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PubMed2006

Targeted retroviral vectors displaying a cleavage site-engineered hemagglutinin (HA) through HA-protease interactions.

We report here a targeting method that exploits the expression pattern of cell surface proteases to induce gene delivery to specific tissues. We describe retroviral vectors harboring modified surface glycoproteins derived from an avian influenza virus hemagglutinin (HA) for which the cell entry properties, dependent on HA cleavage by producer cells, were conditionally blocked at a postbinding step by insertion of matrix metalloproteinase (MMP) substrates. We demonstrate that such vectors induce gene transfer, both in vitro and in mice harboring human tumor xenografts, only through contact with target cells expressing MMPs that cleave the substrate introduced into the recombinant HA. This selective gene transfer in MMP-rich cells was specifically inhibited by 1,10-phenanthroline, a broad-range MMP inhibitor. Importantly, such MMP-activatable vectors selectively transduced MMP-rich cells in heterogeneous populations containing MMP-rich and MMP-poor cells. These vectors will allow useful gene transfer applications into target cells exhibiting specific protease activities.

Amino Acid Sequence↗
PubMed1997

Ganglioside GD3 and GD3-lactone mediated regulation of the intermolecular organization in mixed monolayers with dipalmitoylphosphatidylcholine.

The interactions of dpPC with ganglioside GD3 and two lactones. GD3LacI or GD3LacII, in lipid monolayers occur with reduced, unaltered, or increased molecular area and surface potential/molecule, respectively. dpPC is fully miscible with GD3 and GD3LacI but films with GD3LacII show immiscibility above 75 mol% lactone. At low proportions of GD3 in mixtures with dpPC, GD3 undergoes condensation and depolarization; dpPC is depolarized and its molecular area is reduced above 50 mol% GD3. GD3LacI forms ideally mixed films with dpPC. Mixtures of dpPC with GD3LacII at mole fractions below 0.3 show increased mean molecular area and surface potential/molecule mostly due to lactone alterations. Between mole fractions of 0.3 and 0.75 the surface parameters of dpPC are altered, and above these proportions both lipids are immiscible. Defined variations of molecular properties induced by ganglioside lactonization are selectively transduced to changes of the intermolecular organization and surface electrostatics in mixed interfaces with dpPC. Thus, changes in the relative proportions of a ganglioside and its lactone forms may act as sensitive biotransducers for membrane-mediated cellular functions, without the need for metabolically altering the concentration of gangliosides.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Adenoviral-mediated gene transfer to renal tubular cells in vivo.

The efficient introduction of genetic material into quiescent renal cells is potentially important in the study of renal physiopathology and for gene therapy of kidney related disorders. A replication-deficient adenoviral vector that contained a reporter gene encoding the nuclear beta-galactosidase was either selectively perfused into the renal artery or infused through a retrograde catheter into the pyelic cavity of the left kidney of adult rats. Highly efficient gene transfer was achieved by either route of administration, and nuclear beta-galactosidase activity was detected for two to four weeks following a progressive decline of expression. Genetically-modified cells were identified as proximal tubular cells when the adenoviral vector was selectively perfused via the renal artery, while tubular cells from the papilla and medulla were selectively transduced by retrograde infusion of the viral vector. No obvious cytopathic effect was observed. We conclude that: (i) efficient gene transfer in renal tubular cells can be achieved by adenoviral vectors; (ii) the targeted cell population can be chosen through the route of administration.

Adenoviruses, Human↗
PubMed2002

Herpes simplex virus as a model vector system for gene therapy in renal disease.

The past decade has been marked by significant advances in the application of gene transfer into living cells of animals and humans. These approaches have been tested in a few animal models of inherited and acquired renal diseases, including carbonic anhydrase II deficiency [1] and experimental glomerulonephritis [2, 3]. Gene transfer into proximal tubular cells has been successfully accomplished by intrarenal arterial infusion of a liposomal complex [4] or an adenoviral vector [5]. Tubular cells from the papilla and medulla have been selectively transduced by retrograde infusion into the pelvi-calyceal system of an adenoviral vector containing a reporter for beta-galactosidase [5]. Although the results of these initial studies are promising, further studies to optimize viral vectors, maximize gene delivery, minimize side-effects, and develop cell-specific and long-term regulated gene expression are critical to the success of gene therapy targeted to specific compartments of the kidney. Our recent efforts have focused on defining the cellular pathways responsible for viral entry and infection into renal epithelial cells using herpes simplex virus (HSV) as a model vector. We anticipate that a solid understanding of the basic scientific principles underlying viral entry and gene expression into specific populations of renal cells will facilitate the design of successful therapeutic viral-based gene transfer strategies.

