 2 which is highly expressed on the apical surface of In principle, four general strategies may improve epithelial cells and is stimulated to internalise upon gene transfer efficiency to airway epithelia.

UTP activation. An increase in gene transfer efficiency was observed when adenoviral vectors 3 .2.1. Tight junctions conjugated to biotinylated UTP ligands were used to The barrier function of epithelial tight junctions target the endogenous P2Y -purinoreceptor on well-2 can be transiently disrupted so that vectors can differentiated human airway epithelial cells, known access the basolateral membrane of target cells, to be refractory to adenovirus-mediated gene transfer which are rich in viral receptors and have a higher [51] . Similar results were obtained when GTAs were rate of endocytosis. This can be achieved by using retargeted to bind to the bradykinin receptor [52] , the 21 Ca chelator agents, such as EGTA [41, 42] , non-urokinase plasminogen activator receptor [53] and ionic detergents, such as polidocanol [43] , and the serpin enzyme complex receptor (sec-R), all of antibodies able to block the function of proteins which are expressed on the apical surface of airway involved in the tight junction-complex, such as E-epithelial cells. Administration of plasmid DNA cadherin [44] . In addition, medium-chain fatty acids, carrying the CFTR cDNA condensed with poly-Lsuch as sodium caprate, have been shown to be lysine sec-R ligand to CF mice produced a correction 2 better agents for enhancing gene transfer than of the Cl efflux, a more normal sodium channel EGTA, via disruption of claudin-1, a major structural activity and a reversal of nitric oxide synthase-2 component of the tight junctions [45] . Croyle et al.

downregulation [54] . recently found that a blend of sucrose, mannitol and Investigators are currently trying to characterise Pluronic F68 enhances adenoviral-mediated gene new ligands to target receptors present on the apical expression in both large and small lung airways. This surface of airway epithelia. One method is to use a formulation was shown to increase tight junction phage display library screened for peptides binding permeability and allow the use of 1 / 2 log lower viral with high affinity to airway epithelial cells. This has dose [46] . Airway instillation of perfluorochemical been mainly done in vitro and several promising (PFC) liquid can also transiently open tight junc-candidates have already been identified [55] , but tions, enhancing adenoviral-[47], AAV [48] and attempts to identify more relevant molecules by in cationic lipid-mediated gene transfer [49], although vivo bio-panning are currently under evaluation. this procedure can increase inflammation.

An attractive alternative strategy is to evaluate systematically glycoproteins from other enveloped 3 .2.2. Modification of the host viruses for their ability to efficiently infect airway The apical membrane can be modified so that it epithelia and to use these glycoproteins to pseudobinds vectors. One approach has been to incorporate type recombinant viral vectors. Some viruses have unnatural sugars into membrane glycoproteins and already been shown to infect the polarised airway use them as a molecular handle on which a novel epithelium from the apical surface, including respirareceptor is constructed. The artificial receptor en-tory pathogens, such as human coronavirus 229E hanced adenoviral binding and gene transfer to cells [56] , H1N1, H2N2 and H3N2 influenza A virus that were normally relatively resistant to adenovirus strains [57] and viruses from the Filoviridae family infection [50] . This suggests the feasibility of a gene such as Marburg and Ebola virus. By using an transfer strategy in which the biosynthetic machinery Ebola-pseudotyped HIV vector, Kobinger et al. of the cell is used to engineer novel receptors on the showed efficient and stable transduction of intact cell surface.

airway epithelium from the apical surface in vivo [58] .

The modification of the vector can also be non-The GTA can be modified in order to target a specific. Given the low density of Ad-receptors on receptor on the apical membrane that has the capaci-the apical membrane, several strategies to increase ty to both bind and internalise a vector. One of the cell binding via a non-fibre-dependent pathway have been developed. Complexing Ad vectors with poly-using fusogenic lipids or peptides, such as GALA cations [59] or incorporating Ad in calcium phos-and HA-2 from influenza haemagglutinin, to disrupt phate precipitates [60] have been shown to enhance the endosome membrane. The neutral lipid dioleoylgene transfer to airway epithelia in vitro and in vivo.

phosphatidylethanolamine (DOPE) is generally em-These strategies are thought to neutralise the adverse ployed as a fusogenic helper lipid in a cationic charge interaction between negatively charged Ad lipid-DNA complex. The second approach involves particles and the negatively charged cell surface, using a DNA delivery system with a high buffering resulting in improved binding and uptake of Ad capacity and the flexibility to swell when protonated. vector.

