The use of CpG-free plasmids to mediate persistent gene expression following repeated aerosol delivery of pDNA/PEI complexes

Aerosol gene therapy offers great potential for treating acquired and inherited lung diseases. For treatment of chronic lung diseases such as cystic fibrosis, asthma and emphysema, non-viral gene therapy will likely require repeated administration to maintain transgene expression in slowly dividing, or terminally differentiated, lung epithelial cells. When complexed with plasmid DNA (pDNA), the synthetic polymer, 25?kDa branched Polyethylenimine (PEI), can be formulated for aerosol delivery to the lungs. We show that pDNA/PEI aerosol formulations can be repeatedly administered to airways of mice on at least 10 occasions with no detectable toxicity. Interestingly, peak reporter gene activity upon repeated delivery was significantly reduced by up to 75% compared with a single administration, despite similar pDNA lung deposition at each subsequent aerosol exposure. Although the precise mechanism of inhibition is unknown, it is independent of mouse strain, does not involve an immune response, and is mediated by PEI. Importantly, using a dosing interval of 56 days, delivery of a fourth-generation, CpG-free plasmid generated high-level, sustained transgene expression, which was further boosted at subsequent administrations. Together these data indicate that pDNA/PEI aerosol formulations offer a versatile platform for gene delivery to the lung resulting in sustained transgene expression suitable for treatment of chronic lung diseases.     

The safety profile of a cationic lipid-mediated cystic fibrosis gene transfer agent following repeated monthly aerosol administration to sheep

Clinically effective gene therapy for Cystic Fibrosis has been a goal for over 20 years. A plasmid vector (pGM169) that generates persistent expression and reduced host inflammatory responses in mice has raised prospects for translation to the clinic. The UK CF Gene Therapy Consortium is currently evaluating long-term repeated delivery of pGM169 complexed with the cationic lipid GL67A in a large Multidose Trial. This regulatory-compliant evaluation of aerosol administration of nine doses of pGM169/GL67A at monthly intervals, to the sheep lung, was performed in preparation for the Multidose Trial. All sheep tolerated treatment well with no adverse effects on haematology, serum chemistry, lung function or histopathology. Acute responses were observed in relation to bronchoalveolar cellularity comprising increased neutrophils and macrophage numbers 1 day post-delivery but these increases were transient and returned to baseline. Importantly there was no cumulative inflammatory effect or lung remodelling with successive doses. Molecular analysis confirmed delivery of pGM169 DNA to the airways and pGM169-specific mRNA was detected in bronchial brushing samples at day 1 following doses 1, 5 and 9. In conclusion, nine doses of pGM169/GL67A were well tolerated with no significant evidence of toxicity that would preclude adoption of a similar strategy in CF patients.     

Local gene delivery from ECM-coated poly(lactide-co-glycolide) multiple channel bridges after spinal cord injury

Tissue engineering scaffolds with complex geometries can provide an architecture that directs tissue formation. Drug delivery from these scaffolds to promote regeneration is often challenging due to the complex fabrication processes. Surface-mediated DNA delivery from multiple channel bridges was applied to deliver lipoplexes in vivo to the injured spinal cord. The surface properties of the polymer, DNA deposition with or without drying, and the presence of ECM components were investigated. In vitro studies revealed that fibronectin produced greater expression levels and immobilization efficiencies compared with collagen, laminin, and no coating. In addition, lipoplex incubation on ECM-coated PLG increased expression relative to either of the drying methods. Additionally, the incubation method had more homogeneously distributed lipoplexes and a higher number of transfected cells relative to the dried conditions. Translation to three-dimensional bridges led to high levels of transgene expression in vitro. In vivo, lipoplexes immobilized to the bridge produced transgene expression levels in a rat spinal cord hemisection model that were 2-fold greater than naked plasmid. Additionally, expression with lipoplexes persisted for at least three weeks. Surface-mediated delivery can be applied to scaffolds with complex geometries to promote transgene expression in vivo.     

