Gene therapy vector-mediated expression of insulin-like growth factors protects cardiomyocytes from apoptosis and enhances neovascularization.

IGF-I and IGF-II are single-chain polypeptide growth factors that regulate pleiotropic cellular responses. We have characterized the effect of recombinant IGF proteins, as well as third-generation adenoviral vectors encoding either IGF-I or IGF-II genes, on cardiomyocyte apoptosis and on angiogenesis. We found that endothelial cells cultured in the presence of the extracellular protein laminin exhibit a robust response to IGF-I and -II proteins via enhanced cell migration and angiogenic outgrowth. Furthermore, IGF vectors greatly enhanced neovascularization in an in vivo Matrigel model. Transduction of cardiomyocytes with the IGF adenoviral vectors resulted in a dose- and time-dependent increase in the expression of IGF-I or IGF-II protein. This correlated with abrogation of apoptosis induced by ischemia-reoxygenation, ceramide, or heat shock with optimal inhibition of approximately 80%. We conclude that gene transfer of IGF-I and IGF-II is a plausible strategy for the local delivery of IGFs to treat ischemic heart disease and heart failure by stimulating angiogenesis and protecting cardiomyocytes from cell death.

Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.

Development of adenovirus vectors as potential therapeutic agents for multiple applications of in vivo human gene therapy has resulted in numerous preclinical and clinical studies. However, lack of standardization of the methods for quantifying the physical concentration and functionally active fraction of virions in these studies has often made comparison between various studies difficult or impossible. This study was therefore carried out to define the variables for quantification of the concentration of adenovirus vectors. The methods for evaluation of total virion concentration included electron microscopy and optical absorbance. The methods for evaluation of the concentration of functional virions included detection of gene transfer (transgene transfer and expression) and the plaque assay on 293 cells. Enumeration of total virion concentration by optical absorbance was found to be a precise procedure, but accuracy was dependent on physical disruption of the virion to eliminate artifacts from light scattering and also on a correct value for the extinction coefficient. Both biological assays for enumerating functional virions were highly dependent on the assay conditions and in particular the time of virion adsorption and adsorption volume. Under optimal conditions, the bioactivity of the vector, defined as the fraction of total virions which leads to detected target cell infection, was determined to be 0.10 in the plaque assay and 0.29 in the gene transfer assay. This difference is most likely due to the fact that detection by gene transfer requires only measurement of levels of transgene expression in the infected cell whereas plaque formation is dependent on a series of biological events of much greater complexity. These results show that the exact conditions for determination of infectious virion concentration and bioactivity of recombinant adenovirus vectors are critical and must be standardized for comparability. These observations may be very useful in comparison of data from different preclinical and clinical studies and may also have important implications for how adenovirus vectors can optimally be used in human gene therapy.

Transduction of normal and malignant oral epithelium by an adenovirus vector: the effect of dose and treatment time on transduction efficiency and tissue penetration.

We tested an adenovirus vector that carries a beta-D-galactosidase marker gene for its ability to transduce normal oral mucosa and oral carcinoma cells. Topical application of adenovirus to normal oral mucosa of mice at 1 x 10(10) plaque-forming units (pfu)/mL for 1 minute did not result in transduction of epithelial cells. Similarly, topical application to human oral mucosa ex vivo was not successful. However, systemic administration by intracardiac injection of hamsters did transduce normal oral mucosa effectively. To evaluate transduction of carcinomas, the Tu138 human oral cancer cell line was used. A single application of virus at 1 x 10(8) pfu/mL in vitro resulted in 30% of oral carcinoma cells expressing the marker gene, and 2 x 10(8) pfu/mL transduced 60% of cells. After two applications of virus at 2 x 10(8) pfu/mL with an interval of 18 hours, 100% of oral carcinoma cells expressed the marker gene. Topical application to a raft culture of Tu138 cells for 1 hour produced gene expression that penetrated up to four layers of cells. To emulate the effect of treating a carcinoma, Tu138 cells were implanted subcutaneously in nude mice, allowed to grow to a tumor 1 cm in diameter, and then injected directly with virus. This produced diffuse transduction of around 30% of cells in the tumor, and expression was seen in cells at a significant distance from the injection site. Adenovirus vectors are therefore capable of transferring novel genetic information to both normal and malignant oral mucosa. They may have potential for use in gene therapy in the prevention or treatment of oral squamous carcinomas.

Evaluation of the efficacy and safety of in vitro, adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA.

Cystic fibrosis (CF) is a common, fatal recessive disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene manifested by abnormalities in the regulation of chloride ion (Cl-) secretion across the apical membrane of epithelial cells throughout the body. Adenovirus-mediated delivery of the normal CFTR cDNA and correction of the CF epithelial cell Cl- secretory phenotype suggests the feasibility of gene therapy for CF lung disease. Few studies, however, have focused on the evaluation of the safety of the adenovirus-mediated gene transfer approach. This study presents in vitro data on the efficacy and safety of adenovirus-mediated transfer of the human CFTR cDNA using Av1Cf2. Av1Cf2-mediated transfer of the human CFTR cDNA complemented the abnormal cAMP-regulated Cl- permeability of cells with the CF epithelial phenotype. Av1 vectors did not replicate infectious virus in HeLa cells infected in vitro, although trace vector DNA synthesis was observed at very high multiplicity of infection. Expression of the adenoviral late gene for the hexon capsid protein was observed at trace levels in Av1 vector-infected HeLa cells, but not in freshly isolated human bronchial epithelial cells, consistent with the pattern of DNA synthesis observed in these different target cells. Although, these observations support the efficacy and safety of use of Av1Cf2 for treatment of the fatal pulmonary component of CF.

