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R G Crystal

Publications and source records attributed to R G Crystal.

At least 163 records · Page 9Linked to original sources
PubMed1995

Polarity of secretion of alpha 1-antitrypsin by human respiratory epithelial cells after adenoviral transfer of a human alpha 1-antitrypsin cDNA.

alpha 1-Antitrypsin (alpha AT) deficiency, a hereditary cause of progressive emphysema, can potentially be treated by transfer of a functional human alpha 1AT gene to the respiratory epithelium. For such an approach to be successful, alpha 1AT must be provided to both the interstitium and the epithelial surface--that is, the alpha 1AT directed by the transferred gene must be secreted to both the apical and basolateral surfaces of the epithelial cells. To evaluate this concept, a recombinant, replication-deficient adenoviral vector (Ad-alpha 1AT) containing a human alpha 1AT cDNA driven by an adenovirus major late promoter was used to infect Bet-1A human respiratory epithelial cells. The infected cells expressed Ad-alpha 1AT-directed mRNA transcripts and synthesized and secreted functional human alpha 1AT as shown by [35S]methionine labeling and immunoprecipitation of a 52-kD glycosylated human alpha 1AT molecule capable of interacting with neutrophil elastase, its natural substrate. Bet-1A cells grown on microporous polycarbonate membranes formed tight junctions (resistance > 150 omega x cm2). After infection with Ad-alpha 1AT, [35S]methionine labeling and enzyme-linked immunoassay demonstrated that alpha 1AT was secreted into both the apical and basolateral compartments, with an average apical to basolateral ratio of 1.9 +/- 0.2. Thus, human respiratory epithelial cells infected with a recombinant adenoviral vector containing a human alpha 1AT cDNA secrete alpha 1AT across both the apical and basolateral cell membranes, suggesting that the respiratory epithelium could serve as a target for in vivo gene therapy of alpha 1AT deficiency.

Adenoviridae↗
PubMed1995

Modulation of gene expression after replication-deficient, recombinant adenovirus-mediated gene transfer by the product of a second adenovirus vector.

To regulate gene expression following adenovirus-mediated gene transfer, a strategy was devised utilizing co-infection with two separate adenovirus vectors designed such that the product of one vector modulated the promoter of the second vector. To evaluate this strategy, AdEGR1.TNF, an adenovirus expressing tumor necrosis factor-alpha (TNF) under the control of the early growth response 1 (EGR1) promoter, was used to regulate a transcription unit in AdIL8.beta gal, an adenovirus vector in which the TNF sensitive interleukin-8 (IL-8) promoter drives the expression of beta-galactosidase (beta-gal). Following infection of HS24 cells with AdIL8.beta gal, addition of TNF to the culture induced the expression of beta-gal. Infection of HS24 cells with AdEGR1.TNF resulted in a dose-dependent secretion of TNF. Little beta-gal was produced following co-infection of the cells with the control vector AdCMV.Null (expressing no specific gene) and AdIL8.beta gal. In contrast, co-infection with AdIL8.beta gal and AdEGR1.TNF demonstrated, for a given dose of AdIL8.beta gal, increasing amounts of beta-gal expression dependent on the dose of AdEGR1.TNF. This model suggests control of gene expression in adenovirus-mediated gene transfer can be regulated by utilizing a promoter-gene expression cassette in one vector that modulates the expression of a promoter-gene expression cassette in a second vector.

Adenoviruses, Human↗
PubMed1994

Stimulation of erythropoiesis by in vivo gene therapy: physiologic consequences of transfer of the human erythropoietin gene to experimental animals using an adenovirus vector.