Dextran-spermine-based polyplexes--evaluation of transgene expression and of local and systemic toxicity in mice

Gene delivery using self-assembled polyplexes, formed between negatively charged nucleic acids and cationic polymers, have several drawbacks including low transgene expression and toxicity effects related to their positive charge. Recently, a novel cationic polymer based on dextran-spermine (D-SPM) was developed for gene delivery. This polymer showed systemic biodistribution upon local administration (intramuscular (i.m.) and intranasal (i.n.)) followed by transgene expression in organs remote from the site of injection (liver and lungs). Polyplexes based on D-SPM were administered both i.m. and i.n. to BALB/c female mice. LacZ expression in the liver, lungs, and muscles was assessed using whole-mount 5-bromo-4-chloro-3-indolyl beta-d-galactopyranoside (X-gal) staining and paraffin sectioning. The local toxicity in these organs was evaluated from hematoxylin and eosin stained sections. The systemic toxicity of the polymer and polyplexes was estimated by comparing the mice total weight, major organ weights, blood counts (primarily white blood cells (WBC) and platelets), and serum transaminases, to those of control animals (which received PBS). Transgene expression using D-SPM polyplexes was dependent upon the dosage and the polyplexes (+/-) charge ratio. Using the i.m. and i.n. routes of administration the transfection occurred primarily in the bronchial epithelial cells, pneumocytes, and bronchial alveoli of the lungs; in the muscle's fibrocytes; and in the liver's hepatocytes. Histopathological assays revealed mild toxicity in muscle and no abnormal findings in liver and lung. No systemic toxicity was obtained, as we did not find decrease in WBC count or platelet and no increase in serum transaminases. In addition, mice body weights and major organ weights were similar to the control group at both 2 and 28 days post-administration. This study demonstrates systemic transfection efficacy upon local administration of D-SPM complexes with good tolerability and low toxicity.     

CpG-free plasmid expression cassettes for cystic fibrosis gene therapy

Clinical studies are underway for the aerosol delivery of plasmid DNA complexed with Genzyme Lipid GL67A to the lungs of patients with cystic fibrosis (CF). Plasmid vectors contain several functional elements all of which play a role in determining the efficacy of the final clinical product. To optimise the final plasmid, variations of CpG-free 5' enhancer elements and 3'UTR regions were inserted into a common CpG-free, plasmid backbone containing Luciferase or CFTR transgenes. Plasmids were compared in immortalised cell culture, human airway liquid interface primary cell cultures, and mouse lung models to determine which design directed optimal transgene expression. Following aerosol delivery to mouse lung, plasmids containing the murine CMV enhancer showed higher peak Luciferase activity than the human CMV enhancer, but the human version resulted in persistent expression. In cell culture, the SV40 3'UTR and a novel BGH2 3'UTR exhibited up to 20-fold higher Luciferase activity than the commonly used BGH 3'UTR, but in mouse lung aerosol studies the activity and duration was greater for BGH 3'UTR. Systematic evaluation of each functional component of the plasmid has resulted in an improved design, exhibiting superior levels and duration of lung gene expression.     

Building mosaics of therapeutic plasmid gene vectors

Plasmids are circular or linear DNA molecules propagated extra-chromosomally in bacteria. Evolution shaped plasmids are inherently mosaic structures with individual functional units represented by distinct segments in the plasmid genome. The patchwork of plasmid genetic modules is a convenient template and a model for the generation of artificial plasmids used as vehicles for gene delivery into human cells. Plasmid gene vectors are an important tool in gene therapy and in basic biomedical research, where these vectors offer efficient transgene expression in many settings in vitro and in vivo. Plasmid vectors can be attached to nuclear directing ligands or transferred by electroporation as naked DNA to deliver the payload genes to the nuclei of the target cells. Transgene expression silencing by plasmid sequences of bacterial origin and immune stimulation by bacterial unmethylated CpG motifs can be avoided by the generation of plasmid-based minimized DNA vectors, such as minicircles. Systems of efficient site-specific integration into human chromosomes and stable episomal maintenance in human cells are being developed for further reduction of the chances for transgene silencing. The successful generation of plasmid vectors is governed by a number of vector design rules, some of which are common to all gene vectors, while others are specific to plasmid vectors. This review is focused both on the guiding principles and on the technical know-how of plasmid gene vector design.     

