Lysophosphatidylcholine as an adjuvant for lentiviral vector mediated gene transfer to airway epithelium: effect of acyl chain length

The search for a safe and effective gene therapy for cystic fibrosis (CF) airway disease has been underway for more than 15 years, and throughout this time three issues have underscored the slow progress in this field: the poor efficiency of gene transfer; the short persistence of gene expression; and the abrogation of initial gene expression by host inflammatory and immune responses [1, 2].

Poor efficiency can be seen as a problem in general for gene transfer to airways - evolution has produced a extremely effective series of protective barriers and that efficiently block, remove or destroy the bulk of vector doses delivered into the airway [3, 4]. For a genetic disease like CF, highly-persistent gene correction or complementation is necessary to counter the life-long effects of the disease; in this regard lentivirus vectors are an obvious choice because of their longevity of expression and depending on their pseudotype, their ability to transduce many cell types. Inflammatory and immune responses have limited the therapeutic effectiveness of many other viral vectors such as adenovirus and adeno-associated adenovirus [5‐7]. However, helper-dependent adenovirus have shown enhanced efficiency and surprisingly high levels of expression in mouse [8], rabbit and baboon lungs [9, 10] when delivered in conjunction with lysophosphatidylcholine (LPC). Nevertheless, the long term persistence of expression from these vectors has not been adequately assessed. We have also demonstrated that high level long-lasting lentiviral gene expression can be produced in intact nasal airways of normal and CF mice after LPC pre-treatment [11, 12].

The major action of LPC in all these circumstance appears to be the improvement of vector access to appropriate airway cell surface receptors as well as transient disturbance of epithelial surface barrier function to permit vector particle access to basolateral receptors and to deeper-lying basal cells that may have progenitor-like qualities. Following the success of our pre-treatment studies using a standard and readily available natural form of LPC (derived from egg yolk, a mixture of palmitoyl/stearoyl forms), we report here on studies designed to determine whether molecular variants of LPC could produce more effective enhancement of LV gene transfer and/or reduce the potential for damage to the airway epithelium.

Despite the strong interest in gene therapy as a cure or treatment for the chronic and progressive disease that overtakes the cystic fibrosis lung, no clinically therapeutic benefits have yet appeared due largely to ineffective vector transduction [20] and host immune and inflammatory responses [5, 6]. If the traditionally poor efficiency of gene transfer can be improved there is the potential to produce substantial and positive gene expression outcomes. Increasing the efficiency of gene transduction would permit a reduction of the dose of vector and its contaminants that should automatically help to reduce the intensity of the immune and inflammatory responses. The use of an enhancer such as LPC provides an effective method to reduce the vector dose necessary to achieve a given level of gene expression in vivo.

The efficiency of gene transfer with any vector can be improved by increasing the ability of vector particles to reach and remain near their target cells [21]. In normal circumstances the availability of the majority of vector particles delivered to the airways for transduction process are rapidly and substantially reduced by the natural barriers to airway infection. These include the mucus layer, the mucociliary clearance that transports particles out of the airway [22, 23] and the glycocalyx [24] that can bind vector particles that escape these initial barriers and all can prevent vector contact with the relevant cell-membrane receptors. In terms of vectors that can also target basolateral receptors, such as VSV-G pseudotyped LV vectors, the tight junctions between the cells provide an additional barrier to transduction that must be considered. Although airway gene transfer using a simian LV vector pseudotyped with a Sendai virus envelope [25] or a feline LV vector pseudotyped with a baculovirus GP64 envelope [26] can effectively target the apical receptors in murine nasal airway, a large dose (volume and titre) of the vector and long residence time was needed to overcome these initial barriers. The combination of LPC pretreatment with the VSV-G pseudotyped LV vector permits transduction at apical as well as basolateral sites, providing immediate (apical) and potentially long term transduction via basally located progenitor cells [27].

Since the structural changes present in the molecular variants of LPC are likely to alter their biological function or effectiveness, we examined the effect of different molecular variants of LPC on their ability to enhance LV gene transfer. We used LPC variants based on their differing acyl chain length - from C10:0 decanoyl to C18:1 oleoyl- as well as at a low (0.1%) or a high (1%) concentration. Most biologically occurring LPC molecules possess longer acyl chain lengths, between C16:0 and C22:6 [17, 28].

The lack of effectiveness with decanoyl-C10:0 and other shorter acyl chain LPC molecules mirrors the known lack of effects on cell toxicity [29] and membrane permeability [30] noted elsewhere. At the 0.1% concentration LV gene expression was absent or not significantly different to control (PBS) for the decanoyl-C10:0, lauroyl-C12:0, myristoyl-C14:0 and oleoyl-C18:1 LPC pre-treatment. In contrast, pre-treatment with palmitoyl-C16:0 or stearoyl-C18:0 produced similar levels of gene transfer to our standard LPC mixture (containing both C16:0 and C18:0). Heptadecanoyl-C17:0 pre-treatment produced the strongest LV gene expression of all those tested at the 0.1% concentration. These findings are consistent with those in other studies where 0.1% LPC has produced successful airway gene transfer in mice, rabbits and baboons [8‐10]. This present study also demonstrates that LPC pretreatment can be effective at 1/10^th of the dose previously used in mouse airway to enhance LV gene expression [12]. At the high concentration (1%) every LPC variant except decanoyl-C10:0 produced similar levels of LV gene transfer compared to our standard LPC mixture.