The rationale that endosomes can be ruptured if the Furthermore, it has been reported that both syn-pH drop in the late endosome is inhibited by the thetic [22] and viral vector [61] mediated gene buffering capacity of the formulation led to the use transfer to differentiated airway epithelia can be of pH-sensitive liposomes and polyethylenimine as increased by a prolonged contact time.

gene transfer agents [64] . Because of the great number of secondary amines, PEI behaves as a 3 .2.4. Identification of new viral vectors 'proton sponge', able to buffer the low pH in the Improved binding and entry from the apical endosomes, resulting in the inhibition of the low surface have been reported with both new viral pH-activated nucleases. This leads to a large increase vectors and new serotypes of existing vectors. It has in the ionic concentration inside the endosome, recently been shown that, in contrast to AAV 2, AAV finally resulting in osmotic swelling due to water serotype 5 is able to infect human airway epithelia entry and rupture of the organelle [65] . from the lumenal surface, suggesting that 2,3-linked In contrast to plasmid DNA, viral vectors have sialic acid is either a receptor for AAV 5 or a evolved mechanisms to escape endosome degradanecessary component of a receptor complex [62] .

tion and for some of them it is a prerequisite for Recombinant Sendai virus, a single-stranded RNA subsequent nuclear localisation of the virions. Hanparamyxovirus, is also able to infect airway epitheli-sen et al. have shown that the passage through the al cells from the lumenal surface. Its envelope endosome acidic component exposes AAV to conproteins, F (fusion) and HN (haemagglutinin), have ditions that modify the capsid, allowing the virus to been shown to interact with cholesterol and sialic use the cytoskeleton for subsequent trafficking events acid, respectively, both molecules known to be to the nucleus [66] . Sendai virus uses the F protein to present on the apical membrane of airway epithelial fuse its envelope with the cell plasma membrane, cells [22] . Similarly, a recombinant respiratory allowing the genetic material to be released directly syncytial virus has recently been developed because into the cytoplasm and thus avoiding endosome of its ability to infect ciliated cells via the lumenal degradation. This led to the use of HVJ-liposome as membrane [63] .

a new gene transfer agent in which UV-inactivated SeV particles are mixed together with lipids in a liposome formulation in order to allow the plasmid 4 . Intracellular barriers DNA to be introduced into the cytoplasm of the transfected cell [67] . Viral and nonviral vectors enter cells through endocytosis, a normal process for the internalisation 4 .2. Cytoplasm-based degradation pathways and degradation of extracellular material. Viral vectors and some synthetic vectors, such as 4 .1. Endosome PEI and HVJ-liposome, are characterised by the ability to escape endosome degradation. However, Plasmid-based vectors are quite susceptible to once released in the cytoplasm and before entering endosomal degradation and attempts have been made the nucleus, other hurdles are encountered. Synthetic to enhance transgene escape from the endosome-vector-based strategies are characterised by low gene lysosome pathway (Fig. 1) . One approach involves transfer efficiency because plasmid DNA is quickly 21 degraded by Ca -sensitive cytosolic nucleases, with Several approaches have been taken to improve an apparent half-life of 50-90 min [68, 69] . Duan et nuclear entry of pDNA, including the electrostatic al. showed that in polarised epithelial cells AAV binding of pDNA to NLS-containing proteins, such capsid is ubiquinated after endocytosis and that this as HMG-1, and the covalent attachment of NLSprocess is a barrier to rAAV transduction. motifs to double-stranded DNA. With regard to the Proteasome-dependent degradation of ubiquinated latest approach, most of the studies published so far molecules represents a major pathway for disposal of have used the NLS of the simian virus 40 (SV40) both endogenous and foreign proteins. In vivo appli-large T antigen because its trafficking to the nucleus cation of proteasome inhibitors in mouse lungs is well characterised and has also shown to mediate augmented rAAV-mediated gene transfer from unde-nuclear import of non-karyophilic proteins. By using tectable levels to a mean of 10.461.6% of the this 'piggyback' approach, Sebestyen et al. demonepithelial cells in large bronchioles [70] . strated nuclear accumulation in digitonin-permeabilised cells, but failed to show uptake of the modified 4 .3. Nuclear import DNA in nuclei of intact cells. This suggests that covalent modification of pDNA with a signal peptide Once within the cytoplasm, the transgene must be may alter its behaviour and interaction with other imported into the nucleus to be transcribed. For cellular factors [73] . By capping a luciferase gene plasmid-based expression, nuclear import is a rate-with a single NLS peptide, Zanta et al. showed a limiting step and intracellular trafficking of pDNA, 10-1000-fold increase in gene transfer efficiency either naked or complexed to synthetic vectors, is [74] . However, the major drawback of this technique largely uncharacterised (Fig. 1) . During non-viral is the relatively low amount of NLS-pDNA that can gene transfer, entry of exogenous DNA into the be produced. nucleus occurs only in cells that are actively divid-

