Lentivirus-Mediated Gene Transfer to the Respiratory Epithelium: a Promising Approach to Gene Therapy of Cystic ﬁbrosis

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Gene Therapy (2004) 11, S67–S75 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $30.00 www.nature.com/gt REVIEW Lentivirus-mediated gene transfer to the respiratory epithelium: a promising approach to gene therapy of cystic ﬁbrosis

E Copreni, M Penzo, S Carrabino and M Conese Institute for Experimental Treatment of Cystic Fibrosis, HS Raffaele, Milano, Italy

Gene therapy of cystic fibrosis (CF) lung disease needs logous envelopes are the strategies currently used to highly efficient delivery and long-lasting complementation overcome the paucity of specific viral receptors on the apical of the CFTR (cystic fibrosis transmembrane conductance surface of airway epithelial cells and to reach the basolateral regulator) gene into the respiratory epithelium. The develop- surface receptors. Preclinical studies on CF mice, demon- ment of lentiviral vectors has been a recent advance in the strating complementation of the CF defect, offer hope that field of gene transfer and therapy. These integrating vectors lentivirus gene therapy can be translated into an effective appear to be promising vehicles for gene delivery into treatment of CF lung disease. Besides a direct targeting of respiratory epithelial cells by virtue of their ability to infect the stem/progenitor niche(s) in the CF airways, an alternative nondividing cells and mediate long-term persistence of approach may envision homing of hematopoietic stem cells transgene expression. Studies in human airway tissues and engineered to express the CFTR gene by lentiviral vectors. animal models have highlighted the possibility of achieving In the context of lentivirus-mediated CFTR gene transfer gene expression by lentiviral vectors, which outlasted the to the CF airways, biosafety aspects should be of primary normal lifespan of the respiratory epithelium, indicating concern. targeting of a ‘stem cell’ compartment. Modification of Gene Therapy (2004) 11, S67–S75. doi:10.1038/sj.gt.3302372 the paracellular permeability and pseudotyping with hetero-

Keywords: airway xenografts; CFTR; HIV-1; FIV; progenitor/stem cells; tight junctions; VSV-G

