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

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