The development of peptide nucleic acids (PNAs), ing, i.e., when the nuclear envelope breaks down.

oligonucleotide analogues in which the sugar phos-This is consistent with the observation that well-phate backbone of nucleic acid has been replaced by differentiated, non-dividing airway epithelial cells a synthetic peptide backbone, has led to further show very low transfection efficiency. Pollard et al. developments in this field. PNA is capable of showed that less than 1 / 1000 naked cDNA copies sequence-specific recognition of DNA and RNA microinjected in the cytoplasm were effectively following the Watson-Crick hydrogen-bonding trafficked to the nucleus [71] .

scheme and can be used to link peptides, such as These results may be largely due to the inability of NLS-motifs, to plasmid DNA. The advantage over pDNA to effectively translocate through the nuclear the other strategies is that the NLS peptide can be pore complexes (NPCs). Each NPC is comprised of a linked to very specific regions of plasmid DNA, with large family of proteins (nucleoporins) forming a no effects on the transcription of genes located structure in which a central channel is surrounded by elsewhere on the plasmid. Furthermore, a precise eight peripheral channels. It is thought that the number of NLS-motifs can be attached and large peripheral channels, about 9-10 nm diameter, allow quantities of the modified pDNA obtained [75] . small solutes and proteins up to 50-60 kDa to freely However, up to now, none of the strategies described diffuse in an out the nucleus. Larger proteins need a has been used to enhance nuclear import and gene nuclear localisation signal (NLS) in order to be transfer expression in airway epithelial cells in vivo. actively transported through the central channel of

In addition to this, there are reports suggesting that the pore. Under physiological conditions, a super-some polycations, such as PEI [71] and lactosylated coiled pDNA has a diameter of 10 nm or larger if in poly-L-lysine [76] , may facilitate the nuclear uptake a relaxed conformation, thus suggesting that passive of pDNA and that, unlike lipoplexes, the complexes diffusion of pDNA through the NPC is highly remain intact during nuclear translocation. This unlikely [72] . Therefore, targeting of pDNA to might suggest the existence of nuclear import pathnuclear 'shuttle' proteins has become part of the ways distinct from the conventional NLS one. design of GTAs developed to transfer non-dividing Unlike plasmid-based approaches, viral vectors cells, as in CF gene therapy.

have evolved efficient ways to enter the nucleus. The cytoplasmic movement of viral DNA towards the directed at adenoviral capsid proteins present during nucleus is facilitated by the interaction of viral vector delivery, resulting in the production of neuproteins, such as polymerase or capsid proteins, with tralising antibodies (NAB) that limit repeated vector the microtubular network [77] . It has recently been administration. shown that adenovirus type 2 docks at the CAN / Several approaches have been taken to reduce Nup214 protein of the nuclear pore, then hijacks expression-limiting immune responses in recipients histone H1 and specific H1-import receptors to effect of virus-based gene therapy. a targeted uncoating of its nucleocapsid at the (i) Immunosuppressant drugs such as cyclophosnuclear pore. Consequently, the viral DNA is liber-phamide, cyclosporine and FK506 have been reated near the opening of the pore and positioned for ported to prolong transgene expression and facilitate translocation into the nucleus [78] .

repeated gene transfer in the lung [81] . However, the For most of the viral vectors, but not retroviruses, potential to induce severe systemic side effects may nuclear import does not depend on the mitotic status limit the clinical application of these drugs. Furtherof the cell. It has recently been reported that a more, prolonged general immunosuppression of CF polypurine tract (cPPT) present in the HIV-1 genome patients, where lungs are colonised by pathogenic leads to the formation of a triple-stranded DNA bacteria, could be injurious. structure, the HIV-1 flap, which is recognised by the (ii) Topical corticosteroids, such as budesonide, nuclear import machinery of the host cell. This have allowed successful re-administration of adenoultimately results in the transport of the HIV-1 virus, at least twice. Compared to saline-treated genome into the nucleus across the nuclear pore animals, budesonide treatment resulted in significomplex [79] . Introduction of the cPPT sequence in cantly higher transgene expression and lower recombinant lentiviral vectors has been shown to amounts of NAB both in BALF and serum. Howenhance nuclear import and, therefore, transgene ever, these differences disappeared after five previexpression [80] . For other viruses, such as Sendai ous exposures to the virus [82] . virus, nuclear import is not a rate-limiting step since (iii) Co-administration of interferon-g (IFN-g) or replication and transcription both occur in the cyto-interleukin-12 (IL-12) has been shown to diminish plasm.

