Gene Transfer To Lung Research Articles

627 - Anti-CD20 permits secondary lung gene transfer: implications for gene therapy approaches in CF

627 - Anti-CD20 permits secondary lung gene transfer: implications for gene therapy approaches in CF

608 Anti-CD20 permits secondary lung gene transfer: implications for gene editing approaches in cystic fibrosis

608 Anti-CD20 permits secondary lung gene transfer: implications for gene editing approaches in cystic fibrosis

Long Noncoding RNA FENDRR Exhibits Antifibrotic Activity in Pulmonary Fibrosis.

Abnormal activation of lung fibroblasts contributes to the initiation and progression of idiopathic pulmonary fibrosis (IPF). The objective of the present study was to investigate the role of fetal-lethal noncoding developmental regulatory RNA (FENDRR) in the activation of lung fibroblasts. Dysregulated long noncoding RNAs in IPF lungs were identified by next-generation sequencing analysis from the two online datasets. FENDRR expression in lung tissues from patients with IPF and mice with bleomycin-induced pulmonary fibrosis was determined by quantitative real-time PCR. IRP1 (iron-responsive element-binding protein 1), a protein partner of FENDRR, was identified by RNA pulldown-coupled mass spectrometric analysis and confirmed by RNA immunoprecipitation. The interaction region between FENDRR and IRP1 was determined by cross-linking immunoprecipitation. The in vivo role of FENDRR in pulmonary fibrosis was studied using adenovirus-mediated gene transfer in mice. The expression of FENDRR was downregulated in fibrotic human and mouse lungs as well as in primary lung fibroblasts isolated from bleomycin-treated mice. TGF-β1 (transforming growth factor-β1)-SMAD3 signaling inhibited FENDRR expression in lung fibroblasts. FENDRR was preferentially localized in the cytoplasm of adult lung fibroblasts and bound IRP1, suggesting its role in iron metabolism. FENDRR reduced pulmonary fibrosis by inhibiting fibroblast activation by reducing iron concentration and acting as a competing endogenous RNA of the profibrotic microRNA-214. Adenovirus-mediated FENDRR gene transfer in the mouse lung attenuated bleomycin-induced lung fibrosis and improved lung function. Our data suggest that FENDRR is an antifibrotic long noncoding RNA and a potential therapeutic target for pulmonary fibrosis.

Tetrafunctional Block Copolymers Promote Lung Gene Transfer in Newborn Piglets

Tetrafunctional block copolymers are molecules capable of complexing DNA. Although ineffective in vitro, studies in mice have shown that the tetrafunctional block copolymer 704 is a more efficient lung gene transfer agent than the cationic liposome GL67A, previously used in a phase II clinical trial in cystic fibrosis patients. In the present study, we compared the gene transfer capacity of the 704-DNA formulation and a cationic liposome-DNA formulation equivalent to GL67A in a larger-animal model, the newborn piglet. Our results indicate an efficacy of the 704-DNA formulation well above one order of magnitude higher than that of the cationic liposome-DNA formulation, with no elevated levels of interleukin-6 (IL-6), taken as a marker of inflammation. Transgene expression was heterogeneous within lung lobes, with expression levels that were below the detection threshold in some samples, while high in other samples. This heterogeneity is likely to be due to the bolus injection procedure as well as to the small volume of injection. The present study highlights the potential of tetrafunctional block copolymers as non-viral vectors for lung gene therapy.

In Vivo Genetic Manipulation of Spermatogonial Stem Cells and Their Microenvironment by Adeno-Associated Viruses

SummaryAdeno-associated virus (AAV) penetrates the blood-brain barrier, but it is unknown whether AAV penetrates other tight junctions. Genetic manipulation of testis has been hampered by the basement membrane of seminiferous tubules and the blood-testis barrier (BTB), which forms between Sertoli cells and divides the tubules into basal and adluminal compartments. Here, we demonstrate in vivo genetic manipulation of spermatogonial stem cells (SSCs) and their microenvironment via AAV1/9. AAV1/9 microinjected into the seminiferous tubules penetrated both the basement membrane and BTB, thereby transducing not only Sertoli cells and SSCs but also peritubular cells and Leydig cells. Moreover, when congenitally infertile KitlSl/KitlSl-d mouse testes with defective Sertoli cells received Kitl-expressing AAVs, spermatogenesis regenerated and offspring were produced. None of the offspring contained the AAV genome. Thus, AAV1/9 allows efficient germline and niche manipulation by penetrating the BTB and basement membrane, providing a promising strategy for the development of gene therapies for reproductive defects.

MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

Aberrant proliferation and activation of lung fibroblasts contribute to the initiation and progression of idiopathic pulmonary fibrosis (IPF). However, the mechanisms responsible for the proliferation and activation of fibroblasts are not fully understood. The objective of this study was to investigate the role of miR-101 in the proliferation and activation of lung fibroblasts. miR-101 expression was determined in lung tissues from patients with IPF and mice with bleomycin-induced pulmonary fibrosis. The regulation of miR-101 and cellular signaling was investigated in pulmonary fibroblasts in vitro The role of miR-101 in pulmonary fibrosis in vivo was studied using adenovirus-mediated gene transfer in mice. The expression of miR-101 was down-regulated in fibrotic lungs from patients with IPF and bleomycin-treated mice. The down-regulation of miR-101 occurred via the E26 transformation-specific (ETS) transcription factor. miR-101 suppressed the WNT5a-induced proliferation of lung fibroblasts by inhibiting NFATc2 signaling via targeting Frizzled receptor 4/6 and the TGF-β-induced activation of lung fibroblasts by inhibition of SMAD2/3 signaling via targeting the TGF-β receptor 1. Adenovirus-mediated miR-101 gene transfer in the mouse lung attenuated bleomycin-induced lung fibrosis and improved lung function. Our data suggest that miR-101 is an anti-fibrotic microRNA and a potential therapeutic target for pulmonary fibrosis.

Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene...

Cystic fibrosis gene therapy: a mutation-independent treatment.

Since cloning of the disease-causing gene 27 years ago, the development of cystic fibrosis (CF) gene therapy has been pursued. Here, we will summarize key findings with a particular focus on recent developments. Almost 3 decades of research have highlighted the complexity of lung gene transfer and have generated a body of data that has recently led to the completion of a large phase IIB study. This trial has, for the first time, shown that nonviral gene transfer can, albeit modestly, stabilize lung function in CF and provides the impetus for further development of more potent gene transfer agents. Lentiviral vectors, specifically pseudotyped to enable entry into airway epithelial cells have most recently been developed. Persistent expression after a single dose and the ability to be administered repeatedly suggest that these viral vectors hold promise for the treatment of CF; a first-in-man clinical trial will shortly be initiated. Although the development of CF gene therapy has been slower than initially anticipated, recent progress has been encouraging and has renewed the interest of academics and industry to pursue lung gene therapy.

