The localization of entry pathways for Ad to the basolateral surfaces of airway epithelial cells suggests that a delivery strategy to access these regions would be beneficial to improving gene transfer efficiency. This approach may also allow targeting of the epithelial stem cells (basal cells), resulting in transgene expression in the lung for the lifetime of the individual. This is an important consideration for gene transfer to the airway epithelium since fully differentiated lumenal facing cells (e.g., ciliated cells) have a relatively short lifetime, on the order of 40-100 days, and targeting these cell types specifically will require regular readministration of vectors.

Access to basal cells/basolateral surfaces may possibly be achieved by intravenous administration of vectors if penetration of the blood vessel wall, the connective tissue, and the basal lamina of the basement membrane were achievable. Unfortunately, studies that have attempted intravenous delivery strategies have not been successful since vectors do not appear to gain access to sufficient lung epithelial cells to make this approach feasible [99-102], Barriers functions provided by the blood vessel endothelial cells and connective tissue surrounding the airway passages seems unpenetratable by Ad. Indeed, the particle permeability of the basal lamina alone is thought to exclude inert particles of greater than 10 nm, which would certainly be restrictive to particles the size of Ad (100 nm). In an in vivo experimental mouse model where Ad was externally administered directly to the tracheal basement membrane, efficient gene transfer to the connective tissue fibroblasts adjacent to the basement membrane was observed without gene transfer to the epithelial cells of the juxtaposed epithelium [51].

To date, two main strategies to improve intralumenal delivery of Ad vectors have been focused on. One approach is to access the basolateral surfaces of the epithelial cells by disruption of the epithelial "tight" junctions, and the other is to retarget Ad vectors to nonviral receptors that are present on the apical surface of lumenal epithelial cells that allow for entry of Ad into these cell types. Retargeting has so far been achieved by chemically, immunologically, or genetically modifying the Ad capsid coat by incorporating new receptor ligands that can target candidate receptors.

a. Modification of the Host by Opening Tight Junctions Epithelial cell "tight" junctions (zonulae occludens) are collar-like structures composed of a diverse number of proteins that separate the apical and basolateral domains of the lumenal columnar epithelial cells. As well as functioning as a restrictive barrier to mixing of apical and basolateral membrane components, these intercellular junctions limit the transepithelial transport of solutes across the epithelium. A number of disease states have been shown to alter tight junction permeability (e.g., asthma) and reagents to increase the permeability of the junction are available. The key to successful disruption of tight junctions to allow Ad access to basolateral epithelial cell surfaces will be to use a reagent that opens tight junctions sufficiently for Ad to pass through but that is rapidly reversible to limit the passage of other lumenal contents (e.g., bacteria) or serosal fluid into the airway lumen.

A property exploited for this purpose is the calcium ion dependency of the structural integrity of the junction. Walters et al., have successfully shown that treatment of the apical surface of human WD airway cells with the calcium chelator EGTA or hypotonic solutions (e.g., water) allow for improvements in Ad-mediated gene transfer presumably by allowing Ad access to basolateral receptors [64,103]. The slow reversibility of this effect, however, is problematic; tight junction reformation takes a least a couple of hours, a time period that would be unacceptable in a clinical setting. In vivo studies in mouse airways have confirmed that these treatments improve gene transfer efficiency although parameters of safety were not assessed fully [104, 105],

More specific reagents are available for studying tight junction permeability and the effect on Ad-gene transfer. Parsons et al. used a detergent, polidocanol, in murine airways in vivo to enhance Ad-mediated gene transfer, an effect shown to be due to the ability of this reagent to transiently open tight junctions [86]. The short-chain fatty acid sodium caprate, has also been used to increase Ad-mediated gene transfer to human WD cultures and results in full correction of CF cultures when AdCFTR is subsequently applied to the apical surface. This result is exciting since the effect is rapidly reversible effect and has previously been used clinically for enhancing pharmaceutics absorption across the GI tract, again presumably by an effect on tight junctional permeability.