Animals↗
PubMed2002

T-bet regulates IgG class switching and pathogenic autoantibody production.

A molecular understanding of the regulation of IgG class switching to IL-4-independent isotypes, particularly to IgG2a, remains largely unknown. The T-box transcription factor T-bet directly regulates Th1 lineage commitment by CD4 T cells, but its role in B lymphocytes has been largely unexplored. We show here a role for T-bet in the regulation of IgG class switching, especially to IgG2a. T-bet-deficient B lymphocytes demonstrate impaired production of IgG2a, IgG2b, and IgG3 and, most strikingly, are unable to generate germ-line or postswitch IgG2a transcripts in response to IFN-gamma. Conversely, enforced expression of T-bet initiates IgG2a switching in cell lines and primary cells. This function contributes critically to the pathogenesis of murine lupus, where the absence of T-bet strikingly reduces B cell-dependent manifestations, including autoantibody production, hypergammaglobulinemia, and immune-complex renal disease and, in particular, abrogates IFN-gamma-mediated IgG2a production. Classical T cell manifestations persisted, including lymphadenopathy and cellular infiltrates of skin and liver. These results identify T-bet as a selective transducer of IFN-gamma-mediated IgG2a class switching in B cells and emphasize the importance of this regulation in the pathogenesis of humorally mediated autoimmunity.

Animals↗
PubMedDate not supplied

In vivo perivascular implantation of encapsulated packaging cells for prolonged retroviral gene transfer.

Long-term benefits of coronary angioplasty remain limited by the treatment-induced renarrowing of arteries, termed restenosis. One of the mechanisms leading to restenosis is the proliferation of smooth muscle cells. Therefore, proliferating cells of the injured arterial wall, which can be selectively transduced by retroviruses, are potential targets for gene therapy strategies. A direct single-dose therapeutic application of retroviral vectors for inhibition of cell proliferation is normally limited by too low transduction efficiencies. Encapsulated retrovirus-producing cells release viral vectors from microcapsules, and may enhance the transduction efficiency by prolonged infection. Primary and immortal murine and porcine cells and murine retrovirus-producing cells were encapsulated in cellulose sulphate. Cell viability was monitored by analysing cell metabolism. Safety, stability, transfer efficiency and extent of restenosis using capsules were determined in a porcine restenosis model for local gene therapy using morphometry, histology, in situ beta-galactosidase assay and PCR. Encapsulation of cells did not impair cell viability. Capsules containing retrovirus-producing cells expressing the beta-galactosidase reporter gene were implanted into periarterial tissue or a pig model of restenosis. Three weeks following implantation, beta-galactosidase activity was detected in the pericapsular tissue with a transduction efficiency of approximately 1 in 500 cells. Adventitial implantation of vector-producing encapsulated cells for gene therapy may, therefore, facilitate successful targeting of proliferating vascular smooth muscle cells, and allow stable integration of therapeutic genes into surrounding cells. The encapsulation of vector-producing cells could represent a novel and feasible way to optimize local retroviral gene therapy.

3T3 Cells↗
PubMed2001

Adeno-associated virus and lentivirus vectors mediate efficient and sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons.

Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.

Animals↗
PubMed1998

Development of novel cell surface CD34-targeted recombinant adenoassociated virus vectors for gene therapy.

