Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. II. Transfection efficiency in airway epithelium

A phase I clinical trial was conducted in which recombinant adenovirus containing the cystic fibrosis trans-membrane regulator (CFTR) (Ad2/CFTR) was administered by bronchoscopic instillation or aerosolization to the lungs of cystic fibrosis (CF) patients. In this paper, we evaluate the efficiency of Ad2/CFTR-mediated transduction of bronchial airway cells. The ability of an Ad2/CFTR vector to transduce airway cells was first evaluated in patients to whom the vector was administered by bronchoscopic instillation. Cells at the administration site were collected 2 days after treatment by bronchoscopic brushing. Ad2-specific CFTR DNA was detected in four of five individuals by PCR, and Ad2-specific CFTR RNA was detected in three of five individuals by RT-PCR. Ad2/CFTR-mediated transduction of airway epithelial cells was then determined in CF individuals receiving this vector by aerosol inhalation. Ad2-specific CFTR DNA was detected in 13 of 13 individuals 2 days after aerosolization, and in 3 of 5 individuals 7 days after aerosolization. Ad2-specific RNA was detected in 4 of 13 individuals on day 2, but was not detected in the 5 individuals tested on day 7. The percentage of airway epithelial cells containing nuclear-localized vector DNA was < or =2.4% as determined by fluorescence in situ hybridization (FISH). However, in some cases, a high percentage of nonepithelial mononuclear cells or squamous metaplastic epithelial cells was infected with the adenoviral vector. In conclusion, aerosol administration is a feasible means to distribute adenoviral vectors throughout the conducting airways, but improvements in adenovirus-mediated transduction of airway epithelial cells are necessary before gene therapy for CF will be effective.     

Free Cholesterol Enhances Adenoviral Vector Gene Transfer and Expression in CAR-Deficient Cells

Efficient adenovirus vector-mediated gene transfer depends on the presence of sufficient amounts of the high-affinity coxsackie–adenovirus (Ad) receptor (CAR) on the surface of the target cell leading to receptor-mediated endocytosis of the vector. The present study evaluates the effect of free cholesterol, a lipid component of endocytic vesicles, on Ad uptake into CAR-deficient cells. Infection in the presence of free cholesterol at its maximum solubility in water led to increased binding, uptake, and expression of Ad in human skin fibroblasts and alveolar macrophages, two primary human cells known to be deficient in CAR. The effect of free cholesterol was maximal at its solubility maximum in aqueous solution. Increase of Ad vector-mediated gene transfer with cholesterol was dependent on the lack of CAR receptor expression on the surface and was diminished by overexpression of CAR in CAR-deficient cells. Cholesterol-mediated increase of Ad-mediated gene expression was dependent on coincubation of both cholesterol and Ad and was not dependent on the cholesterol content of the cell. Increased Ad vector-mediated gene expression in the presence of free cholesterol was also observed in murine skin in vivo. Structural analysis of the Ad–cholesterol mixture showed complexation between Ad particles leading to formation of multivirus aggregates due to hydrophobic interaction. The addition of free cholesterol with Ad vectors may be a simple way to increase Ad-mediated gene transfer to cells that are poor targets due to their lack of a sufficient number of Ad receptors.     

Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells.

Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated Cl- secretion, but also increased Na+ absorption and increased Ca(2+)-mediated Cl- secretion. In the present study, we examined whether adenovirus-mediated (Ad5) transduction of CFTR can correct all of these CF ion transport abnormalities. Polarized primary cultures of human CF and normal nasal epithelial cells were infected with Ad5-CBCFTR at an moi (10(4)) which transduced virtually all cells or Ad5-CMV lacZ as a control. Consistent with previous reports, Ad5-CBCFTR, but not Ad5-CMV lacZ, corrected defective CF cAMP-mediated Cl- secretion. Basal Na+ transport rates (basal Ieq) in CF airway epithelial sheets (-78.5 +/- 9.8 microA/cm2) were reduced to levels measured in normal epithelial sheets (-30.0 +/- 2.0 microA/cm2) by Ad5-CBCFTR (-36.9 +/- 4.8 microA/cm2), but not Ad5-CMV lacZ (-65.8 +/- 6.1 microA/cm2). Surprisingly, a significant reduction in delta Ieq in response to ionomycin, a measure of Ca(2+)-mediated Cl- secretion, was observed in CFTR-expressing (corrected) CF epithelial sheets (-6.9 +/- 11.8 microA/cm2) when compared to uninfected CF epithelial sheets (-76.2 +/- 15.1 microA/cm2). Dose response effects of Ad5-CBCFTR on basal Na+ transport rates and Ca(2+)-mediated Cl- secretion suggest that the mechanism of regulation of these two ion transport functions by CFTR may be different. In conclusion, efficient transduction of CFTR corrects hyperabsorption of Na+ in primary CF airway epithelial cells and restores Ca(2+)-mediated Cl- secretion to levels observed in normal airway epithelial cells. Moreover, assessment of these ion transport abnormalities may represent important endpoints for testing the efficacy of gene therapy for cystic fibrosis.     