These studies although fraught with inherent safety issues are beginning to establish that this strategy for delivering transgenes to the lung may be a viable option. The possibility of targeting the basal stem cells by this procedure is reason enough to continue pursuing the usefulness of these strategies.

b. Targeted Ad to Increase Gene Transfer Efficiency Targeted Ad directed against specific receptors have been used to successfully transduce cell types that are usually refractory to Ad infection. The epidermal growth factor receptor, stem cell factor receptor, fibroblast growth factor receptor, ay integrins, and T-cell receptors (CD 3), have all been used as surrogate receptors for Ad entry in a variety of cell types [106-109]. Given the lack of Ad receptors at the apical surface of lumenal airway epithelial cells, a retargeting strategy to receptors known to present on the airway lumen may allow for gene transfer efficiency to be improved. However, a successful targeting strategy to the lung epithelium will require the identification of target molecules that allow for attachment and internalization of AdV across the apical membrane of columnar airway epithelial cells.

The identification of target receptors to which to redirect Ad tropism on the lumen of airway epithelium is difficult because most receptors and entry mechanisms occur on the basolateral surfaces of the cells. Certain members of a specific seven-transmembrane-spanning G-protein-coupled receptor family (i.e., P2Y2-purinoceptors, B2-kinin receptors, and adenosine type 2b receptors) have been identified as putative utile target receptors for redirecting Ad tropism to the surface epithelium of the lung. These receptors have been shown to be present on the lumenal surface of human airway epithelium and internalized into clathrin-coated pits when activated by their respective agonists [110]. The utilization of clathrin-coated pit internalization pathways for native Ad receptors, suggests that the G-protein coupled receptors may provide an ideal surrogate entry pathway for Ad. The high potency of P2Y2 agonists (e.g., ATP, UTP) combined with the low affinity of these agonists for the receptor suggests that the P2Y2 purinoceptors are abundant in number on the lumenal surface of the human respiratory epithelium [111]. Since pharmacological activation of airway epithelial P2Y2 receptors do not result in untoward effects in human airways, this receptor is an ideal target receptor to redirect Ad tropism. However, since the only available ligands for this receptor are low affinity, small organic molecules, certain technical difficulties are associated with conjugating these molecules to Ad. Other receptor types suitable for Ad retargeting exist on the airway, although specific retargeting data for Ad is lacking. The urokinase plasminogen activator receptor, uPA-R and the SEC-2 receptor have also been proposed as target receptors for Ad and AAV, respectively [112, 113].

i. Immunologically modified targeted vectors One immunological approach for targeting gene transfer vectors is using bispecific antibodies linking Ad directly to non-Ad-receptor-types present on the cell surface [108,114]. For example, chemically conjugated antibodies, one of which is directed against an epitope-tagged Ad coat protein and the other against oty integrin membrane proteins have been reported to increase gene transfer efficiency by seven-to ninefold compared to that of nonmodified Ad, indicating that increased Ad-attachment results in increased gene transfer efficiency [114]. In a similar approach, Ad was retargeted to nonviral receptor types in conjunction with ablation of the natural Ad tropism using an anti-fiber-knob protein antibody conjugated to folate [115]. Folate-conjugated antibody was the ligand of choice since the folate receptor is reported to be upregulated on the surface of malignant cells, thus providing a targeted vector for a variety of cancers. Retargeting Ad to cells expressing folate-receptors was shown to be specific and successful with significant increases in gene transfer efficiency.

As "proof of concept" studies, a hemaggluttin (HA)-epitope-tagged P2Y2 receptor expressed at the apical surface of human WD cultures and targeted with bispecific antibodies consisting of antibodies to Ad fiber-knob protein/HA-tag has been shown to facilitate Ad entry into these cell types, shown schematically in Fig. 4 [116, 117]. This effect is enhanced by coadministration of exogenous ATP to activate the receptor, an effect that can be reduced by desensitization of the P2Y2 receptors prior to addition of targeted Ad. Importantly, the apical surfaces of the HA tagged-P2Y2 expressing cultures required a brief exposure to specific proteases before targeting was effective suggesting that the apical surface glycocalyx hindered access of the targeted vector to the target receptors [116]. This approach also relied on the expression of a HA-tagged receptor that may be overexpressed relative to the endogenously expressed P2Y2 receptors in the culture system. The number of target receptors and the affinity of the targeting ligand are both likely to be critical parameters for the success of such a targeting strategy.

ii. Chemically modified targeted vectors Since antibodies to the external domains of P2Y2 receptors are not currently available, a strategy to target Ad to the endogenous P2Y2 receptor was to chemically conjugate small molecule agonists (UTP) to the proteins of the Ad capsid coat. Using chemically reactive

Figure 4 Schematic of targeting strategy used to redirect Ad tropism to P2Y2 receptors on the apical surface of human airway epithelial cells. Bispecific antibodies against the virus and the receptor were used as a targeting link and activation of the receptor results in receptor internalization and entry of Ad with subsequent gene transfer.

