Immunological hurdles to lung gene therapy

Abstract

Gene delivery has the potential to offer effective treatment to patients with life-threatening lung diseases such as cystic fibrosis, α₁-antitrypsin deficiency and lung cancer. Phase I/II clinical trials have shown that, in principle, gene transfer to the lung is feasible and safe. However, gene expression from both viral and non-viral gene delivery systems has been inefficient. In addition to extra- and intracellular barriers, the host innate and acquired immune system represents a major barrier to successful gene transfer to the lung. Results from studies in experimental animals and clinical trials have shown that inflammatory, antibody and T cell responses can limit transgene expression duration and readministration of the gene transfer vector. We will review here how the development of pharmacological and/or immunological agents can modulate the host immune system and the limitations of these strategies. A better understanding of the immunological barriers which exist in the lung might allow for a more sustained expression of the transgene and importantly help overcome the problem of readministration of viral vectors.

Introduction

In vivo delivery of exogenous genes to the lung has the potential to treat, and hopefully cure, diseases such as cystic fibrosis (CF), α₁-antytrypsin deficiency, lung cancer and adult respiratory distress syndrome (ARDS). Gene transfer can be achieved either using recombinant viruses carrying the therapeutic cDNA within their genome, or synthetic vectors complexed to plasmid DNA (lipoplexes and polyplexes). Proof-of-principle studies have shown that gene delivery and expression can correct the defect in diseased cells. However, results from preclinical and clinical studies suggest that current levels of gene transfer efficiency are too low to result in clinical benefit, largely as a result of the barriers faced by gene transfer vectors within the lung [1]. A complex series of extracellular barriers such as mucus, mucociliary clearance and glycocalyx proteins are known to limit the access of gene transfer agents to lung cells. In addition to this, immunological responses directed against the gene transfer agent or the transgene product are known to limit gene expression. Following gene transfer, inflammatory and cellular immune responses are known to limit the duration of gene expression over time, making repeated administrations of the gene transfer vector (GTA) necessary. Successful readministration of GTAs is limited by the formation of neutralizing antibodies (NABs). In this review we will analyse how immune responses limit the efficacy of gene therapy strategies and which solutions have been evaluated, either to increase gene expression duration or to allow for repeated administration.

Hurdles Encountered by Gene Transfer Agents In Vivo

When delivered to the lung in vivo, several hurdles are known to limit gene transfer efficiency and expression.

Innate immune response

The lung itself has evolved innate mechanisms of defence in order to limit effective access of foreign particles (including GTAs therefore) to the target cells. Alveolar macrophages can eliminate the GTAs either via a direct mechanism (phagocytosis) or, in their function as antigen-presenting cells (APCs), through stimulation of the host immune system. In both immunocompetent and immunodeficient mice, resident macrophages have been shown to clear 70% of adenovirus genomes within 24 h of administration [2]. When the lung is targeted by intravenous delivery of GTAs, positively charged lipoplexes and polyplexes are likely to lead to opsonization by complement components and eventually to complement-dependent phagocytosis by macrophages in the reticuloendothelial system [3].

Non-specific inflammation

Once delivered to the lung, both viral and non-viral vectors elicit an antigen-non-specific cytokine-dependent response resulting in acute inflammation. Host cytokines initiate the immune cascade and trigger the activation of B and T cells. Importantly, commonly used viral promoters such as CMV, RSV and SV40 are down-regulated by these cytokines (IFN-γ, TNF-α). Inhibition of gene expression is thought to be at the transcriptional level and cytokines have been shown not to cause vector DNA degradation or inhibit total cellular protein synthesis or to kill transduced cells [4].

Cellular immune response

Cytotoxic (CD8⁺) T-lymphocyte (CTL)-dependent responses directed at cells expressing viral or transgene proteins result in chronic inflammation, and are partly responsible for the lack of persistent transgene expression after each dose of GTA. This is particularly relevant to viral vectors, especially if they are replication-competent, because a constant source of protein is produced and associated with MHC class I molecules for antigen presentation. However, synthetic vectors based on molecular conjugates, in which immunogenic ligands/peptides are used, can lead to similar immune responses.