Augmentation of pulmonary host defense against Pseudomonas by FcγRIIA cDNA transfer to the respiratory epithelium

Fcgamma receptors on the surface of phagocytic cells bind the Fc region of IgG and mediate binding, phagocytosis, and destruction of particulate antigens opsonized by the antigen-specific IgG molecule. The present study evaluates the feasibility of converting lung epithelial cells into phagocytic cells using adenovirus (Ad) vector-mediated gene transfer of FcgammaRIIA cDNA to induce expression of the human FcgammaRIIA receptor. Binding and phagocytosis of opsonized sheep red blood cells (SRBCs) by the A549 human lung epithelial cell line after Ad-mediated FcgammaRIIA gene transfer was demonstrated using light and fluorescence microscopy and phagocytic assays with (51)Cr-labeled SRBCs. When A549 cells were infected with an Ad vector expressing a FcgammaRIIA mutant in which 2 of 3 cytoplasmic tyrosines have been replaced with phenylalanine, only binding, but not phagocytosis, of opsonized SRBCs was observed. In vivo expression of FcgammaRIIA in the lung after intratracheal administration of the AdFcgammaRIIA enhanced clearance of opsonized Pseudomonas aeruginosa from the lung in normal rats and in mice deficient in Fcgamma receptor expression. Similar results were observed with a chimeric FcgammaRIIA construct containing the extracellular domain of FcgammaRIIIA. Together, these data demonstrate that Ad-mediated FcgammaRIIA receptor cDNA expression can mediate the binding and phagocytosis of opsonized particulate antigens by normally nonphagocytic cells, suggesting that gene-transfer strategies might be used to utilize nonphagocytic cells to clear bacteria or other opsonized particulate antigens from the respiratory tract.     

Using a tropism-modified adenoviral vector to circumvent inhibitory factors in ascites fluid

Peritoneal compartmentalization of advanced stage ovarian cancer provides a rational scenario for gene therapy strategies. Several groups are exploring intraperitoneal administration of adenoviral (Ad) vectors for this purpose. We examined in vitro gene transfer in the presence of ascites fluid from ovarian cancer patients and observed significant inhibition of Ad-mediated gene transfer. The inhibitory activity was not identified as either complement or cellular factors, but depletion of IgG from ascites removed the inhibitory activity, implicating neutralizing anti-Ad antibodies. A wide range of preexisting anti-Ad antibody titers in patient ascites fluid was measured by ELISA. Western blot analysis demonstrated that the antibodies were directed primarily against the Ad fiber protein. To circumvent inhibition by neutralizing antibodies, a genetically modified adenoviral vector was tested. The Ad5Luc.RGD vector has an Arg-Gly-Asp (RGD) peptide sequence inserted into the fiber knob domain and enters cells through a nonnative pathway. Compared with the conventional Ad5 vector, Ad5Luc.RGD directed efficient gene transfer to cell lines and primary ovarian cancer cells in the presence of ascites fluid containing high-titer neutralizing anti-Ad antibodies. These results suggest that such modified Ad vectors will be needed to achieve efficient gene transfer in the clinical setting.     