Introduction neutralizing antibodies against capsid3 and remains an important caveat. It has recently been demonstrated Cystic fibrosis (CF) is caused by recessive mutations in the that DNA of bacterial origin produces inflammation CFTR (cystic fibrosis transmembrane conductance regu- when the DNA/cationic lipid complex is instilled in the lator) gene, which encodes a chloride channel residing animal’s lung, highlighting a possible toxicological effect in the epithelium of multiple affected organs. CF lung of multiple applications of lipids.4 disease, characterized by thick mucus and inflammation, A new approach to gene therapy of CF is to target and bacterial infections, is responsible for most morbidity lung airway epithelium with integrating vectors. The and mortality in CF patients.1 Phase I gene therapy trials integration of the therapeutic gene is highly desirable for the treatment of CF lung disease have demonstrated to overcome transient CFTR expression and in the that CFTR cDNA delivery into respiratory epithelial cells perspective of airway ‘stem cell’ targeting. Recombinant is feasible, but clinical effects are still remote.2 In addition, adeno-associated virus (AAV), Moloney murine leuke- duration of gene expression has been shown to be limited mia virus (MLV) and lentiviral vectors address the in time. This means that more efficient gene transfer problem of poor persistence due to their ability to vectors possessing a long-lasting gene expression profile integrate. rAAV-CFTR vectors can achieve long-term should be developed for effective therapy of CF. gene transfer and expression of more than 6 months in Most of the gene transfer systems used in CF patients the airways of rabbits5 and monkeys6 and up to 2 months (cationic liposomes and adenoviruses) result only in in CF patients.7 However, studies in vitro and in vivo episomal maintenance of the vector DNA, which is likely in monkeys indicate that the vector DNA persists as to contribute at least in part to the limited expression concatemers that are episomal, in contrast with the profile of 1–4 weeks seen in man. This lack of genomic naturally occurring form of the virus.8–10 Early attempts integration brings the need for repeated administrations. to accomplish persistent expression with MLV vectors For adenovirus-based vectors, the reduced efficacy on were inefficient because transduction with this vector repeated administrations is likely related in part to system requires cell division and the proliferative activity of the target airway epithelial cells is low.11 Correspondence: Professor M Conese, Institute for Experimental Treat- The development of vectors based on lentiviruses ment of Cystic Fibrosis, HS Raffaele, Via Olgettina 58, 20132 Milano, holds the promise of achieving long-term expression in Italy the airways thanks to integration into both nondividing Lentivirus-mediated gene transfer in CF E Copreni et al S68 cells and cycling progenitor cells. This is due to mito- cells both in vitro and in vivo.27 Moreover, studies sis-independent nuclear import of the preintegration with HIV-1-derived lentiviral vectors to date show no complex.12 evidence of cellular immune response at the sites of The reader is referred to recent state-of-the-art reviews administration in vivo.28 for the design and biosafety of lentiviral vectors.13–17 This FIV-derived vectors hold the property of transducing review will describe some features of lentiviral vectors nondividing cells, do not cause detectable human and will then focus on the results achieved with infection or disease despite prevalent and presumably lentivirus-mediated transduction of the airway epithe- efficient human inoculation, and there is no significant lium in in vivo preclinical models of the CF lung disease. nucleotide-level homology between FIV and HIV-1. Similar to HIV-1, FIV has been modified to achieve biosafety, including removal of accessory genes29 and 30 Lentiviral vectors substitution of rev with heterologous export sequences. FIV-derived vectors have been used to target cells in Although various lentiviruses from different species the brain, eye, hematopoietic system, liver, muscle, have been used to generate gene transfer vectors,14 only and pancreas.15 human immunodeficiency virus-1 (HIV-1) and feline immunodeficiency virus (FIV) have been considered in the context of the airway epithelium and CF gene Gene transfer to the airway epithelium therapy. The prototype for this vector system is HIV-1 and thus Several hurdles limit the successful in vivo application a number of replication-defective versions of HIV-1 have of gene transfer to the airway