431. Lung Antibody Factory for Passive Immunisation Against Influenza

Influenza A is a major global health threat causing >500,000 deaths annually. Neither prior infections nor current vaccines provide lasting protection, mainly due to rapid antigenic evolution of the viral haemagglutinin (HA) protein. Broadly neutralising antibodies (bNAbs) isolated from vaccinated volunteers can provide passive immunity, but this approach is hindered by high antibody manufacturing costs and the relatively short half-life of antibody after delivery to the circulation. We hypothesise that using lentivirus pseudotyped with Sendai virus envelopes (rSIV.F/HN) to deliver genes encoding anti-influenza bNAbs to the lung could provide long-lasting passive immunity to widely divergent strains of influenza. We selected a novel bNAb that we isolated from vaccinated volunteers, namely T1-3B (VH1-69 germline family) that cross-reacts with multiple group 1 influenza A strains including H1 (A/PuertoRico/8/34 & A/Brisbane/59/2007), pandemic H1 (A/California/07/2009) and H5 (A/Vietnam/1203/2004). Delivery of rSIV.F/HN expressing Gaussia luciferase (GLux) reporter protein to the mouse lung (1e7 TU/mouse) resulted in persistent secretion of GLux into both bronchoalveolar lavage fluid (BALF) (1,370,000 RLU/µl; p<0.01) and systemically into the serum (1,000 RLU/µl; p<0.01), representing 18,000-fold, and 25-fold over background, respectively. We then generated rSIV.F/HN vectors expressing T1-3B antibody and showed detection of antibody in the serum (715 ng/ml; p<0.05) for up to 28 days post intranasal delivery of rSIV.F/HN (5e7 TU/mouse). Animals treated with rSIV.F/HN expressing T13B were partially protected against a lethal challenge with either 1,000 or 10,000 TCID50 (~10 and 100 LD50) of A/PuertoRico/8/34 (H1N1) influenza. They experienced a ~2-3 days delay in symptoms and, significantly reduced weight-loss - less than 20% weight loss in 50% (1,000 TCID50) and 20% (10,000 TCID50) treated animals, while all control animals lost >20% weight irrespective of influenza dose (p<0.001 and p<0.05 for 1,000 and 10,000 TCID50 respectively). One major advantage of rSIV.F/HN as a platform for bNAb passive immunization is the proven ability to achieve successful transgene expression after repeated lung vector administration (Mol Ther 18:1173, 2010; Am J Respir Crit Care Med 186:846, 2012), a major hurdle for all other viral vectors we have evaluated, including rAAV (J Virol 81:12360, 2007). Repeat administration of rSIV.F/HN vectors expressing alternative bNAbs, such as those that cross-react with both group 1 and 2 influenza, could provide a truly broad protection against a wide range of influenza strains. We speculate that during the next human influenza pandemic, prophylaxis provided by lung gene transfer may be feasible and more cost-effective and time-responsive than traditional vaccines or parenteral administration of therapeutic antibody.

534. Preparation for a First-in-Man Lentivirus Trial in Cystic Fibrosis Patients

Background: We have recently shown that non-viral gene therapy can stabilise the decline of lung function in cystic fibrosis (CF) patients. However, the effect was modest, and it is important to develop more potent gene transfer agents in parallel. F/HN-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in pre-clinical models. In preparation for a first-in-man CF trial using the lentiviral vector we have undertaken key translational pre-clinical studies. Methods: Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air liquid interface cultures to select the lead candidate; CFTR expression and function were assessed in CF models (knockout mice and human intestinal organoids) using this lead candidate vector. Toxicity was assessed and “benchmarked” against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. Results: A hybrid promoter consisting of the elongation factor 1α promoter and the CMV enhancer was most efficacious in both murine lungs and human air liquid interface cultures. The efficacy, toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector is stable in clinically relevant delivery devices. Conclusions: The data support progression of the F/HN pseudotyped lentiviral vector into a first-in-man CF trial due to start in Q2 2017. Regulatory-compliant toxicology studies are currently being performed.

82. Scalable, Animal-Free, Suspension-Based Production of Siv Lentiviral Vectors

We are developing lung gene transfer vectors to direct abundant, sustained transgene expression in three therapeutic compartments: (i) within lung cells to treat lung disorders such as cystic fibrosis (CF), (ii) secreted from lung cells into the lung lumen to treat viral infections and airway diseases such as alpha1 anti-trypsin deficiency, (iii) secreted from nasal and lung cells into the circulatory system to treat hematologic and a range of other disorders. Such diseases are associated with a high burden of care often involving frequent antibiotic treatments, physiotherapy and supportive systemic infusions; and considerable patient morbidity and mortality. We have developed an efficient lung gene transfer platform based on a European and US Pharmacopoeial compliant, minimal recombinant SIV lentiviral vector which is specifically pseudotyped with the Sendai virus envelope proteins F and HN (rSIV.F/HN). Initially, production of rSIV.F/HN vectors was established in a serum-dependent, calcium phosphate-mediated, transient transfection process. Here we report the development of a scalable, animal-free, cGMP-compliant suspension cell culture based upstream production (USP) method. Serum-free media screening identified a suspension cell growth conditions that support efficient (>70%) multiple plasmid transient transfection of HEK293T cells with a range of cGMP-compliant liposomal and polymeric gene transfer agents. Small volume (40–400mL) studies in Erlenmeyer shake flasks and Design Of Experiments statistical approaches identified robust growth and transfection conditions that yield ≥5e6 TU/mL of unpurified rSIV. F/HN vectors. These parameters supported transition to 1L and 5L scale WAVE bioreactor cultures, where virus yield is maintained and plasmid DNA usage (a high proportion of the cGMP cost of goods) has been reduced to 0.33mg/L. F/HN pseudotype specific DNA removal and virus activation steps are incorporated into the downstream purification (DSP) method which relies on anion-exchange membrane virus capture and hollow-fibre UF/DF for vector purification and final formulation. In 56 independent, pH-controlled WAVE bioreactor cultures, purified yield exceeds 2.0e9 TU/L for a range of reporter gene constructs with a variety of promoters and 1.2e9 TU/L for a range of CFTR constructs (p>0.05; n=42/14 respectively). The final product directs abundant in vivo airway gene transfer, transducing 14.1% epithelial cells in the murine lung (p<0.001; 8e8 TU rSIV.F/HN hCEF EGFP), and directing CFTR transgene expression at ~30% of endogenous CFTR mRNA levels in the ovine lung (p<0.001; 8e8 TU rSIV.F/HN hCEF soCFTR2), exceeding presumed therapeutic levels. While these regulator friendly USP/DSP approaches have been tuned to yield highly purified and potent rSIV.F/HN vectors, the methodology is readily transferrable to VSV-G pseudotype production.

