In this study we establish a method for efficiently and selectively delivering genes to resident AMs in vivo, and we apply this method for gene therapy of emphysema. Although macrophages have been considered difficult to efficiently transfect either in vitro or in vivo (6
), we demonstrate that VSV-G–pseudotyped lentiviral vectors efficiently transduced AMs, which persisted for at least 2 years in the airspaces of mouse lung.
We have adapted our method to durably overexpress hAAT in AMs — a key cell type known to be involved in emphysema pathogenesis (33
). Considerable effort has been invested in attempts to deliver a normal hAAT gene in vivo (7
). Many of these approaches have been limited by either short-lived gene expression or inability to achieve the very high levels of circulating hAAT (1 mg/ml) presumably required to prevent the progression of emphysema in hAAT-deficient patients (7
). An important concept in hAAT gene therapy is the likelihood that localized secretion of hAAT by cells within the lung may provide more effective protection against emphysema at hAAT levels far lower than those required in the circulating blood (7
). Our findings support this concept, since intratracheal delivery of lentivirus provided therapeutic localized secretion of hAAT into the lung ELF in the absence of high circulating concentrations of hAAT protein. Our method achieves an average lung ELF hAAT protein concentration of 142 μg/ml, equivalent to a 42% increase in total AAT over estimated endogenous mouse ELF AAT protein levels (refs. 38
and Supplemental Methods). The vectors presented in our mouse studies should be readily translatable to human cells, as we have observed efficient transduction and gene expression in primary human AMs after in vitro lentiviral infection (46% GFP transduction efficiency; Supplemental Figure 7).
The current standard of care for patients with severe emphysema and hAAT deficiency consists of life-long weekly injections of hAAT protein purified from pooled human plasma (26
). Whether this approach is clinically effective or results in detectable inhibition of lung matrix breakdown in patients remains controversial (41
). In mouse models of emphysema, injected hAAT protein has been shown to ameliorate tobacco smoke–induced emphysema via an anti-inflammatory mechanism related to TNF-α suppression (43
). In addition, in mice exposed to VEGF receptor blockade, hAAT protein has been shown to attenuate airspace enlargement through inhibition of apoptosis and modulation of lung oxidant stress (45
). Although our results did demonstrate altered macrophage recruitment kinetics after elastase injury, further investigations will be required to determine whether this altered inflammatory kinetic or the emphysema amelioration in our studies resulted predominantly from the well-known antiprotease effects or other aforementioned properties of hAAT.
While our protocol offers the prospect of sustained hAAT reconstitution using a single lentiviral-based treatment, our experiments tested augmentation of AAT in mice that already express normal murine AAT. Because complete absence of AAT in mice results in embryonic lethality (46
), a murine model of severe AAT deficiency that resembles the human disease is not currently available. Future tests of the efficacy of our protocol in inducible AAT-knockout mice should help to further define the therapeutic potential of our approach. In addition, before clinical use can be considered, more detailed screens for adverse consequences, including insertional mutagenesis of transduced cells, will be required.
Our findings challenge the widely accepted view of AMs as being short-lived cells which require continual replenishment by circulating bone marrow–derived monocyte precursors (22
). The low BrdU labeling index of AMs in the steady-state lung, the persistence of lentivirally tagged cells on kinetic imaging studies, and the absence of appreciable numbers of transduced circulating cells in our experiments all support the conclusion that a prolonged AM lifespan, rather than proliferation or recruitment of precursor cells, explains the unexpectedly long duration of these cells in uninjured lung tissue. Our findings differ from most prior studies of AM lifespan because the majority of prior reports have relied on myeloablative regimens to examine the kinetics of AM reconstitution with transplanted circulating or bone marrow–derived cells (22
). Those kinetics likely reflect the toxic effects of myeloablative conditioning regimens rather than the true lifespan of AMs. Indeed, we recently reported that when AMs are shielded from radiation exposure in recipient mice being prepared for bone marrow transplantation, there is minimal AM turnover even 8 months after reconstitution of the bone marrow and blood (11
). Because the durable gene expression achieved in our current study depends on the prolonged lifespan of unperturbed murine AMs, it will be important for future studies to determine whether similar longevity applies to AMs in the lungs of humans with or without lung disease. Our results demonstrating durable gene expression in transduced AMs even following PPE- or acute tobacco smoke–induced injuries suggests that resident AMs similarly persist in diseased lungs, although whether AM proliferation in inflamed lungs contributes to this persistence was not assessed in our studies.
The preferential tropism of VSV-G–pseudotyped lentiviruses for AMs in our studies likely results from the comparatively poor tropism of this pseudotype for the other major luminal constituent of the lung, epithelial cells. Receptors for the VSV-G envelope are thought to be sequestered on the basolateral surface of adult lung epithelia, preventing their in vivo transduction in the absence of epithelial injury in most prior reports (13
). However, VSV-G–pseudotyped lentiviral vectors do appear to possess some capacity to transduce lung epithelial cells in vitro (49
), when administered in vivo at early fetal developmental stages (51
), or when delivered together with epithelial mitogens such as KGF (53
). Alternatively, lentiviral vectors pseudotyped with other viral envelope glycoproteins such as Ebola do appear to possess adult lung epithelial tropism in vivo (13
). We have found that Ebola-pseudotyped (NTDL6; ref. 54
) lentiviruses also efficiently transduce AMs in vivo but lack the AM specificity of the VSV-G pseudotype (our unpublished observations).
It cannot be concluded from our studies that lentiviral transduction was entirely specific to AMs. For example, some animals showed persistent gene expression at the tip of the nose (Figure ). In addition, transient reporter gene expression was occasionally observed over the oropharyngeal region, implying that extrathoracic cell types such as pharyngeal lymphoid tissue may have expressed transferred genes. Indeed, the acute intra-alveolar inflammatory response observed 1 week after lentiviral instillation indicates significant inflammatory signaling and activation of a variety of inflammatory cell types, some of which are likely to transiently traffic through the lymph or blood to sites outside the lung. That pulmonary dendritic cells in particular may have been transduced in our studies cannot be excluded because these cells would be expected to migrate to local draining lymph nodes within hours of infection. It is also important to note that we did not find evidence of preferential lentiviral transduction of any particular AM subset (as defined by degree of quiescence, or expression levels of cell surface MHC-II, CD11b, CD11c, or F4/80). However, our results do not entirely exclude the possibility that a particular, as yet unrecognized, subset of lung macrophages may have been preferentially transduced by our method.
In summary, our results suggest the feasibility of selectively targeting differentiated cells resident in a tissue to accomplish prolonged and therapeutic in vivo gene expression. The method presented here should be of considerable interest to those wishing to modulate gene expression in AMs for the study of pulmonary innate immunity or the pathogenesis of a variety of inflammatory lung diseases, including asthma and emphysema.