Leptin receptor JAK2/STAT3 signaling modulates expression of Frizzled receptors in articular chondrocytes.

OBJECTIVE: Differentiated articular chondrocytes express a functional bisoform of the leptin receptor (LRb); however, leptin-LRb signaling in these cells is poorly understood. We hypothesized that leptin-LRb signaling in articular chondrocytes functions to modulate canonical Wnt signaling events by altering the expression of Frizzled (FZD) receptors. METHODS: Human chondrocyte cell lines and primary articular chondrocytes were grown in serum containing growth media for 24h, followed by a media change to Dulbecco's modified Eagle's medium (DMEM) containing 1% Nutridoma-SP to obtain a serum-deficient environment for 24h before treatment. Treatments included recombinant human leptin (10-100nM), recombinant human IL-6 (0.3-3nM), or recombinant human erythropoietin (Epo) (10mU/ml). Cells were harvested 30min-48h after treatment and whole cell lysates were analyzed using immunoblots or luciferase assays. RESULTS: Treatment of cells with leptin resulted in activation of Janus kinase 2 (JAK2) and subsequent phosphorylation of specific tyrosine residues on LRb, followed by dose- and time-dependent increases in the expression of Frizzled-1 (FZD1) and Frizzled-7 (FZD7). Leptin-mediated increases in the expression of FZD1 were blocked by pre-treatment with the protein synthesis inhibitor cycloheximide or the JAK2 inhibitor AG490. Experiments using a series of hybrid Epo extracellular domain-leptin intracellular domain receptors (ELR) harboring mutations of specific tyrosine residues in the cytoplasmic tail showed that increases in the expression of FZD1 were dependent on LRb-mediated phosphorylation of STAT3, but not ERK1/2 or STAT5. Leptin pre-treatment of chondrocytes prior to Wnt3a stimulation resulted in an increased magnitude of canonical Wnt signaling. CONCLUSION: These experiments show that leptin-LRb signaling in articular chondrocytes modulates expression of canonical Wnt signaling receptors and suggests that direct cross-talk between these pathways is important in determining chondrocyte homeostasis.

Topical transfection using plasmid DNA in a water-in-oil nanoemulsion.

Expression plasmids encoding chloramphenicol acetyltransferase (CAT) or human interferon-alpha2 cDNA were formulated in water-in-oil nanoemulsions and applied to murine skin. The histological location of transfected cells was assessed by in situ DNA PCR and showed that the deposition of plasmid DNA was primarily in follicular keratinocytes. Transgene expression in the skin was monitored for 24-72 h, following topical application of either single or multiple daily doses by quantitative RT-PCR and ELISA. It was found that transgene expression was optimal at 24 h following topical application of a single dose of water-in-oil nanoemulsion containing plasmid DNA. Dose-response studies using a total dose of 3, 10 or 30 microg of plasmid DNA suggested that topical transfection using nanoemulsions is subject to both threshold and saturation effects. None of the cationic liposome formulations tested as controls mediated transgenic protein expression at levels higher than background values of the ELISAs used to assay transgenic protein. Single and multiple dose experiments using human interferon-alpha2 as a transgene indicated that the efficiency of nanoemulsion mediated transfection was most effective in the context of normal versus atrophic hair follicles. In addition, the total amount of human interferon-alpha2 present in skin appeared to accumulate as a consequence of multiple dosing. Histologic evaluation of treated skin showed no overt signs of toxicity or irritation associated with the short-term application of the nanoemulsions. The results suggest that water-in-oil nanoemulsions can be used to facilitate transfection of follicular keratinocytes in vivo.

Topical transport of hydrophilic compounds using water-in-oil nanoemulsions.

A variety of water-in-oil nanoemulsions were prepared using sorbitan monooleate (Span80), polyoxyethylene 20 sorbitan monooleate (Tween80), olive oil and water. The nanoemulsions were tested for their ability to facilitate transport of a model hydrophilic solute, inulin, across hairless and hairy mouse skin and hairy rat skin following topical in vitro application. The transport of inulin incorporated in water-in-oil nanoemulsions was found to be significantly higher (5- to 15-fold) than that obtained with micellar dispersions or aqueous controls. The rate and extent of inulin transport across hairy mouse skin was found to be highly dependent on the hydrophile-lipophile balance (HLB) of the surfactant mixture in the nanoemulsion. Nanoemuslions prepared using mixtures with lower HLB exhibited significantly higher rate and extent of transport. It was also found that nanoemulsion-mediated transport was independent of molecular size of the hydrophilic solute and the nature of the aqueous phase. More importantly, transport of inulin from nanoemulsions was independent of animal skin characteristics such as stratum corneum thickness and follicle-type. The combined results suggest that water-in-oil nanoemulsions that are compatible with the lipophilic sebum environment of the hair follicle facilitate efficient transport of incorporated hydrophilic solutes and imply that such transport is predominantly transfollicular in nature.