Humoral immune response

Helper (CD4⁺) T lymphocyte-dependent responses, directed at viral capsid proteins or ligand/peptide on molecular conjugates during vector delivery, result in the production of neutralizing antibodies that limit repeated vector administration. After the first administration NABs recognize the GTA and block its interaction with the target cells, thus causing a decreased transgene expression. This is a critical issue for gene therapy and many of the strategies described later have the readministration of GTAs as their main target.

In addition to the barriers described so far, pathologically abnormal lungs present disease-specific barriers which can limit gene transfer further. This has been studied extensively in the case of CF lungs. Virella-Lowell et al. showed that products of inflammation in CF bronchoalveolar lavage fluid, such as neutrophil elastase and neutrophil α-defensins, are inhibitory to adeno-associated virus (AAV) transduction but that these effects could be reversed by treatment with antiproteinases such as α⁻¹-antitrypsin [5]. Furthermore, the efficacy of adenovirus-mediated gene transfer was reduced in the lungs of a mouse model of bronchopulmonary inflammation induced by Pseudomonas aeruginosa, which is the main cause of infection in CF patients [6].

Pre-existing immunity from antibodies to wild-type viruses, such as adenovirus (Ad) and AAV, could also hinder the vector's ability to reach target cells efficiently. Anti-adenovirus antibodies, contained in the sputum of CF patients, have been shown to inhibit adenovirus vector-mediated transduction [7]. It has also been observed that individuals with a higher baseline anti-Ad neutralizing antibody titre mounted a higher neutralizing antibody response after vector administration [8]. A survey of normal and CF subjects has shown that virtually all subjects had antibodies to Ad5 and to AAV2 (two of the most commonly used vectors for gene therapy), although only 55% and 32%, respectively, were neutralizing antibodies [9].

Solutions to Problems Posed by Viral Vectors

Viral vectors are very efficient in mediating gene transfer and expression in vivo. However, duration of gene expression is limited by CTL-mediated destruction of transduced cells and repeated administration is not feasible partly because of the rise of NABs after the first delivery. Several strategies have been adopted therefore in order to limit the immune response and achieve (i) prolonged transgene expression and (ii) successful repeated administration of the viral vector. The majority, if not all, of these strategies are aimed at indiscriminately blocking both arms of the immune response so that both prolonged expression and repeated administration are achieved. In the lung this can be obtained either through pharmacological interventions aimed at reducing the host immune responses (see below) or through modifications of the viral vector in order to make it less immunogenic (see below).

Host interventions

Pharmacological approaches prolong gene expression and allow some degree of vector readministration.

Because of their ability to block T cell-mediated responses, immunosuppressant drugs such as cyclophosphamide, cyclosporin and FK506 have been reported to prolong transgene expression and facilitate repeated gene transfer in the lung. Intravenous injections of cyclophosphamide prevented the formation of neutralizing antibodies and allowed adenovirus readministration to the lung [10]. It needs to be remembered that treatments with immunosuppressants do not tolerize the recipient to a virus, indicating that the drugs will have to be administered with each vector treatment. Furthermore, a complete block of T cell-mediated response will have to be achieved in order to avoid an increasing immune response each time a viral vector is administered. In addition to this, the potential to induce severe systemic side-effects may limit the clinical application of these drugs. For example, prolonged general immunosuppression of CF patients, where lungs are colonized by pathogenic bacteria, could be injurious.

Similarly, topical corticosteroids have been shown to reduce viral vector-mediated inflammation and concomitantly increase gene transfer efficiency in the lung [11]. Treatment with dexamethasone resulted in five- to 10-fold higher levels of lung transgene expression compared to saline-treated controls [12]. Administration of budesonide allowed readministration of adenovirus vectors to the lung for four consecutive exposures at 2-weekly intervals. However, the concentration of transgene product expressed in the bronchoalveolar lavage fluid (BALF) declined after the second administration and correlated with the appearance of neutralizing antiviral antibodies in BALF and serum. Furthermore, differences between control and treated groups disappeared after five exposures to the virus [13].

Co-administration of interferon-g (IFN- g) or interleukin-12 (IL-12) has been shown to diminish the activity of T_H2 cells and formation of NABs, allowing readministration (at least once) of recombinant virus to the lung [14]. However, a potential drawback of this approach is that T_H2 cells are inhibited at the expense of increased T_H1 activation. Thus, both IFN-g and IL-12, while capable of inhibiting humoral immunity, might enhance the elimination of adenovirus-transduced cells by CTLs.

