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Small, highly absorbing points are randomly present on the surfaces of the main interferometer optics in Advanced LIGO. The resulting nanometer scale thermo-elastic deformations and substrate lenses from these micron-scale absorbers significantly reduce the sensitivity of the interferometer directly though a reduction in the power-recycling gain and indirect interactions with the feedback control system. We review the expected surface deformation from point absorbers and provide a pedagogical description of the impact on power buildup in second generation gravitational wave detectors (dual-recycled Fabry-Perot Michelson interferometers). This analysis predicts that the power-dependent reduction in interferometer performance will significantly degrade maximum stored power by up to 50% and, hence, limit GW sensitivity, but it suggests system wide corrections that can be implemented in current and future GW detectors. This is particularly pressing given that future GW detectors call for an order of magnitude more stored power than currently used in Advanced LIGO in Observing Run 3. We briefly review strategies to mitigate the effects of point absorbers in current and future GW wave detectors to maximize the success of these enterprises.
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HIV-1 infection requires the presence of the CD4 receptor on the target cell surface and a coreceptor, predominantly CC-chemokine receptor 5 (CCR5). It has been shown that individuals who are homozygous for a defective CCR5 gene are protected from HIV-1 infection. A novel self-inactivating lentiviral vector LVsh5/C46 (Cal-1) has been engineered to block HIV-1 infection with two viral entry inhibitors, conferring resistance to HIV-1 infection from both CCR5 and CXCR4 tropic strains. Cal-1 encodes a short hairpin RNA (sh5) to downregulate CCR5 and C46, an HIV-1 fusion inhibitor. Gene therapy by Cal-1 is aimed at transducing CD4+ T cells and CD34+ hematopoietic stem/progenitor cells in an autologous transplant setting. Pre-clinical safety and efficacy studies in vitro and in vivo (humanized mouse model and nonhuman primates) have shown that Cal-1 is safe with no indication of any toxicity risk and acts to decrease viral load and increase CD4 counts. Two clinical trials are underway using Cal-1: a phase I/II study to assess safety and feasibility in an adult HIV-1-positive population not on antiretroviral therapy (ART); and a second Fred Hutchinson Investigator Initiated phase I study to assess safety and feasibility in adults with HIV-1-associated non-Hodgkin or Hodgkin lymphoma.
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Terapia Genética/métodos , Infecciones por VIH/terapia , VIH-1/fisiología , Fusión de Membrana , Receptores CCR5 , Receptores del VIH/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/biosíntesis , Adulto , Animales , Terapia Biológica/métodos , Antagonistas de los Receptores CCR5 , Recuento de Linfocito CD4 , Ensayos Clínicos como Asunto , Regulación hacia Abajo , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , Células Madre Hematopoyéticas , Humanos , Receptores CCR5/biosíntesis , Receptores CXCR4 , Proteínas Recombinantes de Fusión/genética , Carga ViralRESUMEN
We have focused on gene therapy approaches to induce functional cure/remission of HIV-1 infection. Here, we evaluated the safety and efficacy of the clinical grade anti-HIV lentiviral vector, Cal-1, in pigtailed macaques (Macaca nemestrina). Cal-1 animals exhibit robust levels of gene marking in myeloid and lymphoid lineages without measurable adverse events, suggesting that Cal-1 transduction and autologous transplantation of hematopoietic stem cells are safe, and lead to long-term, multilineage engraftment following myeloablative conditioning. Ex vivo, CD4+ cells from transplanted animals undergo positive selection in the presence of simian/human immunodeficiency virus (SHIV). In vivo, Cal-1 gene-marked cells are evident in the peripheral blood and in HIV-relevant tissue sites such as the gastrointestinal tract. Positive selection for gene-marked cells is observed in blood and tissues following SHIV challenge, leading to maintenance of peripheral blood CD4+ T-cell counts in a normal range. Analysis of Cal-1 lentivirus integration sites confirms polyclonal engraftment of gene-marked cells. Following infection, a polyclonal, SHIV-resistant clonal repertoire is established. These findings offer strong preclinical evidence for safety and efficacy of Cal-1, present a new method for tracking protected cells over the course of virus-mediated selective pressure in vivo, and reveal previously unobserved dynamics of virus-dependent T-cell selection.