Substituted beta-cyclodextrins interact with PAMAM dendrimer-DNA complexes and modify transfection efficiency.

The efficiency of PAMAM dendrimer-mediated DNA transfer can be improved by the addition of substituted beta-cyclodextrins (beta-CDs) as formulation excipients. In vitro CAT expression increased approximately 200-fold when dendrimer/DNA/beta-CD formulations were applied on the surface of collagen membranes. The inclusion of beta-CD into the formulations resulted in particles that were smaller and more evenly distributed on the surface of the solid support. The average size of the complex formed at 50 microg/ml and at charge ratio of 1 decreased from 156 nm to 5.8 nm and 21.2 nm in 0.025-0.1% w/vol beta-CDs. Sulfonated beta-CDs bind to dendrimer and in the increased concentration may displace DNA in the dendrimer/DNA complex. High concentrations of amphoteric beta-CD do not dissociate dendrimer/DNA complexes; however, they may decrease their ability to transfect cells. At the optimized formulations the surface-modified beta-CDs may enhance solid support-based transfection in vitro, through modification of dendrimer/DNA complex composition and improved surface distribution.

Reduction of inflammatory response in the mouse brain with adenoviral-mediated transforming growth factor-ss1 expression.

UNLABELLED: Background and Purpose-Chemokines have been shown to play an important role in leukocyte and monocyte/macrophage infiltration into ischemic regions. The purpose of this study is to identify whether overexpression of the active human transforming growth factor-ss1 (ahTGF-ss1) can downregulate expression of monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha), and intercellular adhesion molecule-1 (ICAM-1) and reduce ischemic brain injury. METHODS: -Overexpression of transforming growth factor-ss1 (TGF-ss1) was achieved through adenoviral gene transfer. Five days after adenoviral transduction, the mouse underwent 30 minutes of middle cerebral artery occlusion followed by 1 to 7 days of reperfusion. TGF-ss1, MCP-1, MIP-1alpha, and ICAM-1 were detected by enzyme-linked immunosorbent assay and immunohistochemistry. Infarct areas and volumes were measured by cresyl violet staining. RESULTS: -MCP-1 and MIP-1alpha expression is increased after middle cerebral artery occlusion, and double-labeled immunostaining revealed that MCP-1 is colocalized with neurons and astrocytes. Viral-mediated TGF-ss1 overexpression was significantly greater at measured time points, with a peak at 7 to 9 days. The expression of MCP-1 and MIP-1alpha, but not ICAM-1, was reduced in the mice overexpressing ahTGF-ss1 (P:<0.05). Furthermore, infarct volume was significantly reduced in the mice overexpressing ahTGF-ss1 (P:<0.05). CONCLUSIONS: -This study demonstrates that MCP-1 and MIP-1alpha expressed in the ischemic region may play an important role in attracting inflammatory cells. The reduction of MCP-1 and MIP-1alpha, but not ICAM-1, in the mice overexpressing ahTGF-ss1 suggests that the neuroprotective effect of TGF-ss1 may result from the inhibition of chemokines during cerebral ischemia and reperfusion.

A simple method for the rapid generation of recombinant adenovirus vectors.

Recombinant adenoviruses are useful vectors for basic research. When the vectors are used for delineating protein function, several viruses, each containing a mutated version of the transgene are compared at the same time. However, methods to generate multiple vectors simultaneously within a short time period are cumbersome. In this report, we show that a novel backbone plasmid, when cotransfected with routinely used shuttle vectors into HEK293 cells allowed for production of recombinant viruses in an average of 14 days. The recombinant viruses had no detectable wild-type virus contamination by A549 plaque assay and only three to 300 E1a copies per 109 adenovirus genomes by a sensitive PCR-based assay. Further culturing or serial amplification did not result in wild-type revertants nor did cultures show increased levels of E1a copy number by quantitative PCR. Thus, recombinant adenovirus vectors can be produced very simply, rapidly and with little to no contaminating wild-type particles. This system should facilitate the generation of multiple genetic variants by eliminating the need for time-consuming plaque purification and the need to manipulate and screen very large plasmids. We call this the RAPAd.I system.