epithelium, including been developed as vectors. Naldini et al18 described an anatomical and physical barriers,31 and immune re- HIV-1-derived vector that expressed the core proteins, sponse to vector-encoded proteins.32 In particular, tight enzymes, and accessory factors from heterologous junctions and restricted expression of viral receptors transcriptional signals and the envelope from the are formidable barriers for most vectors.33 It has been vesicular stomatitis virus (VSV)-G protein from a established that the expression of viral receptors is more separate plasmid. VSV-G is used to modulate virus abundant on the basolateral than on the apical surface interaction with the host immune system and to broaden of the airway epithelium. For example, MLV-derived substantially its host range.19 The utility of this vector for vector gene transfer to proliferating airway epithelia transducing neurons in vivo, and mediating persistence was shown to be inefficient from the apical surface in of transgene expression for several months, has been vitro and in vivo, likely due to the paucity of cellular described.18,20 In a second version of the system, the receptors.34,35 In order to overcome this constraint, HIV-1 derived packaging component was reduced to the conditioning of the airway tight junction prior to gag, pol, tat, and rev genes, while the virulence genes env, lentivirus-mediated gene transfer has been carried out. vif, vpu, and nef were deleted.21 At this stage, the system On the other hand, gene transfer to the airways has been was composed of three plasmids: the packaging vector, achieved without conditioning, by switching to other the transfer vector, and the envelope vector. Further pseudotypes. vector development included the replacement of tat-dependent U3 sequence from the 50-long terminal Gene transfer with preconditioning repeat (LTR) by strong heterologous promoter sequences In the model of adult airway xenograft, human airway and expression of rev in trans.22 Finally, a deletion in the epithelial cells from bronchi are seeded onto the surface of U3 region of the 30-LTR allowed to create a so-called self- denuded rat tracheas that are implanted subcutaneously inactivating (SIN) vector,23 by abolishing the transcrip- onto nu/nu mice.11,36–38 The undifferentiated epithelial tional activity of the LTR and thereby increasing the cells initially form a proliferating monolayer of squamous vector biosafety profile. cells with a basal cell phenotype that evolves into a fully In order to increase the efficiency of gene transfer, differentiated pseudostratified epithelium with morpho- some additional cis-acting regulatory sequences have logic and functional features identical to that of native been incorporated into the lentivirus transfer vector human bronchus. This model has been used to evaluate backbone. The introduction of the polypurine tract the biology of gene transfer with a number of vector located in the central position (cPPT), which has been systems, including adenovirus36 and MLV-based retro- shown to facilitate nuclear translocation of preintegration virus.11 Preliminary studies on the efficiency of HIV-1- complexes, into HIV-1-derived vector constructs has led derived vectors in transducing the respiratory epithelium to an increase in the percentage of transduced cells.24 of airway xenografts have shown that a proliferating and The insertion of the post-transcriptional regulatory differentiating epithelium, but not a truly differentiated element of the woodchuck (WPRE) has substantially one, is susceptible to transduction.39 increased the levels of expression from HIV-1-derived Recent studies have confirmed that HIV-1-derived vectors.25 Although WPRE activity was initially shown vectors could not transduce a fully differentiated airway to be promoter-independent,25 recent work indicates epithelium unless it was injured. An HIV-1-derived that WPRE increases gene expression only with a vector transduced polarized tracheal epithelial cells from limited series of promoters in human epithelial and the apical surface only in the presence of the calcium hematopoietic stem cells (HSCs).26 HIV-1-derived vectors chelator ethyleneglycol-tetraacetic acid (EGTA) and, have shown promise in the transduction of several however, at a much lower overall efficiency than was resting cell types such as retinal cells, liver cells, muscle observed for polarized alveolar epithelial cells.40 Johnson cells, pancreatic islets, cells of the central nervous et al41 showed that direct in vivo delivery of VSV- system, or progenitor and differentiated hematopoietic G-pseudotyped HIV-1-derived vector to the nasal