Figure 4 Schematic of targeting strategy used to redirect Ad tropism to P2Y2 receptors on the apical surface of human airway epithelial cells. Bispecific antibodies against the virus and the receptor were used as a targeting link and activation of the receptor results in receptor internalization and entry of Ad with subsequent gene transfer.

biotin derivatives, biotin was coupled to the Ad capsid coat predominately via hexon protein. This strategy is reported to couple 2-300 biotins to a single Ad particle and does not significantly alter the fiber-knob-hCAR interaction. By using commercially available biotin-linked UTP in combination with streptavidin as a "bridge" linking biotin-Ad to biotin-UTP, these molecular conjugates were shown to mediate gene transfer by an interaction specifically with endogenous P2Y2 receptors on the apical surface of WD cultures [110]. Again, the effectiveness of this approach was reduced by the presence of apical surface glycocalyx since gene transfer was only observed in cultures pretreated with agents that degrade this barrier. Regardless, gene transfer efficiency using these conjugates was still inefficient, probably due to the clumsiness of the "streptavidin bridge" and the low affinity of UTP for this receptor. Future experiments using this targeting strategy will require the identification of receptor agonists with higher affinity in addition to improved methods to directly couple the agonist ligands to the Ad capsid coat.

Another method for chemically conjugating receptor ligands to Ad is by the use of polyethylene glycol (PEG) that can be covalently linked directly to the Ad capsid coat. A number of groups have now shown that PEG conjugated viruses can be used to target Ad [112, 118]. For example, Ad conjugated to a 12-amino-acid peptide, identified from phage display assays on the apical surfaces of human WD cultures, resulted in a 10-fold increase in gene transfer efficiency to these cell types [118]. Similarly, Ad conjugated via PEG to a peptide that binds to uPA-R has been shown to target Ad to this receptor type and enhance gene transfer to polarized airway epithelia [112]. An additional bonus of using PEG-conjugated Ad is that these vectors appear to be less immunogenic that non-PEG-conjugated Ad. This effect is due to the masking of antigenic Ad capsid proteins (mainly hexon) from neutralizing antibodies ([119], see below).

iti. Genetically modified targeted vectors The ideal targeted vector would be one in which the target ligand could be incorporated into the capsid coat with minimal disruption of the physical and biological properties of Ad. For targeting strategies in which a peptide ligand is used, the most desirable method would be to generate an Ad vector genetically modified to express a functional peptide ligand on the viral surface. Such an approach for targeting vectors has been reported, where the Ad viral coat has been genetically modified to express multiple polylysine groups on the C-terminus of the Ad fiber-knob protein [70]. This redirects Ad tropism to heparan sulfate moieties that are present on the surfaces of most mammalian cells. With certain nonepithelial cell types, which lack hCAR, this modified vector has been shown to increase gene transfer efficiency 10- to 300-fold in comparison to nonmodified Ad. However, the modified vector will likely not be useful for gene transfer to the airway epithelium since heparan sulfate is not expressed at the apical surface of airway epithelial cells [120]. Targeted Ad in which the fiber-knob protein (responsible for Ad attachment to the hCAR) has been modified to express novel ligands that can interact with other receptor-types are being developed and the feasibility of this approach has now been reported by a number of groups [107, 121, 122]. A recent development in this type of approach was reported by Krasnykh et al. [123], who hypothesized that the HI loop region of the fiber-knob structure can withstand the insertion of heterologous peptide sequences without significantly compromising the tertiary structure of the fiber-knob protein or the production and infectivity of the modified Ad. These authors incorporated the FLAG octapeptide marker sequence into the HI loop region and were able to produce functional Ad. Importantly, they also showed that the sequence contained within intact virions was accessible to a FLAG-specific antibody, suggesting that sequences inserted into this region are capable of interacting with other target substrates such as cell-surface receptors.

