No abstract

Gene transfer to airway epithelia is the most direct approach for treating the progressive lung disease associated with cystic fibrosis. However, the transduction efficiency is poor when viral vectors are applied to the mucosal surface. We reported previously that gene transfer via the apical surface of human airway epithelia in vitro was improved by formulating vectors with ethyleneglycol-bis-(2-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA) in a hypotonic buffer. First, we investigated the mechanism for this enhancement. When 100-nm fluorescent beads were applied to the apical surface in the presence of EGTA, paracellular deposition of the particles was noted. Transmission electron microscopy verified that the epithelial junction complex was disrupted under these conditions. The Ca(2+) chelators EGTA, 1,2-bis (2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ethylenediaminetetraacetic acid all caused a rapid, reversible drop in transepithelial resistance and facilitated gene transfer with retrovirus or adenovirus in vitro. When Ca(2+) chelators were applied to rabbit tracheal epithelia or human nasal epithelia in vivo, the transepithelial voltage decreased, and amiloride sensitivity was lost, suggesting that epithelial junctions opened. Importantly, this novel formulation enhanced both retroviral- and adenoviral-mediated gene transfer to rabbit tracheal epithelia in vivo. This technique may have applications for vector or drug delivery to airway epithelia and other polarized cells.

Gene transfer with recombinant murine leukemia viruses (MuLV) provides the potential to permanently correct inherited lung diseases, such as cystic fibrosis (CF). Several problems prevent the application of MuLV-based recombinant retroviruses to lung gene therapy: (i) the lack of cell proliferation in mature pulmonary epithelia, (ii) inefficient gene transfer with a vector applied to the apical surface, and (iii) low titers of many retroviral preparations. We found that keratinocyte growth factor (KGF) stimulated proliferation of differentiated human tracheal and bronchial epithelia. Approximately 50% of epithelia divided in response to KGF as assessed by bromodeoxyuridine histochemistry. In airway epithelia stimulated to divide with KGF, high-titer ampho- and xenotropic enveloped vectors preferentially infected cells from the basal side. However, treatment with hypotonic shock or EGTA transiently increased transepithelial permeability, enhancing gene transfer with the vector applied to the mucosal surfaces of KGF-stimulated epithelia. Up to 35% of cells expressed the transgene after gene transfer. By using this approach, cells throughout the epithelial sheet, including basal cells, were targeted. Moreover, the Cl− transport defect in differentiated CF airway epithelia was corrected. These findings suggest that barriers to apical infection with MuLV can be overcome.

Gene transfer with integrating vectors such as recombinant retrovirus has the potential to correct inherited lung diseases permanently. As a gene therapy target, the pulmonary epithelium presents several challenges to vector delivery in vivo. Many of the host defenses that have evolved to prevent infection from inhaled bacteria or viruses represent potential barriers to gene transfer to the lung. We performed in vitro studies to determine whether two components of the innate immune system of the lung, airway surface fluid and alveolar macrophages, inhibit retroviral gene transfer to airway epithelia. Human alveolar macrophages obtained by bronchoalveolar lavage from normal subjects were left untreated or activated with lipopolysaccharide (LPS) for 3 hr in the presence of subconfluent human bronchial epithelial cells (HBE); than 4 x 10(5) cfu DA-luciferase retrovirus was added. Three days after infection, luciferase activity was measured in cell lysates. When the epithelial cells were co-cultured with LPS-activated macrophages, retroviral gene transfer to HBE cells was reduced by approximately 60%. Nonactivated macrophages decreased the transfection to approximately 55% of control values. In control experiments with either activated or inactivated macrophages but without epithelia, no luciferase activity was detected, suggesting that terminally differentiated alveolar macrophages are not infected by the recombinant retrovirus. Pretreatment of alveolar macrophages with dexamethasone restored gene transfer to approximately 60% of control values. In contrast, incubation of retrovirus with airway surface fluid had no inhibitory effect on gene transfer. These experiments document that AM inhibit retrovirus-mediated gene transfer to airway epithelia in vitro, and may represent a barrier to retroviral gene transfer in vivo. These barriers may be overcome, at least partially, with pharmacological agents.

Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination.

Background Cell proliferation, vector titer and accessibility of target cells represent hurdles for efficient gene transfer to lung epithelia in vivo using recombinant murine leukemia (MuLV)‐based retroviruses. We tested the hypothesis that the pulmonary epithelium is susceptible to retroviral‐mediated gene transfer when stimulated to proliferate by a mitogen, keratinocyte growth factor (KGF). Methods Rats received keratinocyte growth factor (KGF, 2.5 µg/g×4 doses, two consecutive days) intratracheally followed by high titer amphotropic retrovirus expressing β‐galactosidase. Gene transfer was assessed five days later. Results KGF stimulated transient proliferation in the bronchiolar and alveolar epithelia (30–40% PCNA positive cells at peak) which decreased to background levels seven days after administration. Gene transfer to epithelia (X‐Gal positive cells) occurred more frequently in KGF treated rats, but proliferation exceeded the level of gene transfer. X‐gal positive cells were noted in the alveolar epithelium and occasionally in the bronchiolar epithelium. In order to understand the discrepancy between the number of proliferating and transduced cells, primary rat tracheal epithelium cultured at the air‐liquid interface was infected from either the apical or basolateral side. Gene transfer was achieved only through basolateral application of vector, suggesting that epithelial polarity represents a barrier to MuLV‐based lung gene transfer in vivo. Conclusions KGF transiently stimulates epithelial proliferation in vivo, facilitating MuLV‐based gene transfer. Retroviral vectors may encounter multiple barriers which have evolved to defend the lung from infections. Copyright © 1999 John Wiley & Sons, Ltd.

The generation of a lasting systemic immune response is a primary goal for cancer immunotherapy. Here we examine the ability of high-titer IFN-gamma retroviral vector injected into an accessible tumor to generate significant antitumor responses at a distal untreated site. CT26 or B16F10 murine tumors were inoculated subcutaneously to form solid tumors in BALB/c or C57BL/6 mice. Seven to 10 days postinoculation, high-titer IFN-gamma retroviral vector was directly injected into the subcutaneous tumor nodule, and optimal dose and course of therapy were determined. As a model for disseminated disease, mice were inoculated intravenously with CT26 cells to form pulmonary lesions, at the same time as the subcutaneous injections. Regression of subcutaneous tumor correlated with a systemic response at the distal lung metastases in the IFN-gamma-treated group (p < 0.0005). Splenocytes from mice with completely regressed tumors had a twofold increase in percent specific cytotoxicity in a standard CTL assay as compared with nonresponding mice. CD8+ T cells were shown to be essential for the regional and systemic antitumor response, as determined by in vivo cell depletion experiments. These data demonstrate that IFN-gamma retroviral vector gene therapy delivered intralesionally can generate significant inhibition of pulmonary tumor formation distal to the treatment site. The data from these preclinical studies suggest the potential clinical value of retroviral vector-mediated cytokine gene therapy for systemic cancer.

Introduction of the herpes simplex type I thymidine kinase (HSV-TK) gene into tumor tissue, followed by ganciclovir, initiates a phosphorylation cascade that induces formation of a toxic ganciclovir triphosphate. Animal trials suggest that this ganciclovir triphosphate has antitumor activity. Here we report application of the HSV-TK transfection approach using a retroviral construct. Sixteen patients (median age 61.5 years) with refractory carcinoma (13 melanoma, 1 breast cancer, 1 nonsmall-cell lung cancer, and 1 osteogenic sarcoma) received intratumoral injection of HSV-TK retroviral vector at escalating doses (0.2x10(7) cfu per injection x 5 daily doses) and we evaluated them for toxicity and activity. We observed grade III pain associated with cellulitis in one patient following injection. Analysis of blood samples drawn between 3 and 28 weeks from 14 patients for replication-competent retrovirus by PCR analysis of the amphotrophic envelope revealed no replication-competent retrovirus. We injected 21 lesions. We identified no tumor responses of the injected lesions. Of 13 patients with advanced melanoma, 6 survived over one year. Thus, injection of retroviral delivered HSV-TK in patients with refractory cancer was well-tolerated.