Gene Therapy Lentivirus-mediated gene transfer in CF E Copreni et al S69 epithelium of mice and rats failed to mediate gene transfer unless inhalation exposure to sulfur dioxide

(SO2) was applied. Vector was instilled 1–2 h after SO2 administration. SO2 injury also enhanced HIV-1-derived vector-mediated gene transfer to the tracheas of rodents. In another study, an HIV-1-derived LacZ vector was administered 1 h after instillation of a detergent in the murine airways, 1% lysophosphatidylcholine (LPC), resulting in the most effective pretreatment reagent.42 Preconditioning of the murine airways with LPC permitted sustained gene expression up to 92 days, suggesting that transduction of airway progenitor cells had occurred. Indeed, the cell turnover time of rodent airway epithelium is thought to be in the order of approximately 3 months.43 Similar results were obtained with FIV-derived vectors. Wang et al44 have shown that a VSV-G-pseudotyped FIV-derived vector formulated with EGTA transduced fully differentiated human airway epithelia when applied to the apical surface in vitro. Pretreatment of rabbit tracheas with EGTA solution for 30–60 min prior to vector application resulted in transduction of 1–14% of epithelial cells, mostly of lower airways (basal cells, Clara cells, and alveolar type II cells). Gene transfer persisted in 0.4–5.4% cells for 6 weeks in vivo. Overall, these results may indicate entry block due to the absence or low numbers of VSV-G receptors on the apical membrane of the airway epithelium, as it has been identified for other viral vectors, or postentry block, concerning the endocytosis route and nuclear import of viral genomes.34,35,45–49 However, preliminary studies in our laboratory demonstrate that a last-generation VSV- G-pseudotyped HIV-1-derived vector, which had been shown to be highly efficient due to the presence of cPPT and WPRE elements,24 transduced the murine airway epithelium both at the level of bronchial (Figure 1) and alveolar compartments. These results were confirmed in the alveoli by epifluorescence. A detectable, although Figure 1 Lentivirus-mediated gene transfer to the murine respiratory epi- weaker, green fluorescent protein (GFP) expression was thelium in the absence of tight junction preconditioning. A lentiviral vector found at 1 week postinjection (not shown). Kobinger pseudotyped with the envelope of VSV-G, carrying GFP as reporter gene et al50 have found that animals receiving EboZ-pseudo- and the cytomegalovirus promoter (pRRLsin.cPPT.CMV.EGFP.Wpre),24 typed vector demonstrated minimal expression at day 7, was injected intratracheally in C57BL/6 mice at a dose of 4.5 Â 106 TU. but strong expression in the airway epithelium by day 28 Mice were killed after 48 h. (a) Control mouse injected with PBS. (b) Mouse that persisted at day 63. These results, along with ours, injected with the LV-GFP vector. The arrow points to untransduced cells. Original magnification: Â 400. indicate that the inflammation response to lentiviral infection might have shut-off the CMV promoter at early times, the activity of which recovered at later times. Alternatively, transduction of the airway epithelia in vivo be involved in the transduction of airway epithelial through the apical membrane (without disturbing the cells by these vectors. These studies will be validated by homeostasis of the cell or integrity of the epithelium) investigating the expression of HS and/or other GAGs may result in delayed and/or low expression of the in polarized cells in vitro, as well as in the murine transgene. airways and in human respiratory xenografts. Heparan sulfate (HS) and possibly other glycosamino- glycans (GAGs) have been suggested to act as initial Gene transfer without preconditioning attachment sites for VSV-G-pseudotyped MLV51 and Based on the inability of VSV-G-pseudotyped lentiviral shown to be involved in HIV-152–54 and AAV47 infection. vectors to efficiently infect the airway epithelium in the The expression of this GAG by human respiratory cells absence of injuring agents, researchers have explored and its involvement in lentivirus-mediated gene transfer other heterologous envelope glycoproteins. HIV-1-de- were studied in our laboratory in immortalized human rived vectors packaged using Marburg virus or the Zaire respiratory cells. HS is expressed by 85–88% of cells, subtype Ebola virus glycoproteins mediated infection as shown by cytofluorimetric analysis (Figure 2a). of a comparable wide range of mammalian cell types.55 Preincubation of VSV-G-pseudotyped HIV-1-derived HIV-1-derived vectors pseudotyped with the envelope vector with heparin, a soluble analog of HS, determined from the Zaire strain of the Ebola virus (EboZ) gave a dose-dependent inhibition of transduction up to 65% efficient transduction (70% of positive cells) when of untreated controls (Figure 2b), indicating that HS may applied to the apical side of polarized epithelia.50

Gene Therapy Lentivirus-mediated gene transfer in CF E Copreni et al S70

Figure 2 HS is expressed by respiratory epithelial cells and is involved in transduction of human respiratory epithelial cells by a VSV-G-pseudotyped HIV-1 based vector. (a) Human bronchial (16HBE-S1)106 and tracheal (CFT1-C2)107 cells were stained with FITC-labeled antibody raised against HS and analyzed by means of cytofluorimetry. The filled curve in each panel refers to cells not incubated with the anti-HS antibody and represents background fluorescence. (b) The pRRLsin.cPPT.CMV.EGFP.Wpre vector was preincubated with increasing concentrations of heparin and then incubated with 16HBE- S1 cells in the absence of polybrene. GFP-positive cells were evaluated by cytofluorimetry. The virus was used at 50 MOI (multiplicity of infection) and 2.5 Â 107 TU/ml. Data are expressed as means7s.e. of two experiments. Asterisks denote statistically significant difference between each pretretated virus as compared to untreated virus (Po0.05).