Recombinant adenoassociated virus (rAAV) type 2 vectors have been used to transduce a wide variety of cell types, including hematopoietic progenitor cells. For in vivo gene transfer, it is desirable to have an rAAV vector that specifically transduces selected target cells. As a first step toward generating an rAAV vector capable of targeting delivery in vivo, we have engineered a chimeric protein combining the AAV capsid protein and the variable region of a single-chain antibody against human CD34 molecules, a cell surface marker for hematopoietic stem/progenitor cells. Inclusion of the chimeric CD34 single-chain antibody-AAV capsid proteins within an rAAV virion significantly increased the preferential infectivity of rAAV for the CD34+ human myoleukemia cell line KG-1, which is normally refractory to rAAV transduction. Antibodies against the single-chain antibody and the CD34 protein blocked this transduction. This chimeric vector represents a significant improvement in the host range of rAAV and the first step toward specific gene delivery by rAAV vectors to cells of choice, in this case, hematopoietic progenitor cells, for the treatment of human disease.

Antibodies, Anti-Idiotypic↗
PubMed2001

Suicide gene therapy for chemically induced rat bladder tumor entailing instillation of adenoviral vectors.

The efficacy of an in vivo gene therapy protocol making use of an adenoviral vector in the treatment of bladder cancer was examined. Bladder tumors were induced in rats by oral administration of BBN (N-butyl-N-(4-hydroxybutyl)nitrosamine). Histologically, such tumors resemble those seen in human bladder cancer, and the cells can be selectively transduced using adenoviral vectors. The therapeutic protocol thus entailed instillation of an adenoviral vector containing the HSV-tk suicide gene into rat bladder followed by a regimen of intraperitoneal ganciclovir (GCV) injections. Histological examination after a short-term GCV regimen (3 days) revealed marked vacuolization of the tumor cells. Moreover, TUNEL assays showed that the cytotoxic reaction was mediated by apoptosis. Following a long-term GCV regimen (14 days), tumor growth was significantly inhibited and glandular metaplasia was observed. This is the first report demonstrating the efficacy of in vivo suicide gene therapy in a chemically induced transitional cell carcinoma like that seen in most human bladder cancer. Intravesical instillation is already a well established clinical technique. Our findings indicate that now there is a strong potential for its incorporation into new and useful gene therapies aimed at the treatment of human bladder cancer.

Adenoviridae↗
PubMed1990

Gene transfer into murine hematopoietic stem cells and bone marrow stromal cells.

The use of recombinant retroviral vectors to transfer genetic sequences into hematopoietic stem cells (HSC) is one approach to somatic gene therapy. Two limitations of such retroviral vectors are the degree of efficiency of transfer into the reconstituting hematopoietic stem cells and the loss of reconstituting ability of hematopoietic stem cells when manipulated in vitro during infection and selection. We have investigated the effects on the efficiency of gene transfer of prestimulation of hematopoietic stem cells by growth factors prior to infection. Prestimulation of bone marrow cells in WEHI-3b-conditioned media improved the efficiency of gene transfer into CFU-S stem cells. The majority of animals transplanted with bone marrow infected after prestimulation with a simplified retrovirus, Zip PGK ADA, demonstrated long-term and stable expression of human adenosine deaminase (ADA) after full hematopoietic reconstitution. In separate experiments, retroviral vectors have been used to transfer the SV40 large T antigen sequences into stromal cells making up the hematopoietic microenvironment. Stromal cells expressing large T antigen are immortalized, and some support the maintenance of day 12 CFU-S (CFU-S12) and reconstituting hematopoietic stem cells in vitro for up to 4 weeks. Such immortalized stromal cell lines provide an in vitro hematopoietic microenvironment which may allow prolonged in vitro manipulations during infection and selection of hematopoietic stem cells without loss of reconstituting ability. We are using immortalized stromal cell lines resistant to deoxycoformycin (dCF) to select transduced murine HSC containing human ADA in vitro. The use of recombinant retroviral vectors provides a promising approach to correction of human diseases involving bone marrow cells.

Animals↗
PubMed2004

Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways.