Erythropoietin (Epo), a 30.4-kD glycoprotein, is the principal regulator of erythropoiesis. To evaluate the concept that in vivo gene transfer might be used as an alternative to recombinant human Epo (rhEpo) in applications requiring a 1- to 3-week stimulation of erythropoiesis, the replication-deficient recombinant adenovirus AdMLP.Epo was constructed by deleting the majority of E1 from adenovirus type 5, and replacing E1 with an expression cassette containing the adenovirus type 5 major late promoter (MLP) and the human Epo gene, including the 3' cis-acting hypoxia response element. In vitro studies showed that infection of the human hepatocyte cell line Hep3B with AdMLP.Epo resulted in a 15-fold increase in Epo production in 24 hours that was enhanced to 116-fold in the presence of a hypoxic stimulus. One-time in vivo administration of AdMLP.Epo (7 x 10(9) plaque-forming units/kg) to the peritoneum of cotton rats caused a marked increase in red blood cell production, with a 2.6-fold increase in bone marrow erythroid precursors by day 4, and sevenfold increase in reticulocyte count by day 7. The hematocrit increased gradually, with a maximum of 64% +/- 4% at day 14 (compared with an untreated baseline of 46% +/- 2%), and a level of 55% +/- 1% at day 24. Furthermore, one-time subcutaneous administration of AdMLP.Epo caused an increase in hematocrit that peaked at 14 days (57% +/- 2%) and was still elevated at day 42. Hematocrit level in animals receiving subcutaneous administration of AdMLP.Epo sustained a long-term increase compared with animals receiving intraperitoneal administration. In the context of these observations, gene therapy with a single administration of an adenovirus vector containing the human EPO gene may provide a means of significantly augmenting the circulating red blood cell mass over the 1- to 3-week period necessary for many clinical applications.

Adenoviridae↗
PubMed1994

In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene.

Restenosis, a process characterized in part by excessive smooth muscle cell (SMC) proliferation in areas of vascular injury, occurs in up to 50% of patients undergoing balloon angioplasty. In an effort to develop a treatment strategy for restenosis, we constructed a replication-deficient recombinant adenovirus (AdMLP.HSTK) containing the herpes simplex virus thymidine kinase gene (HSV tk). This viral gene product phosphorylates the prodrug ganciclovir to form a nucleoside analog that inhibits DNA synthesis. Cultured primary rat SMCs infected with AdMLP.HSTK were completely growth-inhibited by incubation in ganciclovir-containing medium. In addition, when only a portion of the SMC population received the HSV tk transgene, an inhibitory effect on neighboring SMCs was evident. Evaluation of this strategy in vivo using a rat carotid balloon injury model demonstrated that local infection of injured arteries with AdMLP.-HSTK followed by 2 weeks of systemic ganciclovir treatment significantly (P < 0.01) reduced injury-induced SMC accumulation. In contrast, there was no suppression of injury-induced SMC accumulation in animals infected with AdMLP.HSTK but not receiving ganciclovir or in those animals infected with a control adenovirus and either treated or not treated with ganciclovir. These results demonstrate the potential utility of adenovirus-mediated gene transfer for treatment of restenosis after balloon injury.

Adenoviridae↗
PubMed1994

Transfer of a constitutive viral promoter-cystic fibrosis transmembrane conductance regulator cDNA to human epithelial cells conveys resistance to down-regulation of cAMP-regulated Cl- secretion in the presence of inflammatory stimuli.

The expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be down-regulated by inflammatory stimuli such as phorbol myristate acetate (PMA). Since the respiratory manifestations of cystic fibrosis (CF) are characterized by intense chronic airway inflammation very early in life, successful gene therapy for CF will require that expression of the transferred normal CFTR gene be resistant to down-regulation by inflammatory mediators. To evaluate the concept that a viral promoter--human CFTR cDNA unit would be resistant to this form of down-regulation, a retrovirus promoter (5' long terminal repeat of the Moloney murine leukemia virus)--human CFTR cDNA unit was transferred to T84 human colon carcinoma cell line using a retrovirus vector. Exposure of the retrovirus-modified T84 cells to PMA resulted in down-regulation of the endogenous CFTR mRNA transcripts (6.5 kb), but did not affect the level of exogenous CFTR transcripts (8.0 kb). Importantly, in parallel with the persistence of the exogenous CFTR transcripts, the modified cells still maintained cAMP-regulated CI- secretion in the presence of PMA. These in vitro data suggest that a constitutive viral promoter--CFTR cDNA unit should be resistant to modulation by inflammatory stimuli, a likely requirement for successful gene therapy for CF.