Evaluation of tetrafunctional block copolymers as synthetic vectors for lung gene transfer

In the present study, we evaluated, in mice, the efficacy of the tetrafunctional block copolymer 704 as a nonviral gene delivery vector to the lungs. SPECT/CT molecular imaging of gene expression, biochemical assays, and immunohistochemistry were used. Our dataset shows that the formulation 704 resulted in higher levels of reporter gene expression than the GL67A formulation currently being used in a clinical trial in cystic fibrosis patients. The inflammatory response associated with this gene transfer was lower than that induced by the GL67A formulation, and the 704 formulation was amenable to repeated administrations. The cell types transfected by the 704 formulation were type I and type II pneumocytes, and transgene expression could not be detected in macrophages. These results emphasize the relevance of the 704 formulation as a nonviral gene delivery vector for lung gene therapy. Further studies will be required to validate this vector in larger animals, in which the lungs are more similar to human lungs.

P203 Development Of An Optimal F/hn Pseudotyped Siv Vector For Cf Gene Therapy

We are developing lung gene transfer vectors to treat acquired and inherited lung disorders such as cystic fibrosis, and have identified two platforms for efficient respiratory gene delivery: one non-viral system based on CpG-free plasmid DNA combined with cationic lipids (pDNA/GL67A), which has recently completed evaluation in a Phase IIb clinical study; and one novel viral system based on a recombinant simian immunodeficiency virus pseudotyped with the F/HN proteins of Sendai virus (rSIV. F/HN) to promote airway cell uptake. Here we report on the development of a “third generation” rSIV. F/HN vector suitable for use in the clinic. The vector is manufactured by transient transfection of cultured human cells using five producer plasmids, all of which have been engineered to be pharmacopoeia compliant. A variety of vector configurations, including a range of enhancers/promoters and transgenes, were evaluated in a panel of airway models. rSIV. F/HN vectors directed high-level, robust gene expression in fully differentiated human airway cells, human nasal brushings and human and sheep lung slices. In the mouse nose and lung, the preferred configuration directed up to x500-fold higher transgene expression than the non-viral platform, for the lifetime of the animal. Transgene expression was observed in 14.1% of lung epithelial cells (p

Delivery of genes into the CF airway

Gene therapy was suggested as a potential treatment for cystic fibrosis (CF), even before the identification of the CFTR gene. Initial enthusiasm has been tempered as it became apparent that...

In Utero Lung Gene Transfer Using Adeno-Associated Viral and Lentiviral Vectors in Mice