Application of membrane-based dendrimer/DNA complexes for solid phase transfection in vitro and in vivo.

In this study a general description of the use of solid support membranes as the device for DNA delivery mediated by PAMAM dendrimers is presented. In contrast to the other DNA carriers, dendrimer/DNA complexes retain the ability to transfect after drying, which enabled coating or incorporation of complexes into poly(DL-lactide-co-glycolide) or collagen-based bioerodable membranes. These studies provide support for the use of this technology for in vitro and in vivo transfection of skin cells. Expression of luciferase or green fluorescent protein from pCF1-Luc and pEGFP1 plasmids indicated that dendrimer/DNA complexes can mediate transfection after dissociation from the solid support and/or when retained on the surface of the membranes. Modification of the membranes by incorporation of an anionic lipid, phosphatidyl glycerol (PG) at 1-5% concentrations, resulted in more efficient in situ transfection, particularly with dendrimer/DNA complexes formed at the low charge ratios (1-5). We also report data supporting the feasibility of membrane-based dendrimer/DNA complexes, particularly formed at lower than neutralizing conditions, for topical in vivo delivery of DNA to hairless mouse skin.

The influence of interleukin-1 receptor antagonist transgene on spiral ganglion neurons.

The cytokine interleukin-1beta (IL-1) has been shown to induce the secretion of NGF and GDNF in several types of neuronal populations. IL-1 has also been shown to mediate immune response following trauma or presence of foreign antigens. We investigated the influence of an IL-1 antagonist on the survival of spiral ganglion neurons in inner ears in which hair cells have been eliminated. We used a replication-deficient adenoviral vector containing the human IL-1 receptor antagonist (IL-1ra) cDNA. Guinea pigs were bilaterally deafened with ototoxic drugs. One week later their left cochleae were inoculated with the IL-1ra vector, designated Ad.IL-1ra. The vector was delivered by injection through the cochlear round window. IL-1ra protein levels within the perilymph of Ad.IL-1ra-injected animals were measured with ELISA and found to be significantly elevated compared to our controls. Spiral ganglion cell counts in experimental ears revealed a lower density of neurons after Ad.IL-1ra inoculation. Taken together, the data suggest that the Ad.IL-1ra-infected cochlear cells synthesized the transgenic human IL-1ra protein, which was then secreted by the cells into the perilymph, resulting in an accelerated neuronal degeneration in hair cell-depleted ears.

Inner ear transgene expression after adenoviral vector inoculation in the endolymphatic sac.

Gene transfer has been performed in a variety of organs. In the mammalian inner ear, viral vectors have been used to introduce exogenous reporter genes via the scala tympani into the cochlea. While scala tympani inoculation is clinically feasible, it is not without risks. Moreover, transgene expression has so far been restricted to the cochlear tissues in the perilymphatic spaces that are contiguous with the scala tympani. To achieve gene transfer of vestibular organs and cells surrounding the endolymphatic space, and to extend the clinical utility of inner ear gene therapy, we developed a new surgical approach for vector inoculation. A replication-deficient adenoviral vector, Ad.RSVntlacZ, was injected into the guinea pig endolymphatic sac. A large number of blue (LacZ-positive) cells was observed in the endolymphatic sac and duct, the vestibule, and the ampulla. Blue cells were also detected in the cochlea, mainly in cells bordering the endolymphatic space: marginal cells in the stria vascularis and supporting cells in the organ of Corti. These findings indicate that inoculation of viral vectors into the endolymphatic sac can provide efficient gene transfer into a variety of cell types that are not accessible via scala tympani inoculation.

In vitro and in vivo assessment of adenovirus 41 as a vector for gene delivery to the intestine.

In order to identify suitable adenoviral vectors for efficient delivery of transgenic proteins and peptides to the intestine, the ability of adenovirus types 5 and 41 (an enterotropic serotype) to bind to and enter undifferentiated and differentiated enterocytes was assessed. FACS analysis showed no significant difference between the virions in their ability to bind to undifferentiated Caco-2 cells as 81.6% of the cellular population bound adenovirus 5 (Ad 5) and 79.8% bound Ad 41. Both virions were also efficiently internalized in this cell type as 99.6% of the cells took up Ad 5, while 95.9% took up Ad 41. In studies with differentiated enterocytes, probable targets for oral gene delivery but rather resistant to adenovirus-mediated gene transfer, 28.4% of the population internalized the Ad 5 vector and less than 10% bound the virus. Adenovirus 41 was efficiently internalized in differentiated enterocytes as 89.6% of the cellular population took up the virus while 37.4% bound the virus. These results were consistent with those observed in vivo in rat jejunum. Thus, molecularly engineered Ad 41-based recombinants could be highly efficient vectors for delivery of transgenic proteins to differentiated enterocytes.