Strategies to block CD4⁺T cell activation.

Both CTL and B cell responses require activation of CD4⁺ T cells of the T_H1 and T_H2 subsets, respectively. The central role of CD4⁺ T cells in the activation of both arms of the immune response suggests that a transient blockade of CD4⁺ T cells at the time of virus administration to the lung might prevent the activation of humoral and cellular immune response to viruses, thus causing prolonged transgene expression and efficient readministration. Several strategies have been adopted, therefore, including the use of non-depleting monoclonal antibodies to the CD4 molecule [15] and the blockade of either CD40–CD40 ligand [16] or CD28-B7 (with CTLA4-Ig) [17], co-stimulatory signals necessary for complete T cell activation. In the lung these treatments have resulted in suppression of cellular and humoral responses, prolonged transgene expression and, in some cases, vector readministration up to four times [15]. However, in non-human primates, treatment with an anti-CD40 ligand monoclonal antibody did not prevent a virus-specific antibody response upon secondary challenge with vector [18]. It is possible that a combination of blocking agents may provide a more complete abrogation of T- and B-cell immune responses.

Inducing tolerance to viral vector antigens as a way to overcome the immunological hurdles.

For many years immunologists have recognized that the ‘rules of immunology’ can be violated and foreign agents can be tolerated. When antigens are encountered, lymphocytes can be activated (immune response), ignored (ignorance) or inactivated/eliminated (tolerance). Recently, many studies have tried to evaluate whether induction of oral [19] or intrathymic [20] tolerance to adenoviral antigens could be used to abrogate the host anti-adenoviral immune response, thus prolonging virus expression and allowing successful viral administration. In these studies adenovirus-mediated gene transfer to the liver of tolerized Gunn rats permitted long-term transgene expression by repeated injection of the recombinant virus. Similarly, prolonged transgene expression and lack of immune response were observed when recombinant adenoviruses were administered during the neonatal period, at a time before T cell maturation had occurred. Interestingly, DeMatteo et al. showed that mice that had received an intrathymic injection of virus as neonates showed expression in the lung up to 70 days postinfection when re-challenged in adulthood via intratracheal instillation [21].

Tolerization protocols are extremely interesting for their potential to prolong transgene expression and allow repeated administration of viral vectors. However, some caveats should be highlighted. First, tolerance requires that virtually all potentially reactive cells are turned off and this depends on the amount of antigen, persistence, route of administration and presence of adjuvants and properties of APC [22]. Secondly, the relatively greater maturity of the human immune system than the rodent at birth would probably require in utero fetal manipulation in order for tolerance to develop if the neonatal injection strategy is to be used. Thirdly, different organs might not behave similarly in their ability to be made tolerant towards a viral antigen. For example, Chirmule et al. showed that Fas–Fas ligand interaction plays a crucial role in CTL-mediated elimination of adenoviral vector-transduced cells in the liver, but not in the lung, thus suggesting that different mechanisms of immune response might exist in the two organs [23].

Gene transfer agent modifications

An alternative to the strategies described above is the modification of the gene transfer vector. This has the advantage of avoiding any immunosuppression treatment, likely to be deleterious in patients already compromised by lung injury.

Strategies to reduce CTL-mediated response.

A general strategy for overcoming the CTL-mediated response against virus-transduced cells is the modification of the vector backbone in order to prevent the expression of viral genes in infected cells. For adenoviral vectors, this has led to the development of vectors devoid of virtually all viral sequences except those required in cis for virus propagation (the terminal repeats and packaging signals). These ‘gutless’ vectors are known as helper-dependent adenoviral vectors (HDAds) because propagation is dependent on co-infection with a helper adenovirus that supplies all replication functions in trans. Although these vectors do not avoid the immune response, many studies have demonstrated both reduced toxicity and prolonged expression in various organs, including the lung [reviewed in 24]. Repeated administrations still remain an issue unless alternative serotypes [25] or immunodeficient animals [26] are used. Furthermore, HDAd vectors remain difficult to produce and purify in clinically relevant quantities and vector preparations are contaminated with low levels of helper Ad virus.