A significant technical advance in Ad targeting strategies evolved from studies that deduced the viral sequences in fiber-knob protein that interact with hCAR. Genetic ablation of these sequences from Ad vectors led to the generation of Ad that no longer binds to hCAR and no longer transduces cells that are permissive for normal Ad transduction [124]. The broad cellular tropism of Ad vectors can now be reduced, and by the addition of targeting moieties to these Ad vectors specific cell-type targeting is possible. Reduced Ad interactions with nontarget cells will lessen the potential for adverse effects with these vectors. In the lung however, the significance of natural tropism ablation is unclear since most of the epithelial cells targeted with delivery strategies do not express Ad receptors at the lumenal surface. However, the loss of transduction to other cell-types that may interact with Ad delivered to the lung (e.g., macrophages, dendritic cells) may benefit from the hCAR-binding ablation mutant.

Recent developments in immunologically, chemically, and genetically modified targeted Ad suggests that "designer" gene transfer vectors will one day be available. Although Ad vectors, in their present form, may not be ideal for a number of gene transfer target tissues, notably the lung epithelium, this vector clearly remains at the forefront of gene therapy research since it is still one of the most efficacious gene transfer vectors available, and will continue to be useful at least in proof-of-concept studies.

w. Screening with other adenoviral subtypes Although over 51 different serotypes of wild-type Ad exist, the predominant serotypes used for gene transfer experiments are serotypes 2 and 5. The reason for this is largely historical since these two serotypes have been extensively studied over the past 30 years and understanding of the viral genome has allowed the manipulations necessary to evolve these viruses into gene transfer vectors. With regard to the airway epithelium, other serotypes have been suggested to be efficacious at delivering transgenes to human WD cultures. Serotypes 17 and 12 have been shown to bind/deliver transgenes 10-fold over Ad2 vectors [125]. However, as of yet no conclusive results have been presented that suggest that the improvements warrant future investigations with these vectors. One approach to determining if any of the other serotypes may be more efficacious in the lung epithelium could be envisioned using a recently reported system of generating an Ad5 capsid-expressing fiber proteins from the other serotypes [126], This system was used to screen vascular endothelial and smooth muscle cells and the efficiency of gene transfer compared against the efficiency of gene transfer with Ad5. This screening procedure identified Ad5 with Adl6 fibers as being significantly more efficient at gene transfer than Ad5 in these particular cell types. It will be of interest to screen these serotypes on human WD cultures relative to Ad5 to determine whether other Ad serotypes may be of benefit to airway epithelial cell gene transfer. A serotype which may be of particular interest is Ad37, since it has been reported that Ad37 utilizes sialic acid residues that are present on the extracellular surfaces of most cells [127], An abundance of sialic acid residues on the lumenal surface of airway epithelial cells as components of glycoconjugates may allow for improved gene transfer. Whether attachment of Ad37 to sialic acid residues located on the airway lumen leads to efficient entry and gene transfer awaits further study.

v. Other methods to increase gene transfer efficiency Nonspecific methods to enhance Ad-mediated gene transfer to airway epithelial cells have been reported [128, 129]. Calcium phosphate coprecipitation has been used to precipitate aggregates of Ad and other vectors to increase gene transfer to airway epithelia both in vitro and in vivo. It has been suggested that in vivo these aggregates increase the rate of nonspecific endocytosis of Ad across the apical membrane of polarized epithelial cells. The possible effects of this technique on cellular and paracellular permeability have not been investigated.

Another method to both improve both the delivery and efficiency of Ad to the lung epithelium in vivo is using the inert perfluorochemicals (PFCs). These compounds are liquid in nature but due to high oxygen saturation capacities can be instilled into the lung for periods of time with maintenance of passive oxygen diffusion. Several studies have now shown that administration of gene transfer vectors (including Ad) with PFC results in increased gene transfer to rodent and nonhuman primate lungs [130-132], The improvements in gene transfer are predominately localized to the alveolar regions with only modest improvements in the efficiency of gene transfer to the respiratory epithelium. The exact mechanism by which PFCs produce these effects remains to be determined, but may be due to prolonged contact time for the vector on the cells and reduced ingestion of Ad by macrophages and/or due to some nonspecific effect on the paracellular permeability. Nonetheless, this method provides an example of a new strategy to deliver transgenes to the lung without the need for direct instillation or aerosolization, which are both inefficient methods for airway epithelium delivery.

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