Beta cyclodextrins enhance adenoviral-mediated gene delivery to the intestine.

PURPOSE: In general, the intestinal epithelium is quite refractory to viral and non-viral methods of gene transfer. In this report, various cyclodextrin formulations were tested for their ability to enhance adenoviral transduction efficiency in two models of the intestinal epithelium: differentiated Caco-2 cells and rat jejunum. METHODS: Transduction efficiency of replication-deficient adenovirus type 5 vectors encoded with either the E. coli beta-galactosidase or the jellyfish green fluorescent protein gene was assessed by X-gal staining or visualization of fluorescence 48 hours after infection. In vivo experiments were performed using an intestinal loop ligation technique. RESULTS: Several formulations of neutral and positively charged beta cyclodextrins significantly enhanced adenoviral-mediated gene transfer in the selected models. The cyclodextrin formulations studied increased adenoviral transduction in the intestine by enhancing both viral binding and internalization. Viral binding was significantly increased on cell membranes treated with positively charged cyclodextrins, as seen with confocal microscopy and rhodamine-labeled virus. Permeability studies and TEER readings revealed that the most successful formulations gently disrupt cell membranes. This enhances internalization of viral particles and results in increased levels of gene expression. CONCLUSIONS: These formulations can be of value in gene transfer to cells and tissues in which adenoviral infection is limited due to a lack of fiber and alpha(v) integrin receptors. They are simple to prepare and do not affect the ability of the virus to transduce target cells.

Overexpression of human intestinal oligopeptide transporter in mammalian cells via adenoviral transduction.

PURPOSE: Our goals are to establish an in vitro screening system and to evaluate a new approach in improving oral absorption of peptides and peptide-like drugs by overexpression of the human intestinal oligopeptide transporter (hPepT1). This study characterizes the expression of hPepT1 in human intestinal Caco-2 cells, rat intestinal epithelial cells (IEC-18), and human cervix epithelial cells (Hela) after adenoviral transduction. METHODS: A recombinant replication-deficient adenovirus carrying the hPepT1 gene was made and used as a vector for the expression of hPepT1. The increase in the uptake permeability of cephalexin and Gly-Sar was determined. The effects of time, dose, apical pH, and substrate specificity were evaluated. RESULTS: A significant increase in the uptake permeability of Gly-Sar and cephalexin was found in all three cell lines after viral transduction. The increase of Gly-Sar permeability in Hela. IEC-18, and Caco-2 cells was 85-, 46-, and 15-fold respectively. Immunoblotting using an antibody against hPepT1 detected high levels of a 85-98-kDa protein in all three infected cell lines. Substrate permeability was dependent on time of infection, inward pH gradients, and multiplicity of infection (MOI). Decreased infectivity and lower hPepT1 expression were observed in differentiated Caco-2 cells. The uptake was inhibited by dipeptides and beta-lactam antibiotics but not amino acids. CONCLUSIONS: Adenoviral infected Hela cells displayed a pronounced level of hPepT1 expression with a low background and high specificity to dipeptides. These features make this system a useful tool for screening of potential substrates. The success of overexpression of hPepT1 in Caco-2 and IEC-18 cells may lead to a novel approach in improving oral absorption of peptides and peptidornimetic drugs.

Development of a highly efficient purification process for recombinant adenoviral vectors for oral gene delivery.

Recently, replication-deficient adenoviruses have received increasing attention as vector for gene delivery and as potential vaccine carriers. With the increased use of the vector in vivo and in clinical trails, the demand for a safe, rapid, and cost effective purification process has been heightened. In this report, a simple and efficient method for the purification of large quantities of live adenoviral vectors was developed. The process involved the replacement of cesium chloride (CsCl) gradients with sucrose gradients. Ultracentrifugation times were reduced and the desalting step eliminated, decreasing total preparation time by 15 hr. A 20-80% linear sucrose gradient provided optimal recovery of infectious viral particles and positioning of the viral band in the gradient. Purification with this gradient system produced a preparation containing 1.39 x 10(14) lac-forming units (lfu)/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the process also removed all associated cellular proteins from the preparation. Studies have shown that direct lyophilization of the vector in sucrose after purification produces a product containing 1.4 x 10(12) lfu/ml. Minimal degradation was seen in the lyophilized preparation. A viral concentration of 6 x 10(11) lfu/ml was detected in the product after 150 days in storage at -20 degrees C. This approach will not only simplify the preparation of adenoviral vectors for in vivo studies and clinical trials, but will facilitate production of stable adenoviral formulations for oral gene delivery.