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We described earlier a dual-combination anti-HIV type 1 (HIV-1) lentiviral vector (LVsh5/C46) that downregulates CCR5 expression of transduced cells via RNAi and inhibits HIV-1 fusion via cell surface expression of cell membrane-anchored C46 antiviral peptide. This combinatorial approach has two points of inhibition for R5-tropic HIV-1 and is also active against X4-tropic HIV-1. Here, we utilize the humanized bone marrow, liver, thymus (BLT) mouse model to characterize the in vivo efficacy of LVsh5/C46 (Cal-1) vector to engineer cellular resistance to HIV-1 pathogenesis. Human CD34+ hematopoietic stem/progenitor cells (HSPC) either nonmodified or transduced with LVsh5/C46 vector were transplanted to generate control and treatment groups, respectively. Control and experimental groups displayed similar engraftment and multilineage hematopoietic differentiation that included robust CD4+ T-cell development. Splenocytes isolated from the treatment group were resistant to both R5- and X4-tropic HIV-1 during ex vivo challenge experiments. Treatment group animals challenged with R5-tropic HIV-1 displayed significant protection of CD4+ T-cells and reduced viral load within peripheral blood and lymphoid tissues up to 14 weeks postinfection. Gene-marking and transgene expression were confirmed stable at 26 weeks post-transplantation. These data strongly support the use of LVsh5/C46 lentiviral vector in gene and cell therapeutic applications for inhibition of HIV-1 infection.
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BACKGROUND: After injection into muscle and peripheral nerves, a variety of viral vectors undergo retrograde transport to lower motor neurons. However, because of its attractive safety profile and durable gene expression, adeno-associated virus (AAV) remains the only vector to have been applied to the human nervous system for the treatment of neurodegenerative disease. Nonetheless, only a very small fraction of intramuscularly injected AAV vector arrives at the spinal cord. OBJECTIVE: To engineer a novel AAV vector by inserting a neuronal targeting peptide (Tet1), with binding properties similar to those of tetanus toxin, into the AAV1 capsid. METHODS: Integral to this approach was the use of structure-based design to increase the effectiveness of functional capsid engineering. This approach allowed the optimization of scaffolding regions for effective display of the foreign epitope while minimizing disruption of the native capsid structure. We also validated an approach by which low-titer tropism-modified AAV vectors can be rescued by particle mosaicism with unmodified capsid proteins. RESULTS: Importantly, our rationally engineered AAV1-based vectors exhibited markedly enhanced transduction of cultured motor neurons, diminished transduction of nontarget cells, and markedly superior retrograde delivery compared with unmodified AAV1 vector. CONCLUSION: This approach promises a significant advancement in the rational engineering of AAV vectors for diseases of the nervous system and other organs.
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Proteínas de Unión al ADN/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/síntesis química , Proteínas Proto-Oncogénicas/genética , Transducción Genética/métodos , Animales , Proteínas de la Cápside/química , Dependovirus/genética , Terapia Genética/métodos , Células HEK293 , Células HeLa , Humanos , Oxigenasas de Función Mixta , Estructura Cuaternaria de Proteína , Ratas , Ratas Sprague-DawleyRESUMEN
Gene transfer has therapeutic potential for treating HIV-1 infection by generating cells that are resistant to the virus. We have engineered a novel self-inactivating lentiviral vector, LVsh5/C46, using two viral-entry inhibitors to block early steps of HIV-1 cycle. The LVsh5/C46 vector encodes a short hairpin RNA (shRNA) for downregulation of CCR5, in combination with the HIV-1 fusion inhibitor, C46. We demonstrate here the effective delivery of LVsh5/C46 to human T cell lines, peripheral blood mononuclear cells, primary CD4(+) T lymphocytes, and CD34(+) hematopoietic stem/progenitor cells (HSPC). CCR5-targeted shRNA (sh5) and C46 peptide were stably expressed in the target cells and were able to effectively protect gene-modified cells against infection with CCR5- and CXCR4-tropic strains of HIV-1. LVsh5/C46 treatment was nontoxic as assessed by cell growth and viability, was noninflammatory, and had no adverse effect on HSPC differentiation. LVsh5/C46 could be produced at a scale sufficient for clinical development and resulted in active viral particles with very low mutagenic potential and the absence of replication-competent lentivirus. Based on these in vitro results, plus additional in vivo safety and efficacy data, LVsh5/C46 is now being tested in a phase 1/2 clinical trial for the treatment of HIV-1 disease.