Ex vivo transduction of human trachea by EboZ- Correction of the CF defect pseudotyped LacZ vector resulted in high levels of b-galactosidase expression as compared to VSV-G-pseu- CFTR is a cAMP-regulated chloride channel expressed dotyped vector. When EboZ-pseudotyped virus was on the apical side of most epithelia, and the conse- injected in the trachea of immunocompetent mice, 30% quences of its loss or altered activity due to mutations are of the entire tracheal epithelium was transduced at day found primarily at the electrophysiological level.58 The 28 and 24% at day 63. Interestingly, high expression was impairment in chloride secretion and sodium absorption observed in submucosal glands (an average of 65% of results in volume depletion in the airway surface fluid cells), while transduction efficiency was lower in the and/or surface liquid tonicity, leading ultimately to a epithelia of more distal lung (airway and alveolar) cells. disruption of innate immune defenses against opportu- In order to increase safety characteristics, EboZ-HIV-1- nistic pathogens.59 derived vector was redesigned by using mutant EboZ A first-generation VSV-G-pseudotyped HIV-1-derived envelopes for pseudotyping. One of those vectors vector was able to transduce wild-type CFTR into poorly showed higher efficiency than the parental vector in differentiated human bronchial xenografts and its ex- transducing murine airways and this result was also pression normalized both the chloride transport defect obtained for an FIV-based vector.56 and reverted the bacterial killing activity.39 Primary Envelopes from influenza virus and respiratory organotypic cultures of human trachea from patients syncytial virus did not promote efficient transduction with CF transduced with a VSV-G-pseudotyped FIV- of human airway cells by HIV-1-derived vectors from derived vector demonstrated ClÀ secretion in response the apical side, although these viruses commonly cause to cAMP agonists in a fashion similar to normal airway severe lung infections.50 A problem with the damage epithelia.44 The correction of ClÀ secretory response of envelopes during ultracentrifugation (a step used to remained stable for 6 months. concentrate virus) was envisioned by the authors as The only study that has so far investigated the efficacy the possible cause for this failure. A preliminary study of CFTR transduced by a lentiviral vector in a murine with Ross River Virus-pseudotyped FIV-derived vectors model for CF is the one by Limberis et al.42 Transduction showed the incapacity of this virus to infect airway cells of the CFTR gene using an HIV-1-derived lentiviral by the apical side in vivo.57 vector formulated with LPC resulted in partial recovery Comparison among different pseudotyped-lentiviral vec- of ClÀ transport defect (evaluated as transepithelial tors in transducing the airway epithelium ex vivo and in vivo potential difference) in the nasal airway epithelium of in human tissues and animal models is shown in Table 1. CF mice for at least 110 days.

Gene Therapy Lentivirus-mediated gene transfer in CF E Copreni et al S71 Table 1 Transduction of the airways ex vivo and in vivo by pseudotyped lentiviral vectors

Virus/pseudotype Route of administration (human or animal model of airway epithelium) Transduction efﬁciency Conditioning treatment Reference

HIV VSV-G Intraparenchymal injection (human xenografts) Low — 39 VSV-G Intratracheal and nasal instillation (mice, rats) High Sulfur dioxide 41 VSV-G Nasal instillation (mice) High LPCa 42 VSV-G Intratracheal injection (human fetal xenografts) High — 69 EboZ Human tracheal explants/intratracheal instillation (mice) High — 50, 56

FIV VSV-G Intratracheal and intrabronchial instillation (rabbits) Low — 44 VSV-G Intratracheal and intrabronchial instillation (rabbits) High EGTAb 44 EboZ Intratracheal instillation (mice) High — 56 RRV Nasal instillation (mice) Low — 57 aLysophosphatidylcholine. bEthyleneglycol-tetraacetic-acid.