Factors that influence stability of recombinant adenoviral preparations for human gene therapy.

This report identifies formulation and processing factors that influence stability of viral preparations such as selection of appropriate buffer systems, cryoprotectants, and cooling rates. Adenovirus type 5 containing the lacZ marker gene was suspended in combinations of trehalose, sorbitol, sucrose, mannitol, glycine, CaCl2, and gelatin. X-gal stains of 293 cells were used to determine the lac-forming units (lfu)/ml of each preparation before and after treatments. Phosphate-buffered solutions (except those containing sucrose or trehalose) demonstrated a drop of 3 pH units upon freezing regardless of cryoprotectant used. Tris-buffered solutions demonstrated a variation in pH which was dependent upon chosen cryoprotectant, with 1 M trehalose exhibiting no change and a 5% mannitol/10 mM CaCl2 combination showing a 3-unit drop in pH. 4-[2-Hydroxyethyl]-1-piperazine ethanesulfonic acid (HEPES)-buffered solutions showed little change in initial pH when frozen regardless of cryoprotectant chosen. In solution, adenovirus was not affected by incubation for 24 hr in buffers ranging from pH 4 to 8. However, when the solutions were frozen, the number of remaining infectious virions was dependent upon the final pH of the suspending medium. Cryoprotectant solutions that significantly maintained viral stability during a single freeze--thaw cycle were 0.5 M sucrose, 0.5 M trehalose, and 10% sorbitol/0.4% gelatin. Long-term stability studies were performed at 4 degrees C with lyophilized sorbital/gelatin and sucrose preparations. Both formulations provided adequate stability for the adenovirus, with 2.6 and 5.6 x 10(11) lfu/ml detected 150 days after drying, respectively.

Role of integrin expression in adenovirus-mediated gene delivery to the intestinal epithelium.

Adenoviral vectors are being developed for oral delivery of therapeutic genes to the intestine. Initial studies in the rat using mucolytics and direct application of adenovirus encoded with the interleukin-1 receptor antagonist gene to the jejunum produced limited gene expression. The goal of this study was to determine the role of integrins in adenovirus-mediated gene delivery to the intestinal epithelium. Integrins are involved in cellular differentiation and tight junction formation and mediate adenoviral internalization. Results from Caco-2 and IEC-18 cells suggest that, as enterocytes differentiate, cell-surface integrin expression decreases. Pretreatment of Caco-2 cells with RGD peptides reduced adenoviral transduction efficiency by 80% in undifferentiated cells and 20% in differentiated cells. Both differentiated and undifferentiated IEC-18 cells showed a 70% drop in transduction when pretreated with the peptide. Infection inhibition studies with monoclonal antibodies further suggest that alpha(v)beta3 and alpha6beta1 integrins play significant roles in adenoviral internalization in the intestine. Expression of integrins in cell culture models of the intestine correlated with in vivo expression in intestinal segments. These results indicate that the ileum is a prime target for efficient adenovirus-mediated gene transfer in the rat. To enhance transduction in differentiated enterocytes (probable targets for oral gene delivery), Caco-2 cells were treated with interleukin-1beta (a cytokine known to increase integrin expression) prior to administration of the virus. Transduction efficiency increased four-fold.

Molecular lysis of synovial lining cells by in vivo herpes simplex virus-thymidine kinase gene transfer.

Herpes simples virus thymidine kinase (HSV-TK) expression plasmid DNA was injected into the joint space of rabbits with antigen-induced arthritis (AIA). Purified plasmid DNA was able to mediate transfection of synovial lining cells and transient overexpression of HSV-TK in the context of active synovial inflammation. The pharmacodynamic distribution of intraarticular expression plasmid DNA was confined to the joint space. Arthritic rabbits treated with intraarticular expression plasmid DNA followed by intravenous ganciclovir (GCV, 5 mg/kg) twice daily for 3 days showed histologic evidence of synovial lining layer cytolysis when articular tissues were examined 21 days posttreatment. There was also a reduction in joint swelling in the TK-treated knees. No untoward clinical effects were observed in the rabbits and no evidence of cytolytic damage specific to the TK-GCV gene therapy was observed either in the articular cartilage or bone. The application of TK-GCV intraarticular gene therapy using purified expression plasmid DNA for the induction of synovial cytolysis may be applicable to the treatment of human inflammatory arthritis.