Blotting, Northern↗
PubMed1994

Enhancer function of a 53-bp repetitive element in the 5' flanking region of the human neutrophil elastase gene.

Expression of the human neutrophil elastase (NE) gene is limited to the early stage of myeloid cell differentiation in bone marrow cells. While NE gene expression is controlled mainly at the transcriptional level during bone marrow cell differentiation, the mechanism of transcriptional control is not fully understood. One motif of interest in the 5' flanking region of the gene is the six tandem repeats of a 53-bp nucleotide sequence (REP53) containing a potential binding site for a basic helix-loop-helix protein located at -1032 to -716. The REP53 sequence can function as a non-cell specific transcriptional enhancer which is capable of augmenting heterologous promoter activity. When the single REP53 element was inserted into the pAZ1037 chloramphenicol acetyltransferase (CAT) expression vector immediately upstream of the chicken beta-actin promoter in either normal or inverted orientation and used to transfect K-562 erythroleukemia or HeLa cervical carcinoma cells, these modified vectors achieved 2 to 3-fold higher CAT activity than the parental pAZ1037 vector irrespective of orientation of the REP53.

Animals↗
PubMed1994

Adenovirus-mediated in vivo gene transfer.

Adenovirus vectors are efficient vehicles for in vivo gene transfer to many different cell types. Recombinant adenovirus vectors containing exogenous genes for transfer are derived from adenovirus type 5 and are made replication deficient by the deletion of the E1 region. Based on the observation that many natural adenovirus infections are targeted to airway epithelial cells, a replication-deficient adenovirus vector was constructed containing the cystic fibrosis transmembrane conductance regulator cDNA for the potential therapy of the respiratory manifestations of cystic fibrosis. Using this vector, the normal human CFTR cDNA has been successfully transferred to airway epithelial cells of experimental animals via the trachea. This finding has led to the development of human gene therapy protocols for the evaluation of the safety and efficacy of adenovirus-mediated CFTR cDNA transfer to lungs of individuals with cystic fibrosis. In addition to the airways, adenovirus vectors have been demonstrated to mediate in vivo gene delivery to cells of the liver, blood vessels, brain, muscle, heart, peritoneum, and salivary glands. Adenovirus vectors containing marker genes have also been demonstrated to transfer genes to human tumor cells in nude mice. Such vectors may be useful for a variety of therapeutic applications for in vivo gene transfer for the therapy of cancer and other diseases.

Adenoviridae↗
PubMed1994

Adenovirus-mediated transfer of human lipase complementary DNA to the gallbladder.

BACKGROUND/AIMS: Despite many improvements in current therapy, exocrine pancreatic insufficiency remains a significant problem in cystic fibrosis. To establish a new therapy for exocrine pancreatic insufficiency, the feasibility of transferring the human pancreatic lipase complementary DNA to the gallbladder as a possible target using a recombinant adenovirus vector was evaluated. METHODS: The adenovirus vector AdCMV. Lip was constructed using the cytomegalovirus immediate early promoter to drive the human pancreatic lipase complementary DNA. In vitro infection of the human gallbladder epithelial cell line HS-181 and ex vivo infection of the sheep gallbladder with AdRSV. beta-gal (an adenovirus vector containing the Escherichia coli lacZ [(beta-galactosidase] gene) or AdCMV. Lip were evaluated. RESULTS: The supernatant from AdCMV. Lip-infected HS-181 showed the secretion of active lipase for at least 2 weeks in vitro. The epithelium of gallbladder infected with AdRSV.beta-gal ex vivo showed the expression of the beta-galactosidase. The fluid from the gallbladder lumen infected with AdCMV. Lip showed the increased lipase activity. CONCLUSIONS: These observations show that an adenovirus vector can transfer a human pancreatic enzyme in vitro and ex vivo, suggesting the feasibility of in vivo gene therapy for exocrine pancreatic insufficiency in cystic fibrosis.