Gefitinib (Iressa, Astra Zeneca Pharmaceuticals) is a tyrosine kinase inhibitor that targets the epidermal growth factor receptor (EGFR) and induces dramatic clinical responses in nonsmall cell lung cancers (NSCLCs) with activating mutations within the EGFR kinase domain. We report that these mutant EGFRs selectively activate Akt and signal transduction and activator of transcription (STAT) signaling pathways, which promote cell survival, but have no effect on extracellular signal-regulated kinase signaling, which induces proliferation. NSCLC cells expressing mutant EGFRs underwent extensive apoptosis after small interfering RNA-mediated knockdown of the mutant EGFR or treatment with pharmacological inhibitors of Akt and STAT signaling and were relatively resistant to apoptosis induced by conventional chemotherapeutic drugs. Thus, mutant EGFRs selectively transduce survival signals on which NSCLCs become dependent; inhibition of those signals by gefitinib may contribute to the drug's efficacy.

Animals↗
PubMed1966

Location of a mutator gene in Salmonella typhimurium by cotransduction.

Kirchner, Carl E. J. (Suffolk County Community College, Selden, N.Y.), and Matthew J. Rudden. Location of a mutator gene in Salmonella typhimurium by cotransduction. J. Bacteriol. 92:1453-1456. 1966.-The LT7 strain of Salmonella typhimurium has been shown to possess a mutator gene which is responsible for an increase in mutation frequency for most loci tested. Preliminary results suggested the gene might be responsible for the production of an abnormal purine or pyrimidine base. Phage prepared on the mutator strain were used to transduce selected purine and pyrimidine LT2 mutants that do not possess this gene. A high frequency (60%) of cotransduction was observed with mutants from only one locus, purA. Transduction of additional mutants from this region gave similar results, except for one mutant (purA1) which showed no transduction of the mutator gene or the purA1 region. The results show that the mutator gene is very closely linked to the purA locus and suggest that it might be part of it.

Chromosome Mapping↗
PubMed2001

Genetic targeting of an adenovirus vector via replacement of the fiber protein with the phage T4 fibritin.

The utility of adenovirus (Ad) vectors for gene therapy is restricted by their inability to selectively transduce disease-affected tissues. This limitation may be overcome by the derivation of vectors capable of interacting with receptors specifically expressed in the target tissue. Previous attempts to alter Ad tropism by genetic modification of the Ad fiber have had limited success due to structural conflicts between the fiber and the targeting ligand. Here we present a strategy to derive an Ad vector with enhanced targeting potential by a radical replacement of the fiber protein in the Ad capsid with a chimeric molecule containing a heterologous trimerization motif and a receptor-binding ligand. Our approach, which capitalized upon the overall structural similarity between the human Ad type 5 (Ad5) fiber and bacteriophage T4 fibritin proteins, has resulted in the generation of a genetically modified Ad5 incorporating chimeric fiber-fibritin proteins targeted to artificial receptor molecules. Gene transfer studies employing this novel viral vector have demonstrated its capacity to efficiently deliver a transgene payload to the target cells in a receptor-specific manner.

Adenoviruses, Human↗
PubMedDate not supplied

US of the peritoneum.

Familiarity with the pathophysiology of peritoneal disease is the basis of successful ultrasound (US) study of the peritoneum. The pouch of Douglas, diaphragmatic surfaces, the paracolic gutters, and the regions of the mesentery and omentum should receive careful scrutiny in the patient at risk for a peritoneal disease process. An optimal US technique requires assessment of the entire peritoneum with a transducer selected to reflect the depth of the region of interest. US may demonstrate minute quantities of free intraperitoneal fluid and is therefore capable of providing sensitive quantitative information about ascites. Qualitative information may also be inferred, as blood, pus, and neoplastic cells demonstrate correlation with particulate ascites on gray-scale US scans. Peritoneal nodules, plaques, and thickening may be detected on the visceral or parietal peritoneal surfaces, especially when high-frequency probes are used. Transvaginal study in women increases the sensitivity of US for detection of peritoneal disease. In women who have unexplained sepsis or are at risk for carcinomatosis, transvaginal scanning should routinely be added to the regular abdominal and pelvic studies regardless of the findings of those studies. Peritoneal carcinomatosis, primary peritoneal neoplasms, pseudomyxoma peritonei, and peritonitis have characteristic appearances at US.