The absence of accessible vitronectin receptors in differentiated tissue hinders adenoviral-mediated gene transfer to the intestinal epithelium in vitro.

PURPOSE: Adenoviral (Ad) vectors have been used as efficient tools for gene therapy in various tissues, whereas in some differentiated epithelium transduction efficiency is almost abolished. METHODS: Caco-2 cell monolayers were chosen as an in vitro model for the differentiated intestinal epithelium. Fluorescence-labeled adenoviral particles were used for binding studies to cell surfaces. Internalization receptors for adenoviral uptake were detected by a fluorescence-labeled vitronectin antibody. Gene expression was studied by using the beta-galactosidase reporter gene. All experiments were done on undifferentiated and differentiated Caco-2 cells. Furthermore, adenoviral particles were allowed to bind to differentiated Caco-2 monolayers followed by a trypsinization step that disintegrates the monolayers and result in a cell suspension. Gene expression was tested after reseeding the cells into dishes. RESULTS: The results from adenoviral binding studies, vitronectin immunofluorescence detection and gene expression are in good agreement and indicate that virion binding as well as the expression of internalization receptors almost disappear in fully differentiated cells. Nonetheless, adenoviral binding to differentiated monolayers seems to be sufficient to cause up to 53% gene expression, but only if internalization of the vector can be induced by disintegrating the monolayers and releasing free vitronectin receptors. CONCLUSIONS: These findings indicate that gene transfer to the intestinal epithelium utilizing adenoviral vectors is poor and ineffective, because of the lack of sufficient internalization receptors. If these receptors can be exposed in differentiated epithelium, transduction can be made more efficient. Alternatively, a viral vector must be developed whose uptake mechanism is independent of integrin receptor expression like the enteral virus Ad40, or Ad5 could be conjugated to ligands that trigger viral internalization by receptor-mediated endocytosis.

Transplantation of adenovirally transduced allogeneic chondrocytes into articular cartilage defects in vivo.

Gene transfer to chondrocytes followed by intra-articular transplantation may allow for functional modulation of chondrocyte biology and enhanced repair of damaged articular cartilage. We chose to examine the loss of chondrocytes transduced with a recombinant adenovirus containing the gene for Escherichia coli beta-galactosidase (Ad.RSVntlacZ), followed by transplantation into deep and shallow articular cartilage defects using New Zealand White rabbits as an animal model. A type I collagen matrix was used as a carrier for the growth of the transduced chondrocytes and to retain the cells within the surgically created articular defects. Histochemical analysis of matrices recovered from the animals 1, 3 and 10 days after implantation showed the continued loss of lacZ positive chondrocytes. The number of cells recovered from the matrices was also compared with the initial innoculum of transduced cells present within the matrices at the time of implantation. The greatest loss of transduced cells was observed in the first 24 h after implantation. The numbers of transduced cells present within the matrices were relatively constant between 1 and 3 days postimplantation, but had progressively declined by 10 days postimplantation. These results suggest that transduction of chondrocytes followed by intra-articular transplantation in this rabbit model may enable us to examine the biological effects of focal transgenic overexpression of proteins involved in cartilage homeostasis and repair.

Viral-mediated gene transfer in the cochlea.

Gene transfer is an exciting new tool in medical therapy and scientific investigation, but only very recently has it begun to be developed in the auditory system. This paper describes in vivo and ex vivo experiments using an adenoviral vector (Ad. RSVntlacZ), which is a replication-deficient virus based on a human adenoviral (serotype 5) genomic backbone. The in vivo experiments demonstrate successful gene transfer into multiple types of cochlear cells. We observed a relatively efficient transduction, several weeks of sustained transgene expression and an absence of major lethal cytotoxicity in spiral ganglion and epithelial cells of the cochlea in adult animals. The ex vivo experiments were performed using fibroblasts transduced in vitro with Ad. RSVntlacZ. Two weeks after inoculation of the fibroblasts into the perilymph, we observed transplanted fibroblasts, which were adherent to the lining of the perilymphatic spaces, and were expressing the lacZ transgene. We speculate that, as the genetic basis of degenerative cochlear diseases is characterized on a mutational level, transgene expression will allow us to test hypotheses regarding the effects of specific genes on cochlear cell biology. Gene transfer will not only increase our understanding of the pathophysiology of hearing loss, but also may provide gene therapy for disease.