An alternative strategy to reduce CTL-mediated destruction of transduced cells is to insert immunosuppressive genes into the viral backbone (Fig. 1). Scaria et al. showed that by using an adenovirus co-expressing both human CFTR and ICP47 (a gene shown to block the transporter associated with MHC class I-mediated antigen presentation to CD8⁺T cells), prolonged expression (up to 21 days) was observed in monkey lungs, even though natural killer cell activity was enhanced [27]. This suggests that viral vectors that down-regulate cell surface class I molecules must also have strategies for blocking the activation of natural killer cells, which can detect decreased expression of class I molecules on the cell surface (as in the murine cytomegalovirus M152). Other immunosuppressive genes, such as CTLA4Ig, have been shown to prolong transgene expression and allow repeated administration of the vector when delivered to liver and muscle [28,29] and need to be tested for their efficacy in the lung [reviewed in 30].

Fig. 1.

The pathway of class I major histocompatibility complex (MHC)-associated antigen presentation with examples of viruses that block different steps. Recombinant viral vectors coexpressing a transgene and one of these genes are under development for their ability to avoid CTL-mediated destruction of the transduced cells. CMV: cytomegalovirus; EBV, Epstein–Barr virus; HSV, herpes simplex virus; TAP, transporter associated with antigen processing.

Strategies to reduce neutralizing antibody-mediated inactivation of viral vectors.

One means by which viral vectors could escape neutralization by the humoral immune response is by coating with polymers such as polyethylene glycol (PEG) [31] and GL67/DOPE-PEG [32], or encapsulating them using bilamellar cationic liposomes [33]. PEGylation of ‘gutless’ adenovectors could produce ‘stealth’ viruses able to avoid both cellular and humoral immune responses, allowing significant gene expression upon readministration in the lung without the need for immunosuppression [34]. This strategy might also fulfil a secondary function of modifying the targeting characteristics of viruses so that a more restricted or entirely distinct population of cells can be transduced, thus avoiding unwanted transduction of macrophages and dendritic cells. Alternatively, effective repeated delivery can be achieved by ‘serotype switching’ where gene therapy is initiated with one virus serotype, then switched to a virus derived from a second serotype for subsequent administration, thereby avoiding neutralizing antibodies induced by the first serotype [35–37]. However, transgene expression may be limited by cross-reactive CTLs that can also target cells infected by the second virus serotype.

Use of less immunogenic viruses.

Beck et al. have suggested recently that vectors based on adeno-associated viruses (AAVs) can escape immunologic surveillance and be delivered repeatedly to rabbit airways, because they are unable to transduce antigen-presenting cells (dendritic cells) [38]. However, other studies have shown that readministration of AAV-based vectors can only be achieved if coupled to AAV ‘serotype switching’ [37] or transient immunosuppression [39,40], which would be impractical in the clinical setting. However, Auricchio et al. showed recently that AAV2-derived vectors packaged in capsids from AAV5 (AAV2/5) can be readministered successfully to the mouse lung 5 months after the first delivery of the same vector and lead to levels of gene expression comparable to those seen in naïve mice. Interestingly, the presence of serum-neutralizing antibodies to AAV5 capsid proteins almost 10 months after vector administration suggests that lung transduction may occur despite the presence of serum antibodies [41]. Lentivirus and retrovirus-based vectors are potentially less immunogenic even if their ability to mediate gene expression in the lung after repeated administration has still to be evaluated.

Synthetic Vectors as an Alternative to Viral Vectors

Non-viral gene delivery strategies are generally regarded as safer and less immunogenic alternatives to viral vectors. However, gene expression is low due to the (i) inability to be endocytosed; (ii) intracellular degradation; and (iii) inability to cross the nuclear membrane. Addition of ligands/peptides able to overcome these barriers has led to ‘virus-like’ particles with both increased gene transfer ability but also increased immunogenicity.

Inflammatory effects

Synthetic vectors can indeed have inflammatory and toxic effects in vivo. Scheule et al. observed a dose-dependent pulmonary inflammation characterized by infiltrates of neutrophils and, to a lesser extent, macrophages and lymphocytes when the cationic lipid GL-67 was topically administered to mouse lungs. Associated with this were elevated levels of the proinflammatory cytokines IL-6, TNF-α and IFN-γ that peaked at days 1–2 postinstillation and resolved by day 14 [42]. Histopathological analysis of lung sections from mice treated with the individual components of the lipoplex suggested that the cationic lipid was the major mediator of the observed inflammation. Further studies reported that the levels of cytokines induced after aerosol delivery in the lung tissue, serum and BALF were much lower than those induced after intravenous delivery of cationic-vector-DNA complexes [43].