The percentage of gene transduction ranged from 1.5% (oleoyl-C18:1) to 15% (heptadecanoyl-C17:0) of the epithelial respiratory cell layer, and if applicable to CFTR gene transfer maybe sufficient for effective correction of the CF gene defect in airways [31, 32]. The majority of respiratory cell types transduced at either concentration were ciliated cells (60-72%), with the only other cell types transduced being unciliated and basal cells. There was a modest increase in the proportion of basal cells compared to unciliated cells after 1% LPC pre-treatment. The increase in basal cell transduction could provide a basis for effective long term LV expression, as this is the niche where progenitor-like stem cells are thought to reside [27].

We sought explanations for these differences in gene transfer via electrophysiological and histological analyses. The magnitude of depolarization of the electrical response (TPD) one hour after 0.1% LPC administration (the time of vector dosing) was strongly correlated with higher gene transfer, as measured by counts of cells expressing LacZ (Fig 4a, c). Since the TPD is reduced as tight junction barrier function is lost [18] this finding supports the idea that tight junctions become permeabilised by LPC after treatment with most of the variants. Such permeabilisation is known to permit viral vector particles to access the relevant basolateral receptors [33], with the ensuing increase in vector particle binding to appropriate basolateral receptors resulting in increased airway gene transfer. Conversely, when little or no depolarization was present 1 hour after the LPC treatment, poor gene expression was observed (Fig. 4: see decanoyl-C10:0 treatment group).

Another example of the influence of epithelial barrier permeability was with 0.1% oleoyl-C18:1 LPC. Here the change in potential difference was brief and transient, with the TPD having returned to baseline by the usual time of vector instillation, and the level of gene transfer produced was low. This relationship between the TPD and LPC pre-treatment suggests that improved gene transduction may have been possible had the time of vector instillation corresponded to the maximum depolarization of TPD (e.g. 30 min for this oleoyl LPC variant). Optimization of successful gene transfer for a particular LPC variant may depend on selecting the best pre-treatment timing and LPC concentration for that variant, and future studies could determine if the shorter timing intervals are more effective for variants such as oleoyl. This notion is further supported by our finding that at the high 1% LPC concentration only the decanoyl-C10:0 variant remained unable to induce necessary membrane permeability as measured electrophysiologically, and this was consistent with the absence of gene transfer.

At the two concentrations tested, the LPC variants displayed differences in the degree of effect on the airway epithelium morphology. We examined both the release of cell-bound mucosubstances and changes to the physical integrity of the epithelium. Mucosubstance changes were particularly evident as a loss of mucin granules from epithelial goblet cells; this is a normal airway response in defense against foreign stimuli and particles and will aid efficient mucus based particle capture and subsequent mucociliary clearance. The lack of effect from control vehicle administrations at the one hour time point (Fig. 5) showed there was no significant release of mucin from the epithelial cells from the delivery process of PBS, and this was also observed after decanoyl-C10:0 administrations. Consequently, the LV vector that is instilled at this time will still encounter a primed mucociliary clearance system (as well as intact tight-junctions), and this may explain in part, the absence of any enhancement of gene transfer by PBS or decanoyl-C10:0 LPC when used as a pretreatment agent. The significant increase in the release of mucin from the epithelial goblet cells in reaction to the remaining LPC variants suggests an innate mucin-based defense exists towards these molecules. Thus, at the time of gene vector instillation most goblet cells will have previously released their mucin. We speculate that the initial loss of mucins in response to LPC delivery may be an additional factor in increasing gene transfer and expression, by leaving the airway temporarily unable to capture and remove the vector gene particles when they are delivered 1 hour later.

All LPC pretreatments that produced effective gene expression displayed physical perturbations of the epithelial layer apparent 1 hour after LPC administration. These included loss of cilia, disruption of cell-cell junctions and even exfoliation of some areas (Fig. 7). Where cellular exfoliation was induced in some regions (typically using the 1% doses of LPC variants) we found that gene expression decreased (significantly for heptadecanoyl-C17:0 and stearoyl-C18:0 only) compared to that produced by the low (0.1%) concentration of LPC. The high LPC concentration produced greater physical disruption than the low LPC concentration, but the level of gene transfer was similar in majority of cases. If the effect of the high dose LPC is mainly to damage the epithelium, the potential for production of gene expression maybe lost or reduced because of cell re-growth [34]. That is, as much gene expression may be lost, as would be regained, by creation and transduction of newly formed epithelial cells. However, these physical disturbances to the epithelial layer were in all cases (even at 1%) temporary, because the gene expression noted at 1 week was within an intact epithelial layer. Our data regarding the level of gene transfer achieved, the effect on mucin release, changes in electrophysiology, and epithelial disturbance supports our selection (and that of others [8, 9]) to use a low dose (0.1%) of LPC for pre-treatment or co-instillation in the production of enhanced LV gene expression [12]. In addition, the widely used C16:C18 mixture remains a good choice of LPC variant. The use of LPC pretreatment also allows for lower volumes and titres of a LV vector to achieve effective gene expression and therefore has the potential to reduce immune responses. However, heptadecanoyl-C17:0 used at the 0.1% concentration (Fig. 1b) can provide significant improvement in gene transfer effectiveness over all other LPC variants tested here. For that reason this LPC variant may be worthy of further investigation, but some caution may be required for in vivo application and/or clinical development as this molecule is an entirely synthetic species not normally present in biological systems.

We have shown that LV gene transduction in murine airways can be significantly enhanced by the optimization and selection of LPC species as a pre-treatment regime. The effect of LPC variants on airway barrier function strongly correlated with gene transduction efficiency, providing further development towards pre-clinical gene therapy protocols.