Adenoviridae↗
PubMed1994

Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis.

We have administered a recombinant adenovirus vector (AdCFTR) containing the normal human CFTR cDNA to the nasal and bronchial epithelium of four individuals with cystic fibrosis (CF). We show that this vector can express the CFTR cDNA in the CF respiratory epithelium in vivo. With doses up to 2 x 10(9) pfu, there was no recombination/complementation or shedding of the vector or rise of neutralizing antibody titres. At 2 x 10(9) pfu, a transient systemic and pulmonary syndrome was observed, possibly mediated by interleukin-6. Follow-up at 6-12 months demonstrated no long term adverse effects. Thus, it is feasible to use an adenovirus vector to transfer and express the CFTR cDNA in the respiratory epithelium of individuals with CF. Correction of the CF phenotype of the airway epithelium might be achieved with this strategy.

Adenoviridae↗
PubMed1994

Gene transfer to freshly isolated human respiratory epithelial cells in vitro using a replication-deficient adenovirus containing the human cystic fibrosis transmembrane conductance regulator cDNA.

Cystic fibrosis (CF) results from mutations of the CF transmembrane conductance regulator (CFTR) gene and subsequent defective regulation of cAMP-stimulated chloride (Cl-) permeability across the apical membrane of epithelial cells. In vitro transfer of normal CFTR cDNA corrects this defect, and studies in experimental animals have shown successful gene transfer to airway epithelium in vivo using a recombinant adenoviral vector containing the human CFTR cDNA (AdCFTR), supporting the feasibility of in vivo AdCFTR-mediated gene therapy for the respiratory manifestations of CF. One step in applying this therapy to CF patients is to evaluate the safety and efficacy of AdCFTR-mediated gene transfer in the actual target for human gene therapy, human airway epithelium. The present study demonstrates that AdCFTR restores cAMP-stimulated Cl- permeability in human CF bronchial epithelial cells. In addition, the study utilizes freshly isolated human airway epithelial cells from the nose and/or bronchi of normal individuals and/or individuals with CF to demonstrate that after in vitro AdCFTR-mediated gene transfer: (i) AdCFTR DNA does not replicate as a function of dose and time; (ii) CF epithelial cells express AdCFTR-mediated normal human CFTR mRNA; and (iii) CF epithelial cells, including terminally differentiated ciliated cells (the most common airway epithelial cell type), express the normal human CFTR protein. Together, these data support the use of AdCFTR in human gene therapy trials and suggest that biologic efficacy should be achievable in vivo.

Adenoviridae↗
PubMed1994

Direct in vivo gene transfer and expression in malignant cells using adenovirus vectors.

To evaluate the ability of replication-deficient, recombinant adenovirus vectors to transfer genes to human tumor cells in vivo, adenovirus vectors containing the Escherichia coli lacZ (Ad.RSV beta gal) gene (coding for beta-galactosidase; used as a cell marker for gene transfer) or the human alpha 1-antitrypsin (Ad-alpha 1AT) cDNA (used as an example of a secreted protein) were administered intraperitoneally to nude mice with human malignant mesothelioma cell (H-MESO-1) malignant ascites. Preliminary in vitro studies showed that both vectors effectively transferred genes to H-MESO-1 cells. Tumor cells recovered from ascites of animals intraperitoneally administered a control adenovirus revealed no evidence of beta-galactosidase (beta-gal) activity 3 or 14 days later. In contrast, beta-gal activity was detected at the same time points in tumor cells from animals receiving intraperitoneal Ad.RSV beta gal. Flow cytometric quantification of beta-gal activity in recovered cells showed < 3% beta-gal-positive cells in animals administered control virus, but in animals administered intraperitoneal Ad.RSV beta gal there was a mean of 71 +/- 18% positive cells at 3 days and 56 +/- 27% at 14 days. Human alpha 1AT was not detected by enzyme-linked immunosorbent assay (ELISA) in ascites of animals receiving a control virus; however, in ascites of animals administered Ad-alpha 1AT, 21,000 +/- 3,800 ng/ml of human alpha 1AT was detected at 3 days and 4,900 +/- 1,700 ng/ml at 14 days. These data demonstrate that replication-deficient recombinant adenovirus vectors can be used to transfer genes to malignant cells in vivo and suggest a new strategy for genetic modification for antitumor therapy.