the activity of T cells and formation of NAB, H2 allowing efficient re-administration (at least once) of recombinant virus [83] . A potential drawback of this 5 . Host immune responses approach is that T cells are inhibited at the H2 expense of increased T activation. Thus, both IFN-H1 As transgene expression is transient, and unless g and IL-12, while capable of inhibiting humoral lung-resident stem cells are targeted, the treatment of immunity, might enhance the elimination of adeno-CF by gene therapy will require repeated administra-virus-transduced cells by CTLs. tions throughout the lifetime of the patient. The (iv) Both CTL and B cell responses require 

prevent the activation of humoral and cellular imbe overcome in order to re-administer the vector. mune response to viruses, thus causing prolonged These include: (i) an antigen-nonspecific, cytokine-transgene expression and efficient re-administration. dependent response resulting in acute inflammation;

Several strategies have been adopted including the 1 (ii) a cytotoxic (CD8 ) T-lymphocyte (CTL)-depen-use of non-depleting monoclonal antibodies to the dent response directed at cells expressing viral or CD4 molecule [84] and the blockade of either CD40transgene proteins, resulting in chronic inflammation CD40 ligand [85] or CD28-B7 (with CTLA4-Ig) and lack of persistent transgene expression; and (iii) [86] , co-stimulatory signals necessary for complete 1 a helper (CD4 ) T lymphocyte-dependent response T-cell activation. These treatments have resulted in suppression of cellular and humoral responses, pro-shortened version of the CFTR gene ('mini-gene') longed transgene expression and, in some cases, [96] . Another approach is to expand rAAV packaging vector re-administration up to four times [84] . How-capacity with trans-splicing or overlapping vectors. ever, in non-human primates, treatment with an anti-

The first reconstitutes gene expression from two CD40 ligand monoclonal antibody did not prevent independent rAAV vectors, each encoding unique, the elicitation of a virus-specific antibody response non-overlapping halves of a transgene. The overlapupon secondary challenge with vector [87] . It is ping vector approach uses homologous recombinapossible that a combination of blocking agents may tion between overlapping regions in two independent provide a more complete abrogation of T-and B-cell vectors [97] . Preliminary results seem to indicate that immune responses.

the trans-splicing approach is 4-10-fold more effi-(v) Antibody neutralisation of the virus can be cient than the overlapping approach [98] . reduced by coating the vector particle with polyethylene glycol (PEG) [88] , GL67-DOPE-PEG [89] 5 .2. Plasmid-based delivery and poly-L-lysine or DEAE-dextran [59] . These treatments have the added advantage of providing a Plasmid-based gene delivery strategies are genermeans to retarget the vector through ligands coupled ally regarded as safer and less immunogenic alterto the coating polymer. Another potential strategy for natives to viral vectors. Repeated administration of effective repeated delivery is 'serotype switching' lipoplexes in mice [99] and CF patients [17] resulted where gene therapy is initiated with one virus in similar levels of transgene expression as observed serotype, then switched to a virus derived from a after a single delivery, thus suggesting that lipoplexsecond serotype for subsequent administration, there-es can be re-administered without apparent loss of by avoiding neutralising antibodies induced by the efficacy. However, the efficacy of repeated adminisfirst serotype. However, transgene expression may be trations is dependent on the dose used and the time limited by cross-reactive CTLs that can also target interval between administrations. Lee et al. reported cells infected by the second virus serotype [90] .

that very little or no loss in efficacy was observed If the use of these strategies were to be combined provided the dose of lipoplexes was low or if the with adenovirus vectors that are devoid of all viral time interval between successive instillations was sequences ('gutless' or helper-dependent adenoviral sufficiently long [100] . It appears that the inflammavectors), thereby avoiding a cell-mediated immune tion elicited by the complexes may affect the efficacy response, it might be possible to repeatedly deliver of repeat administration. the virus. The use of these 'stealth' adenoviruses Non-viral gene delivery agents can indeed have could eliminate the requirement for systemic im-inflammatory and toxic effects in vivo. Scheule et al. munosuppression with repeated administration [91] . observed a dose-dependent pulmonary inflammation Recombinant AAV (rAAV) vectors are generally characterised by infiltrates of neutrophils and, to a less immunogenic in the airways and Beck et al.