Enhancement of adenovirus-mediated gene delivery by use of an oligopeptide with dual binding specificity

The efficiency of human adenovirus serotype 5 (Ad5) transgene delivery was tested on several human and animal cell lines in vitro, by using a bimodular 35-mer oligopeptide carrying two peptide domains with different ligand specificities. One domain mimicked the fiber knob-binding region of the alpha2 domain of human MHC-1 molecules (MH20), and the other corresponded to the gastrin-releasing peptide (GRP). Two synthetic peptides with different configurations were analyzed in Ad-mediated gene transfer assays using Ad5Luc3 vector carrying the luciferase reporter gene. One peptide (GRP-MH20) had the GRP domain on the N-terminal side of MH20, while the other (MH20-GRP), the C-terminally amidified GRP, was on the C-terminal side of MH20. The GRP-MH20 peptide, but not MH20-GRP, was capable of enhancing luciferase gene delivery to Ad-susceptible cells in a GRP receptor-dependent manner. More importantly, GRP-MH20 could also confer susceptibility to Ad infection to normal or cancer cells that lack fiber receptors for the virus. Our data suggested that GRP receptors could function efficiently as alternative attachment receptors for Ad5, but that Ad5 bound to GRP receptors still depended, at least partially, on the penton base-mediated endocytotic pathway for subsequent cell entry. Gene delivery by a human adenovirus serotype 5 (Ad5) vector was assayed with a bimodular oligopeptide carrying two peptide domains of different binding specificities. One domain was a high-affinity peptide ligand of the Ad5 fiber knob (MH20), and the other corresponded to the gastrin-releasing peptide (GRP). The synthetic peptide GRP-MH20 was found to be capable of enhancing Ad-mediated gene transfer to Ad-susceptible cells in a GRP receptor-dependent manner. More importantly, GRP-MH20 could also confer susceptibility to Ad infection to normal or cancer cells that lack fiber receptors. Our data suggested that GRP receptors could function efficiently as alternative attachment receptors for Ad5, but virus bound to GRP receptors still depended partially on the penton base-mediated pathway for cell entry.     

Use of protamine to augment adenovirus-mediated cancer gene therapy.

Improving the therapeutic potential of adenoviral (Ad) suicide gene therapy has become an area of intense investigation since the inception of gene therapy strategies for cancer treatment. Poor efficiency of gene transfer to target tissues has become one of the most important limitations to Ad-based gene therapy. Since polycations have been shown to enhance adenovirus-mediated gene transfer in epithelial cells both in vitro and in vivo, we hypothesized that polycations could augment treatment efficacy in animals with established tumor. To address this hypothesis, protamine sulfate, a polycation already safely administered in humans, was complexed with a recombinant Ad (E1E3-deleted) vector containing the herpes simplex 1 thymidine kinase (HSVtk) suicide gene to treat cancer cell lines in vitro and in animals bearing intraperitoneal tumor. In the presence of 5 microg/ml protamine, the efficiency of gene transfer to a number of cancer cell lines normally resistant to adenovirus was significantly enhanced. Protamine's effect in vitro was found to be inversely proportional to the level of expression of the high affinity Ad binding site, coxsackievirus and adenovirus receptor (CAR), on the sur- face of the various cell lines tested. Ad.tk infected tumor cells were rendered 2.5- to three-fold more sensitive to 20 microM ganciclovir (GCV) in the presence of protamine. Protamine also augmented the in vivo transfer efficiency of the marker gene, LacZ (contained in an Ad vector), on the surface of tumors derived from an intraperitoneal mouse model. Quantitative imaging revealed 50% tumor surface transduced with LacZ when treatment was performed in the presence of 50 microg/ml protamine compared with 12% tumor surface in controls. However, experiments performed utilizing intraperitoneal administration of Ad.tk/GCV in the presence or absence of 50 microg/ml protamine demonstrated no significantly improved median survival in mice bearing established intraperitoneal tumors. Similarly, in Fischer rats bearing intrapleural tumor, no improvement in anti-tumor response was observed when Ad treatment was performed intrapleurally in the presence of protamine. Thus, although protamine induced an enhancement of Ad-mediated gene transfer in vitro and in vivo, its use as an adjunct to intracavitary Ad-based cancer gene therapy in vivo appears to be limited.     