Adenoviruses, Human↗
PubMed1994

Acute responses of non-human primates to airway delivery of an adenovirus vector containing the human cystic fibrosis transmembrane conductance regulator cDNA.

Recombinant human adenovirus (Ad) vectors are leading candidates for human gene therapy for cystic fibrosis (CF) based on demonstration of efficient transfer of exogenous genes to rodent respiratory epithelium in vivo and human respiratory cells in vitro. The safety of Ad-mediated gene transfer to the respiratory epithelium and acute (up to 21 days) clinical responses to airway delivery of a replication-deficient recombinant, E1-, E3- Ad type 5-based vector containing the human cystic fibrosis transmembrane conductance regulator cDNA (AdCFTR) were evaluated in rhesus monkeys. Airway delivery of an Ad vector with the lacZ marker gene demonstrated beta-galactosidase expression in epithelial cells. Animals administered intratracheal AdCFTR demonstrated human CFTR cDNA expression in airway epithelial cells. Animals administered AdCFTR intranasal, and 24 hr later, intrabronchial [2 x 10(7) to 5 x 10(10) plaque-forming units (pfu), n = 12], in a fashion similar to a proposed human protocol, or only intrabronchial (10(11) pfu, n = 3), had no significant changes in clinical parameters compared to vehicle controls (n = 6). Microscopic analysis of the lung by necropsy or bronchoalveolar lavage demonstrated a dose-dependent increase in inflammatory cells, primarily lymphocytes, in the area where AdCFTR was delivered, which persisted for at least 2 months in some animals. Serum anti-Ad type 5 neutralizing antibody titers did not rise and shed Ad was not detected. The presence of AdCFTR DNA, analyzed by the polymerase chain reaction (PCR), was not detected in organs outside the lung. These data demonstrate that AdCFTR is well tolerated in non-human primates, although there is dose-dependent inflammation in the lung not clinically apparent.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗
PubMed1994

Evaluation of the respiratory epithelium of normals and individuals with cystic fibrosis for the presence of adenovirus E1a sequences relevant to the use of E1a- adenovirus vectors for gene therapy for the respiratory manifestations of cystic fibrosis.

Lung disease associated with disorders such as cystic fibrosis (CF) may be amenable to somatic gene therapy in which there is delivery of the normal gene directly to the respiratory epithelium using E1a- adenovirus (Ad) type 2- or 5-based vectors. For safety reasons, the Ad vectors are rendered replication deficient by deletion of the E1a region. Because there is the theoretical possibility of an E1a- replication-deficient vector replicating as a result of recombination or complementation with Ad 2/5 E1a sequences present in the target cell, this study is directed toward evaluating respiratory epithelium of normals and individuals with CF for the presence of E1a sequences. Using Ad 2/5 E1a-specific primers and the polymerase chain reaction to evaluate DNA recovered from freshly isolated nasal and bronchial epithelium recovered by brushing, E1a sequences were detected in respiratory epithelium of 19 of 91 normals (21%). In the E1a-positive samples, the average of E1a copy number was 55 +/- 18/10(3) recovered cells. In CF individuals, 7 of 52 (13%) had detectable E1a sequences in the respiratory epithelium, with E1a copy number in the positive samples of 80 +/- 21/10(3) recovered cells. These results demonstrate that there are detectable Ad 2/5 E1a sequences in the respiratory epithelium of a small percentage of normals and individuals with CF. Because of the theoretical potential of such sequences supporting replication of E1a- Ad vectors, human gene therapy protocols for CF utilizing such vectors should consider evaluating study individuals for the presence of Ad 2/5 E1a sequences in the respiratory epithelium.