Targeting the stem/progenitor cell 6–12 weeks into tracheal or pulmonary structures, including a pseudostratified ciliated and secretory sur- compartment in the CF airways face epithelium, submucosal glands and cartilage as well The possibility of targeting individual cell types in the as alveolar structures and interstitium.68 Using an HIV-1- airways is of particular concern in the CF context. It is derived vector in the human fetal tracheal xenograft believed that bronchioles are the primary site of the onset model, transgene expression was observed in up to 99% of CF lung disease. In this anatomical location, the targets of the surface epithelial cells and the submucosal gland of gene therapy vectors are ciliated and secretory cells of cells up to 9 months after vector administration.69 airway surface epithelium.60 However, the predominant location of CFTR expression in healthy lungs is sub- Bone marrow cells as a source for reconstituting mucosal gland serous cells,61 which are found in the the airway epithelium trachea and bronchi but not in the bronchioles. Finally, it Alternatively to ‘hit’ a local stem cell compartment in the has to be taken into consideration that CFTR is normally lung, it may be conceivable to induce circulating adult expressed by type II pneumocytes,62 but the role of HSCs to home to the damaged respiratory epithelium alveolar CFTR absence/mutation in the CF lung disease during regeneration. This concept has been exploited for is not known yet. It is probably true that one or more muscle regeneration, in view of a cell therapy approach strategies will need to be specifically developed for the to muscular dystrophy.70 So far, some experimental targeting of each cell type in CF. evidences have been provided for HSCs to be able to The final goal of gene therapy of CF is to correct transdifferentiate into epithelial cells. After transplanta- the target cellular compartment(s) involved in the lung tion of bone marrow (BM) or enriched HSCs into disease in a definitive way. This could be obtained by irradiated recipient animals, engraftment of BM-derived targeting the ‘stem’ cell compartment of the respiratory cells in the liver, lung, gut, and skin epithelia71,72 have epithelium with a lentiviral vector bearing the CFTR been detected. Krause et al71 found that the injection of cDNA. The identity of a resident pulmonary multipotent single BM-derived stem cells homed to the columnar ‘stem’ cell is still a challenge; however, it is clear that respiratory epithelium and to the alveoli. Kotton et al73 local stem or precursor cells contribute to repopulate injected BM-derived cells in the tail vein and demon- the injured epithelium in different anatomical regions.63 strated engraftment in recipient lungs as cells possessing Thus, we are now aware of the local repopulating cells of both the morphologic and molecular phenotype of type I trachea and bronchi (basal and mucous secretory), pneumocytes. Moreover, engraftment was facilitated bronchioles (Clara), and alveoli (type II pneumocytes).64 after belomycin-induced lung injury. Lineage analysis using recombinant retroviruses has In humans, evidence from studies in recipients of demonstrated pluripotent progenitor cells within the allogeneic BM transplants shows that BM-derived cells airway epithelium, which also retain the capacity for can differentiate into cells other than blood.74 A recent submucosal gland development.65 Finally, the picture is study on human lung allografts in patients who under- further complicated by the localization of stem cells for went lung transplantation and subsequent organ failure surface airway epithelium to upper tracheal gland ducts has shown that integration of recipient-derived cells in and to distinct foci in the glandless distal trachea.66 the bronchial epithelium, in type II pneumocytes, and in Overall, the studies previously mentioned39,41,42,44 seromucous peribronchial glands had occurred.75 Most indicate that both HIV-1- and FIV-derived vectors notably, analysis of nasal epithelium of patients after mediate long-term gene expression by targeting a gender-mismatched BM transplantation did not show ‘progenitor cell’ compartment. A recent report strongly integration of donor cells at the level of respiratory suggests such a possibility. Human fetal airway xeno- epithelium in the healthy upper airway.76 grafts are obtained by transplantation of embryonic and Overall, these results strongly suggest that HSCs have fetal lung rudiments into xenotolerant severe combined a great potential for replacement of diseased or degen- immunodeficiency (SCID) mice.67 Proximal or distal erating cell populations, tissues, and organs, and show airway primordia grow rapidly and differentiate after promise for a stem cell gene therapy approach.77 CF