Comparative Immunization Study Using RNA and DNA Constructs Encoding a Part of the Plasmodium falciparum Antigen Pf332

Development of nucleic acid-based vaccines against parasitic diseases shows great promise, although certain concerns about safety aspects of conventional DNA vaccines have been raised. This study presents a comparison of antibody responses induced in mice by DNA and RNA-based immunization with vectors encoding a part of the P. falciparum antigen Pf332. Two types of plasmids were used, one conventional DNA plasmid containing a cytomegalovirus promoter and one suicidal DNA plasmid encoding the Semliki Forest virus (SFV) replicase. RNA, encoding the SFV replicase and the relevant antigen, was delivered either as naked RNA or packaged in SFV suicide particles. In general, the antibody responses induced by the DNA plasmids were low and peaking after three injections, the conventional plasmid giving the highest responses. Also the RNA delivered in SFV particles consistently induced antibody responses, although comparatively low. Analyses of the ratio of immunoglobulin (Ig)G1/IgG2a subclasses in the responses indicated that all plasmids resulted in a bias for a Th2-type of response, while the SFV-particles elicited a Th1 type of response. Importantly, all these immunogens induced an immunological memory, which could be efficiently activated by a booster injection with the corresponding protein, with unchanged patterns of IgG subclasses.     

Engineering enhancement of the immune response to HBV DNA vaccine in mice by the use of LIGHT gene adjuvant

DNA vaccines could induce protective immune responses in several animal models. Many strategies have been employed to improve the effect of nucleic acid vaccines. LIGHT is a member of the TNF superfamily and functions as a co-stimulatory molecule for T cell proliferation. In the study, the immunogenicity in the induction of humoral and cellular immune responses by HBV DNA vaccine and the adjuvant effect of LIGHT were studied in a murine model. The eukaryotic expression plasmid pcDNA-L was constructed by inserting mouse LIGHT gene into the vector pcDNA3.1(+). In vitro expression of LIGHT was detected by RT-PCR and indirect immunofluorescence assay in transfected HeLa cells. MLR assay showed that LIGHT-transfected DCs induced markedly higher allogeneic lymphocyte proliferation than pcDNA-transfected DCs and untreated DCs at all dilutions. After BALB/c mice were immunized by three intramuscular injections of the HBV DNA vaccine plasmids alone or in combination with LIGHT expression plasmids, the different levels of anti-HBV immune responses were measured comparable to the control groups immunized with parent plasmid pcDNA or PBS. The HBsAg-specific splenocytes proliferation and specific cytotoxic activities of splenic CTLs in the coinoculation group were both significantly higher than those in the HBV DNA single inoculation group, and an enhancement of antibody response was also observed in the coinoculation group compared with the single inoculation group. Taken together, coimmunization of HBV DNA vaccine plasmids and LIGHT expression plasmids can elicit stronger humoral and cellular immune responses in mice than HBV DNA vaccine plasmids alone, and LIGHT may be an effective immunological adjuvant in HBV DNA vaccination.     

Characterization and in vivo performance of dextran-spermine polyplexes and DOTAP/cholesterol lipoplexes administered locally and systemically

In this study, we compared two systems which can be applied for transfection in vitro and in vivo: polyplexes based on the polymer dextran-spermine (D-SPM) and lipoplexes based on 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol. The carriers differ in (1) solubility in aqueous media, (2) source of the positive charges (quaternary amines for DOTAP and primary plus secondary amines for D-SPM), (3) electrostatics, i.e., for lipid and polymer, respectively: zeta-potential (81.0 and 48.1 mV), surface potential (180 and 92 mV), and surface pH (10.47 and 8.97), and (4) charge distribution (ordered versus non-ordered). The stability of the complex upon interaction with serum proteins was studied by means of fluorescence resonance energy transfer (FRET) between rhodamine-labeled cationic carriers and fluorescein-labeled DNA. Addition of serum increases the lipid-DNA average distance and decreases the polymer-DNA distance. However, FRET efficiency indicates that serum proteins do not induce a major DNA dissociation for either polyplexes or lipoplexes. Comparing the biodistribution of rhodamine-labeled complexes and the transgene expression after intravenous (i.v.), intramuscular (i.m.), and intranasal (i.n.) administration, we found that local administration of lipoplexes resulted in the lipoplexes remaining at the site of injection, whereas the polyplexes showed systemic distribution, accompanied by transgene expression in lungs and liver. It is suggested that the high water-solubility of the polymer combined with its lower positive charge (compared to DOTAP), which makes its association with the cells at the site of injection weaker, enables the polymer to reach and transfect distant organs through the blood stream. Using chemically modified D-SPM, we demonstrated the importance of high density of positive charges and a sufficient level of secondary amines for achieving efficient transgene expression in vivo.     