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Gene therapy has shown clinical efficacy for several rare diseases, using different approaches and vectors. The Gene Therapy for Rare Diseases workshop, sponsored by the National Institutes of Health (NIH) Office of Biotechnology Activities and Office of Rare Diseases Research, brought together investigators from different disciplines to discuss the challenges and opportunities for advancing the field including means for enhancing data sharing for preclinical and clinical studies, development and utilization of available NIH resources, and interactions with the U.S. Food and Drug Administration.
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Enfermedades Raras/genética , Enfermedades Raras/terapia , Ensayos Clínicos como Asunto , Terapia Genética , Humanos , National Institutes of Health (U.S.) , Investigación Biomédica Traslacional , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Human immunodeficiency virus type 1 (HIV-1) infection of target cells requires CD4 and a co-receptor, predominantly the chemokine receptor CCR5. CCR5-delta32 homozygosity results in a truncated protein providing natural protection against HIV infection-this without detrimental effects to the host-and transplantation of CCR5-delta32 stem cells in a patient with HIV ("Berlin patient") achieved viral eradication. As a more feasible approach gene-modification strategies are being developed to engineer cellular resistance to HIV using autologous cells. We have developed a dual therapeutic anti-HIV lentiviral vector (LVsh5/C46) that down-regulates CCR5 and inhibits HIV-1 fusion via cell surface expression of the gp41-derived peptide, C46. This construct, effective against multiple strains of both R5- and X4-tropic HIV-1, is being tested in Phase I/II trials by engineering HIV-resistant hematopoietic cells.
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Antagonistas de los Receptores CCR5 , Terapia Genética/métodos , Infecciones por VIH/terapia , VIH-1/fisiología , Receptores del VIH/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/biosíntesis , Terapia Biológica/métodos , Ensayos Clínicos como Asunto , Infecciones por VIH/virología , VIH-1/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/fisiología , Células Madre Hematopoyéticas/virología , Humanos , Receptores CCR5/biosíntesis , Receptores del VIH/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Intra-articular gene therapy has potential for the treatment of osteoarthritis and rheumatoid arthritis. To quantify in vitro relative gene transduction, equine chondrocytes and synovial cells were treated with adenovirus vectors (Ad), serotype 2 adeno-associated virus vectors (rAAV2), or self-complementary (sc) AAV2 vectors carrying green fluorescent protein (GFP). Using 6 horses, bilateral metacarpophalangeal joints were injected with Ad, rAAV2, or scAAV2 vectors carrying GFP genes to assess the in vivo joint inflammation and neutralizing antibody (NAb) titer in serum and joint fluid. In vitro, the greater transduction efficiency and sustained gene expression were achieved by scAAV2 compared to rAAV2 in equine chondrocytes and synovial cells. In vivo, AAV2 demonstrated less joint inflammation than Ad, but similar NAb titer. The scAAV2 vectors can induce superior gene transduction than rAAV2 in articular cells, and both rAAV2 and scAAV2 vectors were showed to be safer for intra-articular use than Ad vectors.
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Adeno-associated virus (AAV) has become a versatile vector platform. In recent years, powerful -techniques for the generation of tropism-modified vectors (rAAV-targeting vectors) and for investigation of virus-cell interaction were developed. The following chapter describes strategies for insertion of peptide ligands into the viral capsid and the subsequent characterization of capsid mutants, for producing mosaic capsids and for labeling the viral capsid chemically or genetically.