Virus-mediated gene transfer to the fetal lung epithelium holds considerable promise for the therapeutic management of prenatally diagnosed, potentially life-threatening inherited lung diseases. In this study we hypothesized that efficient and life-long lung transduction can be achieved by in utero gene therapy, using viral vectors. To facilitate diffuse entry into the lung, viral vector was injected into the amniotic sac of C57BL/6 mice on embryonic day 16 (term, ∼ 20 days) in a volume of 10 μl. Vectors investigated included those based on adeno-associated virus (AAV) (serotypes 5, 6.2, 9, rh.64R1) and vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-1-based lentivirus (LV). All vectors expressed green fluorescent protein (GFP) under the transcriptional control of various promoters including chicken β-actin (CB) or cytomegalovirus (CMV) for AAV and CMV or MND (myeloproliferative sarcoma virus enhancer, negative control region deleted) for LV. Pulmonary GFP gene expression was detected by fluorescence stereoscopic microscopy and immunohistochemistry for up to 9 months after birth. At equivalent vector doses (mean, 12 × 10(10) genome copies per fetus) three AAV vectors resulted in long-term (up to 9 months) pulmonary epithelium transduction. AAV2/6.2 transduced predominantly cells of the conducting airway epithelium, although transduction decreased 2 months after vector delivery. AAV2/9-transduced cells of the alveolar epithelium with a type 1 pneumocyte phenotype for up to 6 months. Although minimal levels of GFP expression were observed with AAV2/5 up to 9 months, the transduced cells immunostained positive for F480 and were retrievable by bronchoalveolar lavage, confirming an alveolar macrophage phenotype. No GFP expression was observed in lung epithelial cells after AAV2/rh.64R1 and VSV-G-LV vector-mediated gene transfer. We conclude that these experiments demonstrate that prenatal lung gene transfer with AAV vectors engineered to target pulmonary epithelial cells may provide sustained long-term levels of transgene expression, supporting the therapeutic potential of prenatal gene transfer for the treatment of congenital lung diseases.

Rapid identification of novel functional promoters for gene therapy

Transcriptional control of transgene expression is crucial to successful gene therapy, yet few promoter/enhancer combinations have been tested in clinical trials. We created a simple, desktop computer database and populated it with promoter sequences from publicly available sources. From this database, we rapidly identified novel CpG-free promoter sequences suitable for use in non-inflammatory, non-viral in vivo gene transfer. In a simple model of lung gene transfer, five of the six promoter elements selected, chosen without prior knowledge of their transcriptional activities, directed significant transgene expression. Each of the five novel promoters directed transgene expression for at least 14 days post-delivery, greatly exceeding the duration achieved with the commonly used CpG-rich viral enhancer/promoters. Novel promoter activity was also evaluated in a more clinically relevant model of aerosol-mediated lung gene transfer and in the liver following delivery via high-pressure tail vein injection. In each case, the novel CpG-free promoters exhibited higher and/or more sustained transgene expression than commonly used CpG-rich enhancer/promoter sequences. This study demonstrates that novel CpG-free promoters can be readily identified and that they can direct significant levels of transgene expression. Furthermore, the database search criteria can be quickly adjusted to identify other novel promoter elements for a variety of transgene expression applications.

The significance of plasmid DNA preparations contaminated with bacterial genomic DNA on inflammatory responses following delivery of lipoplexes to the murine lung

Non-viral gene transfer using plasmid DNA (pDNA) is generally acknowledged as safe and non-immunogenic compared with the use of viral vectors. However, pre-clinical and clinical studies have shown that non-viral (lipoplex) gene transfer to the lung can provoke a mild, acute inflammatory response, which is thought to be, partly, due to unmethylated CG dinucleotides (CpGs) present in the pDNA sequence. Using a murine model of lung gene transfer, bronchoalveolar lavage fluid was collected following plasmid delivery and a range of inflammatory markers was analysed. The results showed that a Th1-related inflammatory cytokine response was present that was substantially reduced, though not abolished, by using CpG-free pDNA. The remaining minor level of inflammation was dependent on the quality of the pDNA preparation, specifically the quantity of contaminating bacterial genomic DNA, also a source of CpGs. Successful modification of a scalable plasmid manufacturing process, suitable for the production of clinical grade pDNA, produced highly pure plasmid preparations with reduced genomic DNA contamination. These studies help define the acceptable limit of genomic DNA contamination that will impact FDA/EMEA regulatory guidelines defining clinical grade purity of plasmid DNA for human use in gene therapy and vaccination studies.