Modulating polymer chemistry to enhance non-viral gene delivery inside hydrogels with tunable matrix stiffness

Non-viral gene delivery holds great promise for promoting tissue regeneration, and offers a potentially safer alternative than viral vectors. Great progress has been made to develop biodegradable polymeric vectors for non-viral gene delivery in 2D culture, which generally involves isolating and modifying cells in vitro, followed by subsequent transplantation in vivo. Scaffold-mediated gene delivery may eliminate the need for the multiple-step process in vitro, and allows sustained release of nucleic acids in situ. Hydrogels are widely used tissue engineering scaffolds given their tissue-like water content, injectability and tunable biochemical and biophysical properties. However, previous attempts on developing hydrogel-mediated non-viral gene delivery have generally resulted in low levels of transgene expression inside 3D hydrogels, and increasing hydrogel stiffness further decreased such transfection efficiency. Here we report the development of biodegradable polymeric vectors that led to efficient gene delivery inside poly(ethylene glycol) (PEG)-based hydrogels with tunable matrix stiffness. Photocrosslinkable gelatin was maintained constant in the hydrogel network to allow cell adhesion. We identified a lead biodegradable polymeric vector, E6, which resulted in increased polyplex stability, DNA protection and achieved sustained high levels of transgene expression inside 3D PEG-DMA hydrogels for at least 12 days. Furthermore, we demonstrated that E6-based polyplexes allowed efficient gene delivery inside hydrogels with tunable stiffness ranging from 2 to 175 kPa, with the peak transfection efficiency observed in hydrogels with intermediate stiffness (28 kPa). The reported hydrogel-mediated gene delivery platform using biodegradable polyplexes may serve as a local depot for sustained transgene expression in situ to enhance tissue engineering across broad tissue types.     

HIV-1重组核酸疫苗质粒诱导小鼠免疫应答的研究

目的：研究白细胞介素6（IL－6）对人类免疫缺陷病毒（HIV）gp120基因免疫小鼠的调节作用。方法：构建表达中国流行株HIV－1B亚型gp120基因的核酸疫苗质粒pGP及共表达中国流行株HIV－1B亚型gp120基因与IL－6基因的核酸疫苗质粒pGPIL-6，检测其在哺乳动物细胞中的表达并 将上述两种质粒注射Balb/c鼠，检测小鼠产生的CD4^ 和CD8^ T细胞数情况及诱导CTL反应能力。结果：以间接免疫荧光法（IFA）检测到gp120在哺乳动物细胞中得到表达。pGPIL-6免疫鼠，小鼠产生的CD4^＋和CD8^ T细胞数及诱导产生CTL反应的能力明显高于单纯注射gp120的小鼠。结论：协同应用IL－6基因可明显加强HIV－1gp120基因免疫效果，为中国流行株HIV－1核酸疫苗的可行性提供了重要实验依据。     

Construction and evaluation of a eukaryotic expression plasmid for stable delivery using attenuated Salmonella

An approach to enhancing the stability of eukaryotic expression plasmids for delivery using attenuated Salmonella has been evaluated. The expression apparatus and beta-galactosidase gene from the expression plasmid, pCMVbeta, was cloned into the low copy number plasmid pLG339. The resulting construct, pLGbetaGAL, was shown to have a lower copy number than pCMVbeta in Salmonella enterica var Typhimurium aroA strain SL7207. Furthermore, beta-galactosidase-specific antibody was induced in mice following intramuscular inoculation with pLGbetaGAL as naked DNA. Following oral administration of mice with SL7207/pCMVbeta, recombinants could not be detected in tissues 3 days after inoculation. In comparison, SL7207/pLGbetaGAL recombinant bacteria could be detected in the Peyer's patches and spleens indicating that the Salmonella strain was stable. However, both SL7207/pCMVbeta and SL7207/pLGbetaGAL failed to induce beta-galactosidase-specific IgG in vivo. The mechanism by which attenuated Salmonella are able to release heterologous DNA for antigen processing and presentation is not yet understood. These results suggest that the mechanism needs to be further elucidated in order to rationally improve the system.     