lesser extent, macrophages and lymphocytes when have recently suggested that AAV can evade immu-the cationic lipid GL-67 was administered to mouse nological surveillance and can be repeatedly de-lungs in vivo. Associated with this were elevated livered to rabbit airways, because they are unable to levels of the pro-inflammatory cytokines IL-6, TNFtransduce antigen presenting cells (dendritic cells) a and IFN-g that peaked at day 1-2 post-instillation, [92] . However, this possibility has recently been but resolved to normal limits by day 14 [101] . ruled out unless different serotypes [93] or some Histopathological analysis of lung sections from form of immunosuppression [94, 95] is used. Further-mice treated with the individual components of the more, the small packaging size limit of the virion has lipoplex suggested that the cationic lipid was the restricted the use of genetic control elements that can major mediator of the observed inflammation. Howdrive CFTR expression compared to the relatively ever, results of clinical studies in which CF patients weak promoter activity of the native viral inverted were subjected to either aerosolised liposomes alone terminal repeat (ITR) sequence. Recent progress to [102] or cationic lipid-pDNA complexes indicated overcome this problem has included the use of a that bacterially derived pDNA may also be inflam-matory [16, 19] . Each of the cationic lipid-pDNA-tion, Scaria et al. showed that by using an adenovirus treated patients, but not the liposome-treated con-co-expressing both human CFTR and ICP47 (a gene trols, exhibited mild flu-like symptoms (including shown to block the transporter associated with MHC 1 fever, myalgia and a reduction in FEV of approxi-class I-mediated antigen presentation to CD8 T 1 mately 15%) over a period of 24 h. cells) a prolonged expression (up to 21 days) was One possible explanation for this response may be observed in monkey lungs, even though natural killer related to the presence of unmethylated CpG di-cell activity was enhanced [107] . nucleotide sequences in bacterially derived pDNA.

However, several lines of evidence suggest that Compared with DNA of eukaryotic origin, bacterial attenuation of promoter function may be the most genomic DNA contains a 20-fold higher frequency significant factor in the lack of persistence of transof the dinucleotide sequence CpG. Further, unlike gene expression. The transcriptional activity of the eukaryotic DNA, in which approximately 80% of the widely used cytomegalovirus immediate early gene cytosines are methylated, bacterial DNA is relatively promoter (CMV) is highly robust, but prone to unmethylated. Instillation of bacterial DNA or oligo-inactivation over time. Cytokines induced by adenonucleotides containing immunostimulatory CpG virus-or plasmid-mediated gene delivery have been motifs into mouse lungs resulted in inflammation of shown to down-regulate CMV-driven expression. For the lower respiratory tract [103] . Several strategies this reason, many investigators have evaluated alterhave been employed to decrease the immuno-native promoters, with many showing increased stimulatory properties of pDNA, including (i) meth-persistence. These include the polyubiquitin C proylation of CpG sequences [104] , (ii) reduction of the moter (high-level transgene expression for up to 8 CpG frequency by eliminating non-essential regions weeks and still detectable after 6 months), the or by site-directed mutagenesis [105] and (iii) the elongation factor 1a promoter (expression up to 4 use of inhibitors of the CpG signalling pathway, such weeks) [108] and the CMV-ubiquitin B hybrid as chloroquine or quinacrine [105] . Independent of promoter (expression up to 3 months, with 50% of the strategy used, the CpG-reduced pDNAs were day 2 levels remaining at day 84) [109] . Similar found to be less pro-inflammatory. However, meth-prolonged transgene expression was obtained when ylation of the CpG motifs can severely reduce the the E4 region from adenovirus 2 or simply the open expression of the transgene [104] .