Effect of the CpG motifs.

Indications that bacterially derived plasmid DNA (pDNA) may also be inflammatory came from the results of clinical studies in which CF patients were subjected to either aerosolized liposomes alone [44] or cationic lipid/pDNA complexes [45,46]. Each of the cationic lipid/pDNA-treated patients, but not the liposome-treated controls, exhibited mild flu-like symptoms (including fever and myalgia) over a 24-h period. One explanation for this response may be related to the presence of unmethylated CpG dinucleotide sequences in bacterially derived pDNA (Fig. 2). Compared with DNA of eukaryotic origin, bacterial genomic DNA contains a 20-fold higher frequency of the dinucleotide sequence CpG. Further, unlike eukaryotic DNA, in which approximately 80% of the cytosines are methylated, bacterial DNA is relatively unmethylated. In B cells and plasmacytoid dendritic cells, CpG motifs are recognized by toll-like receptor (TLR) 9, which triggers signalling pathways leading to production of Th1-like proinflammatory cytokines, interferons and chemokines [reviewed in 47]. Instillation of bacterial DNA or oligonucleotides containing immunostimulatory CpG motifs into mouse lungs resulted in inflammation of the lower respiratory tract which was not seen using mammalian DNA [48].

Fig. 2.

Induction of innate and acquired immune responses by plasmid DNA containing CpG motifs.

Several strategies have therefore been employed to decrease the immunostimulatory properties of pDNA, including (i) methylation of CpG sequences [49]; (ii) reduction of the CpG frequency by eliminating non-essential regions or by site-directed mutagenesis [50]; and (iii) the use of inhibitors of the CpG signalling pathway, such as chloroquine or quinacrine [50]. Independent of the strategy used, the CpG-reduced pDNAs were found to be less proinflammatory. However, methylation of the CpG motifs can severely reduce the expression of the transgene [49].

Repeated administration.

Repeated administration of lipoplexes in mice [51] and patients with CF [52] resulted in similar levels of transgene expression as observed after a single delivery, thus suggesting that lipoplexes can be readministered without apparent loss of efficacy. The efficacy of repeated administrations is dependent on the dose used and the time interval between administrations. Very little, or no, loss in efficacy was observed provided the dose of lipoplexes was low or if the time interval between successive instillations was sufficiently long [53,54]. It appears that the inflammation elicited by the complexes may affect the efficacy of repeat administration.

This picture might be altered once non-viral vectors are conjugated with peptides or other molecules able to elicit an immune response. Thus, Ferkol et al. showed that DNA complexed with a molecular conjugate based on Fab fragments of antibodies to the polymeric immunoglobulin receptor expressed on the apical surface of epithelial cells can lead to an escalating antibody response after repeated administration to the lung in vivo. Transgene expression was significantly lower in mice that received three administrations of the DNA complexes. The serological response was directed exclusively against the Fab antibody fragment component of the molecular conjugate and not against either the plasmid DNA or poly l-lysine [55].

Solutions might involve the use of (i) immunosuppressant as previously described for viral vectors or (ii) immunologically inert peptides/ligands. However, there are no reports to date of studies evaluating the efficacy of repeated administrations.

Clinical Trials

Preclinical studies suggest that the lack of persistence of expression is secondary to innate and adaptive immune responses. Similarly, clinical trials of gene therapy for lung disorders have shown that (i) transgene expression is limited and declines over time (with both viral and non-viral vectors) and (ii) readministration of viral vectors leads to a less pronounced degree of correction [56]. Unlike viruses, lipolexes can be readministered successfully without apparent loss of efficacy [52], even if inflammatory effects due to CpG motifs in plasmid DNA are observed (Table 1 and http://www.wiley.co.uk/genmed/clinical for a detailed list of clinical trials and immunological problems observed).

Table 1.