In vivo evaluation of the safety of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA to the lung.

Cystic fibrosis (CF) is a common, fatal hereditary disease resulting from mutations of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene in which epithelial cells throughout the body manifest altered regulation of apical membrane chloride secretion. Although the disease affects multiple organs throughout the body, over 90% of patients die of complications of the lung involvement. The feasibility of adenovirus-derived vectors for in vivo delivery of the human CFTR cDNA to treat the pulmonary component of CF is currently being evaluated using in vitro and in vivo approaches. Defining the therapeutic window between biological efficacy and toxicity is an important part of this work. Here we present data regarding the preclinical evaluation of the safety of in vivo delivery of the human CFTR cDNA to the cotton rat airway epithelium using the replication-deficient adenoviral vector Av1Cf2 or a similar vector, Av1LacZ4, expressing the Escherichia coli LacZ gene as a histologic marker. Gene transfer to the respiratory epithelium was efficient, as demonstrated by in situ hybridization and histochemical staining. Administration of these vectors resulted in a mild, transient, dose-dependent cellular inflammatory response similar in character to that seen with adenovirus 5 (Ad5), but far less in intensity, which was not associated with structural lung damage or mortality. Av1Cf2 DNA sequences were easily detected in the lung after pulmonary administration, but could not be demonstrated in organs other than the lung. These preclinical observations suggest that adenovirus-mediated gene transfer to the airway epithelium can be achieved efficiently, but is accompanied by a dose- and time-dependent inflammation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenovirus-mediated gene transfer for cystic fibrosis: quantitative evaluation of repeated in vivo vector administration to the lung.

Adenoviral vectors have an important role as in vivo gene delivery vehicles in developing human gene therapy for the fatal pulmonary component of cystic fibrosis. In this study we evaluated the immune responses to wild-type adenovirus and replication-deficient, first generation adenoviral (Av1) vectors in the cotton rat (Sigmodon hispidus) and then quantitatively evaluated the efficiency of gene delivery and expression of single and repeated in vivo administration of Av1 vectors to the respiratory tract. Av1 vector reporter gene expression was quantitatively evaluated by employing a luciferase expression vector (Av1Luc1) and measuring luciferase activity in whole lung tissue homogenates by routine luminometry. Gene transfer and expression in naive animals (e.g. first Av1 vector dose) was efficient. A repeat dose also resulted in successful gene transfer and expression, although at a significantly reduced level (p < 0.01) compared with naive animals. This reduction inversely correlated with serum human adenovirus neutralizing antibody (HANA) titers. Importantly, increasing doses of Av1Cf2, an Av1 vector expressing the human CFTR cDNA, resulted in a graded HANA response consistent with a lack of in vivo replication. These observations have significant implications for repeated administration of adenoviral vectors to the lungs of individuals with cystic fibrosis.

Factors affecting retroviral vector function and structural integrity.

Recombinant retroviruses are widely used for gene transfer into eukaryotic cells and exhibit significant potential for human gene therapy. Despite the utility of retroviral vectors, their design is still essentially empirical. We have constructed a series of reciprocal, double-gene vectors to compare the dual expression of beta-galactosidase (beta-gal) and neomycin phosphotransferase (neor) in a retroviral delivery system. The first gene of the pair was driven by the viral LTR promoter and the internal gene was regulated by either the SV40 virus early promoter or the cytomegalovirus (CMV) major late promoter. Clones of vector producer cells were isolated either by G418 selection for expression of neor, or by fluorescence-activated cell sorting for expression of beta-gal, and the activity of both genes was evaluated. In general, vectors using the SV40 promoter performed better than those with the CMV promoter, regardless of whether the selected gene was regulated by the LTR or the internal promoter. Southern analysis of clones indicated that loss of beta-gal gene function was related to significant rearrangements and deletions in vector structure. We also found that the arrangement of genes within the vector was important. When beta-gal preceded neor, gene expression and vector stability were markedly enhanced relative to vectors containing these genes in the inverse order.

A human rel proto-oncogene cDNA containing an Alu fragment as a potential coding exon.

Two rel-containing cDNA clones were isolated from a library derived from the Daudi human cell line, which is known to express c-rel mRNA. Clone #1 appeared to contain the entire c-rel coding sequence, which differs from v-rel in having three additional N-terminal residues and 111 additional C-terminal residues. In addition, Clone #1 had an internal 32 amino acid exon not found in v-rel or in turkey c-rel. Clone #2 was truncated at its 5' end and did not contain this new exon. Analysis of a genomic clone of human c-rel revealed that the new exon was a portion of an inverted Alu repeat. The occurrence of potential splice sites and of open reading frames in the inverted consensus Alu sequence suggests that the incorporation of Alu fragments as potential coding exons could be a relatively common event in human mRNAs. Whether such messages can be translated is unknown: antiserum raised against a peptide at the predicted C-terminus of the c-rel protein precipitated p82hc-rel, but antiserum raised against a peptide located in the Alu exon did not.