Adenoviridae Infections↗
PubMed1994

Increasing expression of the normal human CFTR cDNA in cystic fibrosis epithelial cells results in a progressive increase in the level of CFTR protein expression, but a limit on the level of cAMP-stimulated chloride secretion.

Cystic fibrosis (CF) results from mutations of the CF transmembrane conductance regulator (CFTR) gene and the consequent defective regulation of cAMP-stimulated Cl- permeability across epithelial cell apical membranes. Given that in vitro transfer of normal CFTR cDNA corrects this defect and that recombinant adenovirus (Ad) vectors can transfer the normal human CFTR cDNA in vivo, Ad vectors have significant potential in the development of effective strategies for CF gene therapy. One concern is whether CFTR overexpression achievable with Ad vectors may have untoward effects on cAMP-stimulated Cl- efflux. To address this, the CF pancreatic epithelial cell line CFPAC-1 was infected with increasing doses of AdCFTR, a recombinant Ad containing the normal CFTR cDNA, and analyzed for CFTR mRNA and protein levels and CFTR function. As the AdCFTR dose increased [multiplicity of infection (moi) 0-1,000], CFTR mRNA and protein levels increased. However, while CFTR function measured by cAMP-stimulated 36Cl- efflux was observed with low doses of the vector (moi 20), there was no further increase in CFTR function with increasing doses of AdCFTR (moi from 20 to 1,000). These data suggest that after AdCFTR-mediated gene transfer, epithelial cells limit the level of cAMP-stimulated Cl- secretion despite increasing levels of CFTR protein.

Adenocarcinoma↗
PubMed1994

Ex vivo and in vivo gene transfer to the skin using replication-deficient recombinant adenovirus vectors.

The skin has the potential for a variety of gene therapy applications. In addition to local delivery, it is the largest organ of the body, and highly vascular, and thus is an ideal site for systemic delivery of gene products. To evaluate the potential for adenovirus-mediated skin gene transfer, the replication-deficient recombinant adenovirus vectors Ad.RSV beta gal (coding for Escherichia coli beta-galactosidase) and Ad alpha 1AT (coding for human alpha 1-antitrypsin) were used in both ex vivo and in vivo approaches. Following in vitro infection with Ad.RSV beta gal, murine keratinocytes expressed beta-galactosidase. Parallel in vitro studies with Ad alpha 1AT documented de novo synthesis and secretion of human alpha 1AT as shown by [35S]methionine labeling and immunoprecipitation. Quantification of human alpha 1AT in the culture supernatants demonstrated 0.1-0.3 microgram human alpha 1AT secreted/ml-24 h. Evaluation of the serum of mice receiving transplants (10(5) cells/mouse) of Ad alpha 1AT-infected syngeneic keratinocytes demonstrated human alpha 1AT for at least 14 d with maximum levels of 41 ng/ml. To demonstrate the feasibility of direct adenovirus-mediated in vivo transfer of genes to the skin, Ad.RSV beta gal or Ad alpha 1AT were administered subcutaneously to mice. Histologic evaluation after 4 d demonstrated expression of beta-galactosidase in various types of skin cells. Quantification of human alpha 1AT in serum of animals infected subcutaneously with Ad alpha 1AT showed levels of 53 ng/ml at day 4, with human alpha 1AT detectable for at least 14 d. These observations support the feasibility of ex vivo and in vivo gene transfer to the skin mediated by replication-deficient adenovirus vectors.