Immune responses following lung gene delivery in clinical trials of gene therapy^*

Trial	Vector used	Disease	Major immunological findings	Reference
Zabner et al. 1996	Adenovirus	Cystic fibrosis	Repeated, five escalating doses to the nasal epithelium of subjects with pre-existing antibody to Ad. After the 5th dose correction of chloride efflux defect was not significant, due probably to increased neutralizing antibody titre	[56]
Alton et al. 1999	Cationic lipid/plasmid DNA	Cystic fibrosis	Lipid/DNA-treated patients, but not liposome-treated controls, exhibited mild flu-like symptoms over a period of 24 h due probably to CpG motifs in plasmid DNA	[45]
Bellon et al. 1997	Adenovirus	Cystic fibrosis	No increase in anti-adenovirus antibodies levels in blood and BALF with respect to those at baseline. No significant deviation from baseline for lymphocyte proliferation	[57]
Harvey et al. 1999	E1, E3-deleted Adenovirus	Cystic fibrosis	Low, medium and high doses repeatedly given in three cycles, each of 90 days. Vector-derived CFTR cDNA expression after 1st administration, 2nd (but only at medium dose), but not after the 3rd administration. At anti-Ad titres of ≤1 : 40 there was no relationship between expression and antibody titre. At anti-Ad titres of ≥ 1 : 80 no vector derived expression at all doses or cycles	[58]
Zuckerman et al. 1999	Adenovirus	Cystic fibrosis	Ad delivered by bronchoscope. Increase in Ad-specific lymphoproliferative response after vector administration in all subjects. Neutralizing antibody responses were stimulated only to a modest range or not at all	[59]
Ruiz et al. 2001	Cationic lipid/plasmid DNA	Cystic fibrosis	Lipoplexes, but not lipid alone or DNA alone, shown to elicit proliferation of immune mononuclear cells (CpG motifs as a cause?). No antibodies against lipid or plasmid DNA. Serum IL6 levels increased	[46]
Aitken et al. 2001	AAV-2	Cystic fibrosis	tgAAV-CFTR (1010−1013 DNase-resistant particles = DRP) aerosolized to four cohorts of three subjects each. One patient in the 1012 DRP cohort and three subjects in the 1013 DRP cohort had significant fourfold or greater increase in serum neutralizing antibody. No neutralizing antibody in BALF	[60]
Brigham et al. 2000	Cationic lipid/plasmid DNA	α1-antitrypsin deficiency	Significant decrease of IL8 in nasal lavage fluid of transfected nostrils, compared to untransfected, 5 days after gene delivery	[61]

^*Lung cancer gene therapy trials have been omitted because immunogenicity is not a critical issue or obstacle.

Very recently, differences have been identified with regard to how the immune system reacts towards GTAs between experimental animals and humans. Harvey et al. have shown that repeated administrations of Ad vectors to the respiratory epithelium of normal [62] and CF individuals [8,58] resulted in minimal anti-Ad neutralizing antibodies. Similar to what has been observed in experimental animals, the levels of Ad genome decreased progressively over time, but in contrast minimal or no humoral and cellular responses were observed. There are various possibilities to explain the differences in responses observed in animals and humans. First, the amount of vector administered to humans could be below the threshold required to induce a significant humoral and cellular response. Secondly, the responses observed in experimental animals may be more intense because the majority of the vector is delivered to the alveoli, a site that may incite a much stronger host responses. In contrast, in human trials vectors are generally delivered to a limited portion of the bronchial wall (by bronchoscope) or aerosolized. Thirdly, human responses to Ad-vectors may be different from those of experimental animals. This suggests that factors other than vector-specific adaptive immune responses also play a significant role in modulating gene expression. Future studies in larger animal models of lung disorders might help elucidate whether factors such as host species, vector dose or delivery route can explain the difference in host responses observed.

Conclusions

Both physical and immunological defence mechanisms can limit gene delivery and expression to the lung. The strategies to modulate the host immune system described herein have shown that prolonged transgene expression can be achieved, even if their clinical applicability remains an open issue. However, the real challenge is that of repeated administration, especially for lung disorders such as cystic fibrosis and alpha-1 antitrypsin. Collaboration between gene therapists, virologists and immunologists will be important to address these issues. Tolerance or strategies used by viruses and parasites to escape immune surveillance may help to design treatments to overcome these hurdles.