reading frame 3 (ORF3) of E4 were cloned upstream of the CMV promoter on a plasmid backbone [110] . However, a potential disadvantage of this approach 6 . Expression of the therapeutic gene is the immunogenicity of the E4 ORF3 product once expressed in the transfected cell, thereby limiting its One of the main obstacles to the development of usefulness in vivo. In addition, there is growing gene therapy for the airways is the inability of evidence that genomic sequences, either within or current viral and nonviral gene transfer vectors to flanking the gene, might be essential to provide in direct sustained expression of a therapeutic trans-vivo long-term expression [111] . gene. This may be due to several causes including An alternative approach to achieve prolonged loss of the vector (especially if present in an episom-transgene expression is to use artificial chromosome al form), transcriptional silencing of the transgene vectors or integrating viruses, since they are stable promoter, loss of the transfected cell through cell over time and will propagate to daughter cells should turnover, or the generation of an immune response to cell division occur. Huertas et al. have recently the transgene product or the transfected cell itself.

developed a circular yeast artificial chromosome Several approaches have therefore been taken to (YAC) carrying the human CFTR sequence and the achieve longer-term expression following each gene oriP and EBNA-1 genes from Epstein-Barr (EBV) transfer treatment. As outlined in Section 5.1, im-virus [112] . Plasmids carrying these two EBV genes munosuppressant drugs have been reported to have been shown to allow long-term episomal prolong transgene expression [106] , because of their maintenance of the DNA, being able to replicate and ability to block T cell-mediated response. In addi-segregate in the daughter cells. However, unless lung-resident stem cells are targeted, these vectors integrated in the host genome will eventually be lost are unlikely to have greater advantages over conven-due to cell turnover. tional plasmids, since the airway epithelium is An alternative would be to target lung resident mainly composed of non-dividing cells. Furthermore, stem cells so that the integrated transgene is continutheir size makes in vivo delivery and subsequent ously propagated to the daughter cells when cell nuclear trafficking quite difficult. division occurs. There has been a long-standing With regard to integrating viruses, AAV and, more debate regarding the identity of airway epithelial recently, lentiviruses have been considered as good stem cells and two theoretical models of cell lineage candidates for prolonged expression. Wild-type AAV in the pseudostratified airway epithelium have been persists by site-specific integration into human chro-suggested. The 'stem cell niche' model suggests that mosome 19. However, recombinant AAVs persist airway epithelial stem cells are localised to distinct predominantly in an episomal form and integrate and potentially inaccessible compartments of the randomly in the host genome at a much lower lung. However, there is evidence for great plasticity frequency than wild-type AAV, probably because of in growth and differentiation potential of airway the lack of rep gene products [113] . Furthermore, epithelial cells and earlier studies showed that both results demonstrate that, at least in the liver extra-basal and non-basal cells could regenerate a comchromosomal, not integrated genomes, are the pri-plete mucociliary epithelium in tracheal grafts. This mary source of rAAV-mediated gene expression observation led to an alternative 'unlimited plastici- [114] .

ty' model where many non-terminal cells with ample Lentiviral vectors from different strains including progenitorial capacity are scattered throughout the human, feline and equine immunodeficiency virus epithelium [115] . A better understanding of this issue have been used to transduce airway epithelial cells is also likely to benefit any gene therapy strategy because of their potential to provide long-term using integrating vectors, considering the different expression through the integration of the provirus accessibility of stem cells in the two models. into the host cell genome. Kobinger et al. recently used an HIV-based vector pseudotyped with the envelope from the Ebola (EboZ) virus to efficiently 7 . How many and which cells should be transduce airway epithelia in vivo. Animals receiving corrected to achieve clinical benefit? EboZ-pseudotyped HIV vector demonstrated minimal expression at day 7, but strong expression in the A key issue is to distinguish between two conairway epithelium, including submucosal glands, by cepts: 'percent of cells corrected' and 'level of CFTR day 28 that persisted at day 63. On average, 30% of transduced / cell'. the entire tracheal epithelium was transduced by the vector at day 28 and 24% at day 63 [58] . 7 .1. Percent of cells corrected Despite these results, integrating vectors still have some problems. The first is that chromosomal posi-An in vitro study has shown that approximately tion and structure can negatively affect transgene 6-10% of 'corrected' cells are needed to restore 2 expression, thus leading to host shut-off of the normal Cl transport function [116] . The amplifica-2 transferred expression cassette, a problem that has tion of functional correction reflects the fact that Cl plagued retrovirus gene transfer vectors. This effect, can move via gap junctions from non-corrected cells known as transcriptional silencing, can be mitigated into adjacent corrected cells for secretion. An in vivo through the use of chromatin insulators, which are study has shown that 5% of the normal level of Cftr protein-binding DNA elements that lack intrinsic gene expression can correct the chloride abnormality promoter / enhancer activity, but shelter genes from (50% of normal) and, importantly, the intestinal transcriptional influence of surrounding chromatin.