Cellular immune responses of healthy individuals to intradermal administration of an E1-E3- adenovirus gene transfer vector

In animals, Ad-mediated gene transfer initiates anti-Ad host immune responses that vary, depending on vector design, dose, host, and transgene. To begin to understand whether the anti-Ad vector responses in humans simulate those in animals, Ad(GV)CD.10, an E1-E3- Ad5 vector encoding the E. coli cytosine deaminase gene, was administered by the intradermal route to six normal individuals (8 x 10(7) to 8 x 10(9) particle units, each dose administered to two sites; n = 2 per group). No adverse events were observed. Polymerase chain reaction/Southern analysis demonstrated vector genome in the skin through 28 days in all individuals except one of two at the lowest dose. Local induration, independent of vector dose and baseline systemic anti-Ad5 neutralizing antibodies, developed in all subjects (6 to 17 mm, peak by day 3). Biopsies revealed a mild to moderate T cell (CD3+, CD4+, CD8+), B cell, and macrophage infiltrate at day 3, all decreased by day 28. Langerhans cells accumulated primarily in the papillary dermis. The day 3 cellular response was dose independent. On day 28, CD4+ and CD8+ T lymphocytes and macrophages showed dose dependency. There was minimal systemic Ad5-specific lymphocyte proliferation induced by Ad vector administration in three individuals studied, and no Ad5-specific cytotoxic T lymphocytes (evaluated in two subjects) could be detected. Thus, intradermal administration of an E1-E3- Ad vector to normal subjects induces mild/moderate local cellular responses, even in Ad-immunized individuals. These observations provide a baseline to determine if these human anti-Ad vector host responses can be circumvented by using "stealth" vectors and/or immunosuppression.     

Enhanced gene transfer to mouse dendritic cells using adenoviral vectors coated with a novel adapter molecule.

Adenovirus (Ad)-mediated transduction of dendritic cells (DC) is inefficient because of the lack of the primary Ad receptor, CAR. DC infection with Ad targeted to the CD40 results in increased gene transfer. The current report describes further development of the CD40-targeting approach using an adapter molecule that bridges the fiber of the Ad5 to CD40 on mouse DC. The adapter molecule, CFm40L, consists of CAR fused to mouse CD40 ligand via a trimerization motif. A stable cell line that secretes CFm40L at high levels was generated. Gene transfer to mouse bone marrow-derived DC (mBMDC) using CFm40L-targeted Ad was over 4 orders of magnitude more efficient than that for the untargeted Ad5. Gene transfer was achieved to over 70% of the mBMDC compared to undetectable transduction using untargeted Ad5. In addition to dramatically enhanced gene transfer, the CFm40L-targeted Ad5 induced phenotypical maturation and upregulated IL-12 expression. Most importantly, the CFm40L-targeted Ad5 elicited specific immune response against a model antigen in vivo. The results of this study demonstrate that Ad-mediated gene transfer to DC can be significantly enhanced using nonnative transduction pathways, such the CD40 pathway, which may have important applications in genetic vaccination for cancer and infectious diseases.     

Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo.

Adenovirus (Ad)-mediated gene transfer to airway epithelia is inefficient because the apical membrane lacks the receptor activity to bind adenovirus fiber protein. Calcium phosphate (CaPi) precipitates have been used to deliver plasmid DNA to cultured cell lines. However, such precipitates are not effective in many primary cultures or in vivo. Here we show that incorporating recombinant adenovirus into a CaPi coprecipitate markedly enhances transgene expression in cells that are resistant to adenovirus infection. Enhancement requires that the virus be contained in the precipitate and viral proteins are required to increase expression. Ad: CaPi coprecipitates increase gene transfer by increasing fiber-independent binding of virus to cells. With differentiated cystic fibrosis (CF) airway epithelia in vitro, a 20-min application of Ad:CaPi coprecipitates that encode CF transmembrane conductance regulator produced as much CF transmembrane conductance regulator Cl- current as a 24-h application of adenovirus alone. We found that Ad:CaPi coprecipitates also increased transgene expression in mouse lung in vivo; importantly, expression was particularly prominent in airway epithelia. These results suggest a new mechanism for gene transfer that may be applicable to a number of different gene transfer applications and could be of value in gene transfer to CF airway epithelia in vivo.     

Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus

We sought to evaluate the ability of an E1(-), E3(-) adenovirus (Ad) vector (Ad(GV)CFTR.10) to transfer the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the airway epithelium of individuals with cystic fibrosis (CF). We administered Ad(GV)CFTR.10 at doses of 3 x 10(6) to 2 x 10(9) plaque-forming units over 9 months by endobronchial spray to 7 pairs of individuals with CF. Each 3-month cycle, we measured vector-derived versus endogenous CFTR mRNA in airway epithelial cells prior to therapy, as well as 3 and 30 days after therapy. The data demonstrate that (a) this strategy appears to be safe; (b) after the first administration, vector-derived CFTR cDNA expression in the CF airway epithelium is dose-dependent, with greater than 5% endogenous CFTR mRNA levels at the higher vector doses; (c) expression is transient, lasting less than 30 days; (d) expression can be achieved with a second administration, but only at intermediate doses, and no expression is observed with the third administration; and (e) the progressive lack of expression with repetitive administration does not closely correlate with induction of systemic anti-Ad neutralizing antibodies. The major advantage of an Ad vector is that it can deliver sufficient levels of CFTR cDNA to the airway epithelium so that CFTR expression protects the lungs from the respiratory manifestations of CF. However, this impressive level of expression is linked to the challenging fact that expression is limited in time. Although this can be initially overcome by repetitive administration, unknown mechanisms eventually limit this strategy, and further repetitive administration does not lead to repetitive expression.     

Signaling antibodies complexed with adenovirus circumvent CAR and integrin interactions and improve gene delivery

Current adenoviral (Ad) vectors cannot be targeted to specific cell types due to the widespread distribution of the Ad receptor (CAR). Moreover, CAR and/or internalization receptors (alphav integrins) are absent or present at low levels on some cell types, rendering them resistant to Ad-mediated gene delivery. To address these problems, we have developed a novel vector targeting approach that takes advantage of the common cell signaling pathways initiated by ligation of alphav integrins and growth factor receptors. Recombinant growth factor/cytokines (TNF-alpha, IGF-1, EGF) which trigger phosphatidylinositol-3-OH kinase (PI3K) activation, a signaling molecule involved in adenovirus internalization, were fused to a monoclonal antibody specific for the viral penton base. Ad vectors complexed with these bifunctional mAbs increased gene delivery 10 to 50-fold to human melanoma cells lacking alphav integrins. The bifunctional mAbs also enhanced gene delivery by fiberless adenovirus particles which cannot bind to CAR. Improved gene delivery correlated with increased virus internalization and attachment as well as PI3K activity. The use of bifunctional mAbs to trigger specific cell signaling pathways offers a widely applicable method for bypassing the normal Ad receptors in gene delivery and potentially increasing the selectivity of gene transfer.     

Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells

Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.     

Adenovirus vectors based on human adenovirus type 19a have high potential for human muscle-directed gene therapy

Until recently, adenovirus-based gene therapy has been almost exclusively based on human adenovirus serotype 5 (Ad5). The aim of this study was to systematically compare the efficiency of transduction of primary muscle cells from various species by two adenoviral vectors from subgroups C and D. Transduction of a panel of myoblasts demonstrated a striking specificity of an Ad19a-based replication-defective E1-deleted vector (Ad19aEGFP) for human cells, whereas the Ad5-based vector had high affinity for nonhuman primate myoblasts. Transgene expression correlated well with cell-associated vector genomes. Up to 6.59% of the initially applied Ad19aEGFP vector particles were taken up by human myoblasts, as compared with 0.1% of the corresponding Ad5 vector. Remarkably, Ad19aEGFP but not Ad5EGFP efficiently transduced differentiated human myotubes, an in vitro model for skeletal muscle transduction. Uptake of Ad19aEGFP vector particles in human myotubes was 12-fold more efficient than that of Ad5EGFP. Moreover, both vectors demonstrated an early block at the level of vector uptake in mouse myoblasts and rat L6 cells. Investigation of the underlying mechanism for binding and uptake of the two vectors by human myoblasts showed high susceptibility for Ad19a to neuraminidase and wheat germ agglutinin (WGA) lectin, whereas Ad5-mediated transduction was dependent on binding to the coxsackie-adenovirus receptor (CAR) and sensitive to soluble RGD peptide and heparin. Our study offers insights into species-dependent factors that determine Ad tropism and, moreover, provides a basis for application of the novel Ad19a-based vector for gene transfer into human skeletal muscle.     