Diagnosis, Differential↗
PubMed2004

Converse relationship between in vitro osteogenic differentiation and in vivo bone healing elicited by different populations of muscle-derived cells genetically engineered to express BMP4.

UNLABELLED: In this study, we compared the use of primary muscle-derived osteoprogenitor cells (PP6 cells) for the delivery of BMP4 to improve bone healing to that of muscle-derived non-osteoprogenitor cells (PP1 cells). Surprisingly, the use of PP1 cells resulted in an improved outcome because of the lack of adverse responses to BMP4 involving cell differentiation, proliferation, and apoptosis. INTRODUCTION: Although researchers frequently opt to use osteogenic cells for osteogenic bone morphogenetic protein (BMP)-based ex vivo gene therapy to improve bone healing, it remains unclear whether the osteogenic potential of a cellular vehicle affects the outcome of bone healing applications. Here we compared the use of muscle-derived non-osteoprogenitor cells (PP1 cells) to that of primary muscle-derived osteoprogenitor cells (PP6 cells) for the delivery of BMP4 to improve the healing of bone defects. MATERIALS AND METHODS: Two distinct populations of primary rat muscle-derived cells--PP1 and PP6--were selected, transduced with retroviral vectors to express BMP4 or a marker gene (LacZ), and implanted into critical-sized calvarial defects created in syngeneic rats. The bone healing was monitored radiographically and histologically at 7 and 14 weeks after implantation. Cellular responses to BMP4 were evaluated by alkaline phosphatase histochemical staining and RT-PCR of another osteogenic marker to indicate osteogenic differentiation, a cell proliferation assay and BrdU (bromodeoxyuridine) labeling to assess cell proliferation, and the TUNEL assay to determine apoptosis. RESULTS AND CONCLUSIONS: In all animals (nine rats per group), transduced PP1 cells expressing BMP4 demonstrated significantly advanced healing compared with PP6 cells expressing BMP4 and control cells expressing LacZ. We found that constitutive BMP4 expression negatively impacted the in vitro proliferation and in vivo survival rates of PP6 cells, but not PP1 cells. BMP4 exposure also directly inhibited the proliferation and induced the apoptosis of PP6 cells, but not PP1 cells. The impairment in PP6 cell proliferation was directly associated with the osteogenic differentiation of these cells. These results indicate that PP1 cells are better suited than osteoprogenitor cells for use as cellular vehicles to deliver osteogenic BMP4 to improve bone healing and that cellular behavior in response to a particular gene can be used to predict the cells' performance as delivery vehicles in ex vivo gene therapy.

Animals↗
PubMed1999

Specific galpha11beta3gamma5 protein involvement in endothelin receptor-induced phosphatidylinositol hydrolysis and Ca2+ release in rat portal vein myocytes.

In this study, we identified the receptor subtype activated by endothelin-1 (ET-1) and the subunit composition of the G protein coupling this receptor to increase in cytosolic Ca2+ concentration in rat portal vein myocytes. We used intranuclear antisense oligonucleotide injection to selectively inhibit the expression of G protein subunits. We show here that the endothelin receptor subtype A (ETA)-mediated increase in cytosolic Ca2+ concentration was mainly dependent on Ca2+ release from the intracellular store. ETA receptor-mediated Ca2+ release was selectively inhibited by antisense oligonucleotides that inhibited the expression of alpha11, beta3, and gamma5 subunits, as checked by immunocytochemistry. Intracellular dialysis of a carboxyl terminal anti-betacom antibody and a peptide corresponding to the Gbetagamma binding region of the beta-adrenergic receptor kinase-1 had no effect on the ETA receptor-mediated Ca2+ release. In contrast, a synthetic peptide corresponding to the carboxyl terminus of the alphaq/alpha11 subunit, heparin (an inhibitor of inositol 1,4,5-trisphosphate receptors), and U73122 (an inhibitor of phosphatidylinositol-phospholipase C) inhibited, in a concentration-dependent manner, the ETA receptor-mediated Ca2+ responses. Accumulation of [3H]inositol trisphosphate evoked by norepinephrine peaked at approximately 15 s, whereas that evoked by ET-1 progressively increased within 2 min. In myocytes injected with anti-alphaq antisense oligonucleotides, both amplitude and time course of the norepinephrine-induced Ca2+ release became similar to those of the ET-1-induced Ca2+ response. We conclude that the ETA receptor-mediated Ca2+ release is selectively transduced by the heterotrimeric G11 protein composed of alpha11, beta3, and gamma5 subunits, and that a delayed stimulation of phospholipase C occurs via the alpha11 subunit.