Adenoviridae↗
PubMed1994

Mechanism of enhancement of DNA expression consequent to cointernalization of a replication-deficient adenovirus and unmodified plasmid DNA.

Given the knowledge that replication-deficient adenoviruses can mediate the delivery of unlinked plasmid DNA into eukaryotic cells (K. Yoshimura, M. A. Rosenfeld, P. Seth, and R. G. Crystal, J. Biol. Chem. 268:2300-2303, 1993), this study focuses on the role of receptor-mediated endocytosis in this process. AdCFTR (an E1- E3- adenovirus type 5-based replication-deficient adenovirus containing the 4.5-kb human cystic fibrosis transmembrane conductance regulator cDNA) was added to Cos-7 cells together with plasmid pRSVL (containing the Rous sarcoma virus long terminal repeat promoter followed by the luciferase cDNA), and luciferase activity was quantified as a measure of the expression of the plasmid DNA. When AdCFTR was bound to Cos-7 cells at 4 degrees C and the cells were subsequently incubated at 37 degrees C in the presence of pRSVL, the expression of luciferase activity was increased in proportion to the amount of AdCFTR added, reaching > 10(4)-fold at 3,000 PFU per cell. AdCFTR-mediated increase in pRSVL was inhibited by addition of purified adenovirus fiber but not hexon, suggesting cell surface adenovirus receptors were involved in the cointernalization process. Cell lines with a high number of adenovirus receptors (Cos-7 and HeLa) showed significant AdCFTR-dependent pRSVL expression, while cell lines with low numbers of adenovirus receptors (NIH 3T3 and U-937) showed little. AdCFTR-mediated increase in the expression of pRSVL was prevented when AdCFTR was heat treated and exposed to antibody against adenovirus or when the cointernalization process was evaluated in the presence of chloroquine, conditions all known to prevent adenovirus-mediated disruption of endocytic vesicles. In contrast, the uptake of AdCFTR into Cos-7 cells was not affected by any of these conditions. When AdCFTR was exposed to UV light, its ability to grow in 293 cells was obviated, but AdCFTR-dependent increase in pRSVL expression was minimally reduced. Finally, empty capsids of AdCFTR were able to enhance the delivery and expression of plasmid pRSVL into Cos-7 cells, suggesting that the adenovirus genome is not required for AdCFTR-mediated plasmid cointernalization. Together, these observations suggest that the ability of a replication-deficient recombinantly adenovirus to mediate the cointernalization and expression of plasmids is mediated by the receptor-mediated endocytosis pathway.

Adenoviruses, Human↗
PubMed1994

Direct in vivo adenovirus-mediated gene transfer to salivary glands.

Gene transfer to the salivary glands holds the potential for the therapy of salivary gland disorders and for delivery of therapeutic proteins to the mouth and upper gastrointestinal tract. Administration of the recombinant adenovirus vectors Ad.RSV beta gal [coding for the intracellular protein beta-galactosidase (beta-Gal)] and Ad alpha 1AT [coding for human alpha 1-antitrypsin (alpha 1-AT), a secreted protein] to salivary gland cell lines in vitro demonstrated exogenous gene expression. Retrograde ductal injection of the Ad.RSV beta gal vector to rat salivary glands in vivo resulted in beta-Gal expression in acinar and ductal cells. Exposure of submandibular glands in vivo to Ad alpha 1AT resulted in expression of alpha 1-AT mRNA transcripts, de novo synthesis of alpha 1-AT, and secretion in the saliva. To evaluate the feasibility of adenovirus-mediated gene transfer to human glands, human minor salivary glands were infected ex vivo with Ad.RSV beta gal, and implanted into severe combined immunodeficient mice. Evaluation of the human tissue demonstrated beta-Gal activity. These observations demonstrate that adenovirus vectors are capable of direct delivery of genes to the salivary glands, suggesting a variety of possible gene therapy applications.

Adenoviridae↗