Hybrid Nonviral/Viral Vector Systems for Improved piggyBac DNA Transposon In Vivo Delivery

The DNA transposon piggyBac is a potential therapeutic agent for multiple genetic diseases such as cystic fibrosis (CF). Recombinant piggyBac transposon and transposase are typically codelivered by plasmid transfection; however, plasmid delivery is inefficient in somatic cells in vivo and is a barrier to the therapeutic application of transposon-based vector systems. Here, we investigate the potential for hybrid piggyBac/viral vectors to transduce cells and support transposase-mediated genomic integration of the transposon. We tested both adenovirus (Ad) and adeno-associated virus (AAV) as transposon delivery vehicles. An Ad vector expressing hyperactive insect piggyBac transposase (iPB7) was codelivered. We show transposase-dependent transposition activity and mapped integrations in mammalian cells in vitro and in vivo from each viral vector platform. We also demonstrate efficient and persistent transgene expression following nasal delivery of piggyBac/viral vectors to mice. Furthermore, using piggyBac/Ad expressing Cystic Fibrosis transmembrane Conductance Regulator (CFTR), we show persistent correction of chloride current in well-differentiated primary cultures of human airway epithelial cells derived from CF patients. Combining the emerging technologies of DNA transposon-based vectors with well-studied adenoviral and AAV delivery provides new tools for in vivo gene transfer and presents an exciting opportunity to increase the delivery efficiency for therapeutic genes such as CFTR.     

Adenovirus vector-mediated gene transfer to regional lymph nodes

Regional lymph nodes (RLNs) possess important immune functions and represent a major pathway of metastasis for solid tumors. Given these facts, the ability to transfer exogenous genes to the RLNs with the goal of manipulating the local immunological milieu would be desirable. On the basis of the hypothesis that a significant proportion of adenovirus (Ad) gene transfer vectors traffic through the lymphatics, E1-E3- Ad vectors were injected into the hind footpad of C3H/He mice and the RLNs assessed for vector trafficking and transgene expression. A low dose (10(9) particles) of an Ad vector encoding the firefly luciferase gene (Ad-CMV.Luc) resulted in luciferase expression only in the injection site and RLNs, with no detectable systemic (liver, spleen, lung) expression. At a higher dose (10(11) particles), some expression could be detected systemically in addition to the RLNs, but at levels in liver 14-fold less than in the RLNs. Transgene expression in the RLNs was transient, peaking at 1 day, decreasing markedly by 7 days. At high doses (10(11) particles), interruption of draining lymphatics decreased the amount of systemic dissemination 22-fold, suggesting that a large proportion of the vector trafficks through the lymphatics before reaching the systemic circulation. Administration of a vector encoding the jellyfish green fluorescent protein gene (AdCMV.GFP, 10(11) particles) showed that transgene expression in the RLNs was primarily in the cortical area. After footpad injection of a fluorescent-labeled Ad vector (Cy3-AdCMV.Null), fluorescent virions were visualized in the draining lymph. Regional lymph collected from animals injected in the footpad with AdCMV.Luc (10(11) particles) contained functional vector. Augmentation of local immune function in the RLNs was achieved by footpad administration of an Ad vector encoding murine IL-12, resulting in high mIL-12 and IFN-gamma levels in the regional, but not distant, nodes. These data demonstrate that expression of exogenous genes in RLNs is easily accomplished with Ad vectors, Ad vector dissemination occurs primarily via the lymphatics after footpad administration in mice, and basic immune functions in the RLNs can be manipulated by Ad-mediated gene transfer in vivo.     

Prevention of posterior capsule opacification by the induction of therapeutic apoptosis of residual lens cells

Posterior capsule opacification (PCO) is a common complication of cataract surgery. Using adenovirus(Ad)-mediated gene transfer, we overexpressed the proapoptotic molecules p53, procaspase 3, Bax, and TRAIL to induce therapeutic programmed cell death of residual lens cells to prevent PCO. Overexpressed TRAIL did not induce apoptosis in cultured rabbit lens cells or in human lens cells. Overexpressed p53 induced apoptosis of lens cells in vitro and ex vivo, but was unable to prevent PCO in vivo. Overexpressed procaspase 3 was associated with engagement of many components of the apoptotic pathway, including cleavage of intracellular caspase targets such as PARP and inter-nucleosome DNA fragmentation. Even when only slightly overexpressed, Bax caused apoptosis of transduced rabbit and human lens cells by engaging the mitochondrial pathway, including catalytic activation of the caspases. A single in vivo injection of Ad vectors expressing either Bax or procaspase 3 into the capsular bag at the end of phacoemulsification prevented PCO in rabbits. These experiments show that Ad-mediated Bax or procaspase 3 overexpression is capable of inducing therapeutic programmed cell death in vitro and in vivo in residual lens cells and preventing PCO in a rabbit model of PCO. Manipulation of proapoptotic molecule expression could be a novel gene therapy approach for prevention of PCO.