Lifelong reporter gene imaging in the lungs of mice following polyethyleneimine-mediated sleeping-beauty transposon delivery

Polyethyleneimine (PEI) is a cationic polymer that is effective in gene delivery in vivo. Plasmid DNA incorporating the Sleeping-Beauty (SB) transposon has been shown to induce long-term transgene expression in mouse lungs after PEI-mediated delivery. In the current report, we followed the reporter gene expression mediated by PEI/SB delivery in lungs of mice using the non-invasive bioluminescent imaging (BLI) technology. After delivery, the reporter gene signal showed a rapid decay in the first two weeks to a nearly undetectable level, but then the signal augmented gradually in the following weeks and finally reached a stable level that maintained until the natural death of animals. The stabilization of transgene expression is associated with the multiplication of a small number of PEI/SB-labeled alveolar cells, which proliferated both under normal conditions and in response to acute local injury for epithelia repair, and may play a role in long-term homeostatic maintenance in alveoli. The data presented here suggests that systemic delivery of PEI/SB induces stable transfection specifically in a small population of alveolar progenitor cells. The technique provides a promising platform for future research in distal lung biology and tissue regenerative therapy.     

Development of S/MAR minicircles for enhanced and persistent transgene expression in the mouse liver

We have previously described the development of a scaffold/matrix attachment region (S/MAR) episomal vector system for in vivo application and demonstrated its utility to sustain transgene expression in the mouse liver for at least 6?months following a single administration. Subsequently, we observed that transgene expression is sustained for the lifetime of the animal. The level of expression, however, does drop appreciably over time. We hypothesised that by eliminating the bacterial components in our vectors, we could improve their performance since bacterial sequences have been shown to be responsible for the immunotoxicity of the vector and the silencing of its expression when applied in vivo. We describe here the development of a minimally sized S/MAR vector, which is devoid of extraneous bacterial sequences. This minicircle vector comprises an expression cassette and an S/MAR moiety, providing higher and more sustained transgene expression for several months in the absence of selection, both in vitro and in vivo. In contrast to the expression of our original S/MAR plasmid vector, the novel S/MAR minicircle vectors mediate increased transgene expression, which becomes sustained at about twice the levels observed immediately after administration. These promising results demonstrate the utility of minimally sized S/MAR vectors for persistent, atoxic gene expression.     

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.     

Prophylactic and therapeutic intervention in IgE responses by biolistic DNA vaccination primarily targeting dendritic cells

BACKGROUND: Allergen gene transfer represents an alternative approach to specific immunotherapy with allergen extracts. Gene gun-mediated DNA immunization with plasmid vectors expressing a transgene under control of the promoter of the fascin gene (pFascin) allows for antigen production predominantly by dendritic cells and resulted in the generation of CD8(+) cytotoxic T lymphocytes as well as in the development of a type 1 immune response. OBJECTIVE: We compared the in vivo efficiency of biolistic transfection with pFascin and plasmids containing the cytomegalovirus promoter (pCMV) in a mouse model of type I allergy. METHODS: BALB/c mice were sensitized with the model allergen beta-galactosidase to induce a distinctive type 2 immune response. The effect of prophylactic as well as therapeutic biolistic transfection with beta-galactosidase-encoding plasmids on the development of antibody titers was followed, and anaphylactic potential of sera was determined. Spleen cells were stimulated in vitro to analyze cytokine production and induction of CD8(+) effector T cells. RESULTS: Protective allergen gene transfer with pFascin efficiently prevented specific IgE production accompanied by immune deviation toward a T(H)1-polarized immune response as well as by the induction of IFN-gamma-producing CD8(+) effector T cells, being comparable to vaccination with pCMV. In a therapeutical setting, biolistic DNA vaccination with pFascin or pCMV transiently protected allergen-sensitized mice against the strong increase in specific IgE production caused by subsequent allergen challenge. CONCLUSION: We demonstrate for the first time that restricting transgene expression primarily to dendritic cells after DNA vaccination suffices to cause inhibition of IgE production prophylactically and therapeutically.     