Jaagsiekte sheep retrovirus pseudotyped lentiviral vector-mediated gene transfer to fetal ovine lung

Viral vector-mediated gene transfer to the postnatal respiratory epithelium has, in general, been of low efficiency due to physical and immunological barriers, non-apical location of cellular receptors critical for viral uptake and limited transduction of resident stem/progenitor cells. These obstacles may be overcome using a prenatal strategy. In this study, HIV-1-based lentiviral vectors (LVs) pseudotyped with the envelope glycoproteins of Jaagsiekte sheep retrovirus (JSRV-LV), baculovirus GP64 (GP64-LV), Ebola Zaire-LV or vesicular stomatitis virus (VSVg-LV) and the adeno-associated virus-2/6.2 (AAV2/6.2) were compared for in utero transfer of a green fluorescent protein (GFP) reporter gene to ovine lung epithelium between days 65 and 78 of gestation. GFP expression was examined on day 85 or 136 of gestation (term is ∼145 days). The percentage of the respiratory epithelial cells expressing GFP in fetal sheep that received the JSRV-LV (3.18 × 10(8)-6.85 × 10(9) viral particles per fetus) was 24.6±0.9% at 3 weeks postinjection (day 85) and 29.9±4.8% at 10 weeks postinjection (day 136). Expression was limited to the surface epithelium lining fetal airways <100 μm internal diameter. Fetal airways were amenable to VSVg-LV transduction, although the percentage of epithelial expression was low (6.6±0.6%) at 1 week postinjection. GP64-LV, Ebola Zaire-LV and AAV2/6.2 failed to transduce the fetal ovine lung under these conditions. These data demonstrate that prenatal lung gene transfer with LV engineered to target apical surface receptors can provide sustained and high levels of transgene expression and support the therapeutic potential of prenatal gene transfer for the treatment of congenital lung diseases.

The AAV9 receptor and its modification to improve in vivo lung gene transfer in mice

Vectors based on adeno-associated virus (AAV) serotype 9 are candidates for in vivo gene delivery to many organs, but the receptor(s) mediating these tropisms have yet to be defined. We evaluated AAV9 uptake by glycans with terminal sialic acids (SAs), a common mode of cellular entry for viruses. We found, however, that AAV9 binding increased when terminal SA was enzymatically removed, suggesting that galactose, which is the most commonly observed penultimate monosaccharide to SA, may mediate AAV9 transduction. This was confirmed in mutant CHO Pro-5 cells deficient in the enzymes involved in glycoprotein biogenesis, as well as lectin interference studies. Binding of AAV9 to glycans with terminal galactose was demonstrated via glycan binding assays. Co-instillation of AAV9 vector with neuraminidase into mouse lung resulted in exposure of terminal galactose on the apical surface of conducting airway epithelial cells, as shown by lectin binding and increased transduction of these cells, demonstrating the possible utility of this vector in lung-directed gene transfer. Increasing the abundance of the receptor on target cells and improving vector efficacy may improve delivery of AAV vectors to their therapeutic targets.

A recombinant adenovirus for targeted gene therapy of pulmonary inflammation (155.23)

Abstract Inflammation is a pathological component of many debilitating lung diseases. Thus, therapeutics that suppress the inflammatory response should also minimize inflammation-induced airway destruction. In this regard, Adenovirus type 5 (Ad5) vectors have a long history as therapeutic agents, due to their in vivo stability and amenability to genetic manipulation. Since circulating leukocytes infiltrate and mediate damage to the sites of pathology, we proposed to modulate Ad5 tropism to the dense leukocyte pool in the lung microvasculature for localized therapeutic intervention of inflammation. However, leukocytes do not express the coxsackie and adenovirus receptor (CAR) and are thus resistant to Ad5 infection. Therefore, we utilized phage display to identify a myeloid-binding peptide (MBP) and then genetically incorporated this sequence into an Ad5 fiber lacking the native CAR-binding domain (knob). Non-replicative Ad reporter vectors containing these MBP-fibers (Ad.MBP) maintained myeloid-binding specificity, and upon intravenous delivery the altered tropism enhanced lung gene transfer by five orders of magnitude over that obtained with unmodified Ad5. Disparity between cell types bound and those transduced suggest Ad.MBP is first sequestered by circulating leukocytes and subsequently “handed-off” to transduce pulmonary endothelium. These studies highlight the importance of pulmonary leukocytes as a target for therapeutic delivery in inflammatory lung disease.