pathology seen in mice with CF [117] . However, The second problem is that the majority of the different levels of correction may be required to epithelium is comprised of differentiated cells that restore the various functions of CFTR. The relationare replaced every few months, so that a transgene ship between efficiency of gene transfer and normali-sation of sodium transport is likely to be linear, gene, will have to be used to drive CFTR expression. reflecting the fact that CFTR directly regulates ENaC

To produce more physiological CFTR expression channels within individual cells. This suggests that other approaches such as the use of (i) genomic virtually every affected cell (100%) should be cor-context vectors in which CFTR expression is driven rected [116] . The level of transfection required for by its natural promoter and regulatory sequences and other functions of CFTR to be restored, e.g., sulpha-(ii) 'gene targeting' molecules, such as RNA-DNA tion / sialylation defects or transport of other mole-chimeric oligos and small DNA fragments for cules, remains unknown and appears to depend on homologous replacement may be needed. This latter the cell type transduced and the GTA used. Zhang et strategy would allow the mutation within the CFTR al. showed that cationic liposome-mediated CFTR gene to be 'surgically' modified without altering the transfer achieved very low transgene expression with promoter and the regulatory sequences of the CFTR insignificant correction of the chloride defect, but gene. mucus sulphation was reduced to levels seen in non-CF airways. The converse was seen with adenovirus that, despite higher levels of expression, did not 8 . Non-conventional approaches transduce goblet cells [118] . In addition, the route of administration itself seems to have a role with regard Because of the hurdles encountered by GTAs in to the localisation of the delivered gene. Instillation getting into airway epithelial cells, many less conof GTAs into the mouse lungs results predominantly ventional strategies than those described above have in transfection of the alveolar and terminal bronchial been developed and will be reviewed here. cells, while, when they are aerosolised, a higher level of transfection is observed in the airway epithelium 8 .1. Oligonucleotide-mediated strategies [119] . Aerosolisation is more likely to lead to a more even deposition of the GTAs throughout the lung Because of their size, oligonucleotides have the than could be attained by instillation, which pre-potential to enter the cell and the nucleus much more sumably primarily deposits the complexes in the easily than plasmid DNA. Goncz et al. have deparenchyma.

veloped a new strategy based on gene targeting by small fragment homologous replacement (SFHR).

Specific genomic sequences are targeted with small fragments of exogenous DNA (400-800 bp) that are A delivered gene would ideally be expressed in a homologous to the targeted endogenous DNA semanner similar to the normal pattern of the defective quences except for the particular base pairs that gene that it is replacing. To counterbalance the poor encode the desired modification. Gene targeting is gene transfer efficiency with current GTAs, very thought to have several advantages over classic gene strong non-specific viral promoters such as RSV and complementation, including long-term and tissue CMV have been used to drive transgene expression. specific expression of the functional gene, no intro-However, because the number of CFTR molecules duction of foreign sequences and no immune reper respiratory epithelial cell is low (20-100 channel sponse. For the first time, Goncz et al. showed the proteins / cell) and that the CFTR protein regulates modification of specific genomic sequences in exon other ionic channels implicated in water and salt 10 of the mouse CFTR after small DNA fragments secretion, it is possible that high levels of CFTR were delivered to the lungs of normal mice [122] . expression may perturb the function of other proteins

Recently, conversion of wild-type CFTR to the or alter physiological properties of the cell. It has G551D mutation in primary rat hepatocytes has been been reported that a high level of CFTR expression reported by using a different molecule, RNA-DNA can cause growth arrest and increased cell volume chimeraplasts [123] . [120, 121] , thus suggesting that either regulatable Another strategy is to use oligonucleotides as expression cassettes [121] or epithelium-specific antisense molecules. Friedman et al. used antisense promoters, such as that for the human cytokeratin 18 oligoribonucleotides to correct the CFTR splicing mutation 3849 1 10 kb C → T in human and mouse uptake. The feasibility of these methods is very epithelial cells [124] . In another report, antisense organ-and tissue-specific and for the lung represents inhibition of B cell antigen receptor-associated pro-a challenge with many unknown aspects. Gersting activated Cl currents in [DPhe ]CFTR-expressing fected with plasmid DNA mixed with superparamag-CHO cells [125] . Antisense strategy might suggest a netic nanoparticles (magnetofection) resulted in more new way to correct the defects present in CF, such as than 100-fold increase in gene transfer [128] . The mucin production or sodium hyperabsorption.