Gene Therapy Lentivirus-mediated gene transfer in CF E Copreni et al S72 is characterized by chronic respiratory infections by effective toxicity in vivo101 and on the possibility of opportunistic pathogens that cause remodeling78 and disrupting the tight junction integrity transiently.34,102,103 proliferation79 of the airway epithelium. Thus, the Additional studies are necessary for establishing challenge will be to unveil and characterize those cellular whether lentivirus-based vectors can be considered compartments that are involved in these processes and adequate for human trials. First, it is not known which target them with HSCs transduced with a CFTR immune responses, if any, are generated in response to lentivirus. A recent report has demonstrated that this transduction with these viruses. Kobinger et al50 have is feasible with retrovirally transduced BM cells. Trans- reported that in vivo delivery of EboZ-pseudotyped gene-expressing lung epithelial cells were present in all HIV-1-derived vector in tracheas of C57Bl/6 mice did recipients analyzed at 2, 5, or 11 months after transplant not induce inflammatory infiltrates at any time points and 1–7% of cytokeratin-positive cells in the lung were postinfection. Moreover, they50 and others44 observed eGFP-positive.80 In this study, BM cells were cultured persistent expression in mice and rabbits, which suggests in growth factor-supplemented medium and irradiated ‘immunotolerance or ignorance’ of the transgene in those viral producer cells for 72 h prior to transplantation, animal models. Humoral immune response to the and this protocol may have determined that only envelope protein is another concern in virus-mediated pneumocytes of recipient mice were transduced. gene transfer, and this should be taken into consideration in the lentivirus field and may dictate whether read- Lentiviral versus oncoretroviral vectors in transducing ministration will be possible. Other safety concerns such HSCs as generation of replication-competent HIV-1-derived Most HSCs are in a quiescent state,81 are relatively slow vectors, mutagenesis following integration, or recombi- to respond to stimulation,82–84 and when induced to nation during wild-type HIV infection will have to be divide tend to lose long-term repopulating capacity.85–90 addressed. In addition, the relative paucity of viral receptors on the Finally, although lentivirus vectors could be highly surface of HSCs may limit binding of virus and further efficient in transducing the airway epithelium, they prevent efficient gene transfer.91 LV vectors pseudotyped might not be evaluated for their efficacy unless adequate with VSV-G offer a potential solution to the dual problem surrogate end points for CF will be studied. It is of quiescence and low viral receptor expression inherent currently unknown whether increased CFTR expression to transduction of HSCs with MLV.92 or improved electrophysiology in airway epithelium Successful transduction of human CD34+ HSCs and constitutes successful gene therapy. Besides chloride and long-term engraftment of non-obese diabetic (NOD)/ sodium transport across the airways, surrogate end SCID mice with these cells, using a short-term exposure points should be used in evaluating vector efficacy, to lentiviral vector in the absence of cytokines, has been comprising inflammatory and infection parameters. shown with both early-generation93 and late-genera- Recently, it has been shown that in an AAV-CFTR gene tion24,94 HIV-1-derived vectors. While MLV vectors were therapy trial, a decrease in IL-8 level in the sputum of 104 unable to transduce unstimulated cells or were less treated CF patients could be determined. Cftr knock- efficient in the transduction of the cytokine-treated cells, out mice receiving a helper-dependent adenoviral vector HIV-1-derived vectors efficiently transduced purified in the airways 7 days before respiratory bacterial 93,95–98 challenge had less severe histopathology and recovered cells in G0/G1. The possibility of transducing 105 quiescent cells may enable BM-derived HSCs to maintain the bacterial clearance activity. These end points their pluripotency. This is particularly rewarding when should be assessed in the future in the context of considering that different cell types of the lung and lentivirus-mediated CFTR gene transfer. airway epithelium in the CF context may be eligible targets of gene therapy. Acknowledgements We thank A Follenzi and L Naldini for providing us with Concluding remarks and perspectives the lentiviral vector used in this study. This work was supported by Italian Cystic Fibrosis Research Foundation Transduction of the airway epithelium by lentiviral and Associazione Lombarda Fibrosi Cistica – ONLUS. vectors has been proved to be limited by the paucity of viral receptors on the apical surface. 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