Animals↗
PubMed2004

Establishment of life-span extended bovine fibroblast cells carrying the characterization of primary cells.

Although primary bovine embryonic fibroblast (BEF) cells have previously been used as nucleus-donors for nuclear transfer (NT), it has now been proposed to use BEF cells to generate cloned cows that were genetically modified by transgenic or a knock-out system. A major limitation to gene targeting somatic cells, however, is the overall life-span of the cell. In this study, we first examined in vitro life-span of primary BEF cells. Primary BEF cells were found to be replicative senescent at passage 10th-12th, similar to primary murine embryonic fibroblast cells. To overcome this short in vitro life-span, we have optimized culture conditions to extend the life-span and determined growth characteristics of BEF cell lines. Two life-span extended BEF cell lines (designated CGFR -BO-1 and CGFR-BO-2) were shown to grow much faster than their parental primary counterparts. Both cell lines did not display any potential for abnormal growth such as foci formations in either soft-agar or confluent culture condition. In cloning experiments using these cell lines as a nuclear donor, the reconstructed karyoblasts underwent apoptosis, reprogramming and development in the blastocyst stage, at a similar frequency to those observed with parental as well as adult primary fibroblasts. Furthermore, these cell lines targeted with green fluorescence protein (GFP) were successfully transduced, selected and reprogrammed by NT to develop into a blastocyst stage with GFP expression. Our results suggested methods to extend life-span of donor cells with tremendous implications for the genetic engineering of bovine fibroblast cells.

Animals↗
PubMed2005

[Characterization of a canine model of dystrophic bullous epidermolysis (DBE). Development of a gene therapy protocol].

The main human forms of epidermolysis bullosa (EB), namely EB simplex, junctional EB and dystrophic EB, have also been described in domestic animals (small and large ruminants, and horses) and companion animals (cats and dogs). A recent description of dystrophic epidermolysis bullosa (DEB) in Golden Retriever dogs provided details of the principal clinical, morphological and genetic features. The disease is characterized by blisters and erosions in the oral and esophageal epithelia, together with milia, nails dystrophy and growth retardation. The cutaneous lesions regress spontaneously in adult dogs, whereas the epithelial lesions persist, aggravate and spread, notably to the cornea. Classical microscopic studies (light and electron microscopy, indirect immunofluorescence) have revealed anchoring fibril abnormalities and very low-level and heterogenous expression of collagen type VII. The culprit mutation (G1906S) in the canine gene COL7A1 (87.8% nucleotide sequence identity to the human counterpart) involves the replacement of guanine 5716 by adenine, leading to glycine substitution by serine at amino acid position 1906. Transmission in kennels occurs in recessive mode (RDEB). These features recall certain human forms of DEB, and particularly those with a phenotype intermediate between gravis (the so-called Hallopeau-Siemens form) and mitis. No curative treatment of human EB is currently available, and efforts are therefore being made to develop a gene therapy protocol in animals. The first steps have already been successfully achieved, namely the development of a recombinant virus vector able to insert the wild-type gene into the keratinocyte genome, and grafting of artificial skin containing transfected canine keratinocytes in nude mice. The recombinant vectors are Moloney-type retroviruses (MMLV-PCMV), and the Zeocin resistance gene is used to select transduced cells. The artificial skin reconstructed in vitro is of the full-thickness type. Despite the large size of the transduced (9 kb), 95% of cells are transduced and produce large amounts of wild-type collagen. Two key issues remain, however: the possible immunogenicity of the transgene product and the persistence of transgene expression in individuals with a functional immune system. Golden Retriever dogs will provide a suitable animal model for these studies.

Animals↗