challenge is now to see whether this very promising technique [129] can be applied to the airway epi-8 .2. Spliceosome-mediated RNA trans-splicing thelium in vivo. (SMaRT) 8 .4 . In utero gene transfer A very recent technology developed by Mitchell and collaborators takes advantage of the cell's Because of the hurdles and barriers involved with endogenous splicing machinery as a strategy for conventional gene transfer, several groups have modifying pre-mRNA. The SMaRT process uses begun to look into in utero gene delivery as a new pre-therapeutic RNA molecules (PTMs) that are way potentially to increase the efficacy and duration designed to base pair with the intron of a targeted of transgene expression. CF is a particularly inviting pre-mRNA to suppress target cis-splicing while target disease for the development of in utero gene enhancing trans-splicing between the PTM and therapy because amniotic fluid circulation provides target. The aim is to repair mutant pre-mRNA vector exposure to pulmonary, gastrointestinal and molecules and generate full length repaired mRNA sinus epithelia, all primary sites of CF pathology. that is translated and processed into mature CFTR Viral vector introduced into amniotic fluid of mice, protein [126] . In an in vivo model of DF508 CF rats, sheep and rabbits results in reporter gene airway epithelia, Liu et al. showed that human CF expression in both pulmonary and gastrointestinal bronchial xenografts infected with a recombinant epithelia. Transgene expression was also demonadenovirus encoding a PTM targeted to CFTR intron strated in pulmonary epithelia after intratracheal 9 demonstrated partial correction of CFTR-mediated instillation of vector in utero. Persistence of trans-2 Cl permeability to 22% of that seen in non-CF gene expression in the lung ranged from 14 to 30 xenograft [127] . This strategy would allow a more days post-infection. Most of these studies have been physiological expression of CFTR, as previously carried out with adenoviral vectors, and many have discussed. Furthermore, as PTM expression cassettes led to substantial inflammation and subsequent foetal can be much smaller than those encoding full-length loss. Fewer adverse events have been observed when cDNAs, SMaRT also allows for the use of smaller AAV is used. Retroviral vector use for applications and less immunogenic vectors, such as rAAV with in utero has been limited because the amniotic fluid limited packaging capacity. However, the very high reduces infectivity (see Ref. [130] for an extended titres required to achieve correction and the potential overview). of trans-splicing into non-CFTR mRNAs are dis-

The potential advantage of in utero gene transfer advantages which should not be ignored. over other approaches is that, as the host immune system is not fully developed, it may be possible to 8 .3. Physical methods tolerise the organism to viral vectors. However, recent reports have ruled out the possibility of Because of the inefficiency of currently available successful repeated administration of viral vectors GTAs, newer ways of increasing gene transfer have during adult life despite previous in utero exposure to be developed. Physical methods such as magnet- [131] . In humans this approach would be even less ism, electroporation and ultrasound have been em-successful since the immune system becomes responployed by several groups as a means to enhance gene sive by midgestation.

Larson et al. have recently presented an interesting future a feasible option to detect gene expression in a and controversial report, showing that treatment of non-invasive way in human airways. primate foetuses with an adenovirus expressing the cftr gene resulted in accelerated differentiation of the lung [132] . Furthermore, after in utero gene transfer 1 0. Conclusion with an adenovirus containing the cftr gene, a permanent reversion of the lethal phenotype in CF

In conclusion, in the 13 years since the cloning of knockout mice was observed [133] . This might the CFTR gene and after about 20 clinical trials, suggest that CF is a developmental disease that could some of the crucial barriers limiting gene transfer be prevented by transient in utero cftr gene expreshave become clearer. The combination of newly sion at the proper time of lung and intestine differendeveloped GTAs and technologies, better models to tiation.

test gene-based therapies and new detection systems will hopefully allow these barriers to be overcome.

Clinical trials have shown that there is clearly a This work was supported by the Cystic Fibrosis requirement for newer approaches to improve deliv-Research Trust (SF) and a Wellcome Trust Senior ery and efficiency, increase duration of expression Clinical Fellowship (EWFWA). The authors are and permit repeated administration of GTAs. In members of the UK Cystic Fibrosis Gene Therapy addition there is an increasing need by the scientific Consortium (www.cfgenetherapy.org.uk). community that all these new GTAs and technologies are tested on relevant airway test systems (i.e., only highly differentiated epithelial cells in R eferences vitro and a spectrum of in vivo systems) before entering clinical trials. CF mice have been a great

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