pH-sensitive polymer-liposome-based antigen delivery systems potentiated with interferon-γ gene lipoplex for efficient cancer immunotherapy

Potentiation of pH-sensitive liposome-based antigen carriers with IFN-γ gene lipoplexes was attempted to achieve efficient induction of tumor-specific immunity. 3-Methylglutarylated poly(glycidol) (MGluPG)-modified liposomes and cationic liposomes were used, respectively, for the delivery of antigenic protein ovalbumin (OVA) and IFN-γ-encoding plasmid DNA (pDNA). The MGluPG-modified liposomes and the cationic liposome-pDNA complexes (lipoplexes) formed hybrid complexes via electrostatic interactions after their mixing in aqueous solutions. The hybrid complexes co-delivered OVA and IFN-γ-encoding pDNA into DC2.4 cells, a murine dendritic cell line, as was the case of MGluPG-modified liposomes for OVA or the lipoplexes for pDNA. Both the lipoplexes and the hybrid complexes transfected DC2.4 cells and induced IFN-γ protein production, but transfection activities of the hybrid complexes were lower than those of the parent lipoplexes. Subcutaneous administration of hybrid complexes to mice bearing E.G7-OVA tumor reduced tumor volumes, which might result from the induction of OVA-specific cytotoxic T lymphocytes (CTLs). However, the hybrid complex-induced antitumor effect was the same level of the MGluPG-modified liposome-mediated antitumor immunity. In contrast, an extremely strong antitumor immune response was derived when these liposomes and lipoplexes without complexation were injected subcutaneously at the same site of tumor-bearing mice. Immunohistochemical analysis of tumor sections revealed that immunization through the liposome–lipoplex combination promoted the infiltration of CTLs to tumors at an early stage of treatment compared with liposomes, resulting in strong therapeutic effects.     

Genetic delivery of an anti-RSV antibody to protect against pulmonary infection with RSV

Respiratory syncytial virus (RSV) is a common cause of severe lower respiratory tract infections. Protection against infection with RSV can be achieved by monthly administration of the humanized monoclonal antibody palivizumab. The present study analyzes if genetic delivery of a murine version of palivizumab by single administration would achieve high-level and sustained antibody expression to protect mice against pulmonary infection with RSV. A murine version of the palivizumab antibody was constructed by replacing the human sequences with sequences from the constant region of a murine IgG1 antibody, while preserving the complementarity-determining region. As a proof-of-principle to test the validity of the strategy, the coding sequence for the heavy and light chains were cloned into a replication-defective serotype 5 human adenovirus vector (AdalphaRSV). Antibody expression and specificity for RSV was confirmed by Western analysis. To determine if AdalphaRSV would mediate production of anti-RSV antibodies in vivo, 5x10(10) particle units of AdalphaRSV or a control vector without transgene (AdNull), were administered intravenously to BALB/c mice. RSV neutralizing antibodies were detected in the serum after 4 days in mice receiving AdalphaRSV but not in AdNull-infected or naive mice (p<0.05). The mice that had received AdalphaRSV had at least 5.4-fold lower RSV titers in the lung 4 days following intranasal challenge with RSV compared to the AdNull or naive group (p<0.01). To evaluate long-term protection, the antibody construct was expressed in a non-human primate serotype rh.10 adeno-associated virus vector (AAVrh.10alphaRSV). RSV neutralizing antibodies were detected in serum and bronchoalveolar lavage fluid for up to 21 wk following intrapleural administration of AAVrh.10alphaRSV, but not with a control AAV vector expressing an unrelated transgene (AAVrh.10alpha1AT). Following challenge with RSV at 7 or 21 wk, 14.3-fold and 10.6-fold lower RSV titers were observed after 4 days in the lungs of mice that had received AAVrh.10alphaRSV compared to AAVrh.10alpha1AT (p<0.05). Together these data demonstrate that a gene transfer strategy for delivery of an anti-RSV antibody can generate protective immunity in mice against RSV infection in the respiratory tract and may provide an alternative to the administration of the antibody itself.