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Dependovirus/genética , Vectores Genéticos/genética , Cápside/química , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Reacción en Cadena de la Polimerasa , Virión/químicaRESUMEN
This study quantified and compared the transduction efficiencies of adenoviral (Ad), Arg-Gly-Asp (RGD)-modified Ad, adeno-associated viral serotype 2 (AAV2), and self-complementary AAV2 (scAAV2) vectors within full-thickness osteoarthritic (OA) and unaffected canine cartilage explants in vitro. Intraarticular administration of Ad and scAAV2 vectors was performed to determine the ability of these vectors to transduce unaffected guinea pig cartilage in vivo. Following explant exposure to vector treatment or control, the onset and surface distribution of reporter gene expression was monitored daily with fluorescent microscopy. At termination, explants were divided: one half was digested for analysis using flow cytometry; the remaining portion was used for histology and immunohistochemistry (IHC). Intact articular joints were collected for real-time RT-PCR and IHC to detect reporter gene expression following injection of selected vectors. Ad vector transduced focal areas along the perimeters of explants; the remaining vectors transduced chondrocytes across 100% of the surface. Greater mean transduction efficiencies were found with both AAV2 vectors as compared to the Ad vector (p < or = 0.026). Ad and Ad-RGD vectors transduced only superficial chondrocytes of OA and unaffected cartilage. Uniform reporter gene expression from AAV2 and scAAV2 was detected in the tangential and transitional zones of OA cartilage, but not deeper zones. AAV2 and scAAV2 vectors achieved partial and full-thickness transduction of unaffected cartilage. In vivo work revealed that scAAV2 vector, but not Ad vector, transduced deeper zones of cartilage and menisci. This study demonstrates that AAV2 and scAAV2 are reliable vectors for use in cartilage in vitro and in vivo.
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Adenoviridae/genética , Dependovirus/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Osteoartritis/terapia , Transducción Genética , Animales , Cartílago Articular/patología , Modelos Animales de Enfermedad , Perros , Expresión Génica , Genes Reporteros/genética , Vectores Genéticos/uso terapéutico , Cobayas , Osteoartritis/patología , Resultado del TratamientoRESUMEN
AIM: The aim of our study was to develop an effective gene delivery system for ovarian cancer gene therapy. METHODS: The expression of heparin sulfate proteoglycan (HSPG) and integrins alpha(upsilon)beta(3) and alpha(upsilon)beta(5) were analyzed with flow cytometry on 2 human ovarian cancer cell lines (OVCAR-3 and SKOV-3ip). The gene transduction efficiencies were evaluated with recombinant adeno-associated viral vector (rAAV)2-green fluorescent protein or rAAV2-lactase Z followed by flow cytometry or cytohistochemistry staining. The effect of 17beta-estradiol on ovarian cancer cell proliferation, HSPG, the expressions of integrins alpha(upsilon)beta(3) and alpha(upsilon)beta(5), and adeno-associated viral vector (AAV)2-mediated gene transduction were determined. RESULTS: In the present study, we found: (1) a variation in HSPG and the expressions of integrins alpha(upsilon)beta(3) and alpha(upsilon)beta(5) between OVCAR-3 and SKOV-3ip; (2) that 17beta-estradiol was shown to significantly stimulate cell proliferation and integrin beta(5) expression in certain ovarian cancer cell lines; and (3) integrintargeted A520/N584RGD-rAAV2, which has alternative interactivity with integrins and abrogates the binding capacity HSPG, showed much higher gene transduction efficiency in ovarian cancer cells than rAAV2 in the presence/absence of 17beta-estradiol. Moreover, this RGD-modified rAAV2 exerted more efficient transduction in ovarian cancer cells in response to 17beta-estradiol. CONCLUSION: Our findings implied that A520/N584RGD-rAAV2 may offer great potential for ovarian cancer treatment in vivo.
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Dependovirus , Estradiol/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Animales , Antivirales/farmacología , Muerte Celular/fisiología , Línea Celular Tumoral/efectos de los fármacos , Dependovirus/genética , Dependovirus/metabolismo , Femenino , Ganciclovir/farmacología , Terapia Genética , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Heparina/análogos & derivados , Heparina/metabolismo , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Neoplasias Ováricas/terapia , Proteoglicanos/metabolismo , Receptores de Vitronectina/genética , Receptores de Vitronectina/metabolismo , Transducción GenéticaRESUMEN
We have developed a highly specific and robust new method for labeling adeno-associated virus (AAV) vector particles with either biophysical probes or targeting ligands. Our approach uses the Escherichia coli enzyme biotin ligase (BirA), which ligates biotin to a 15-amino-acid biotin acceptor peptide (BAP) in a sequence-specific manner. In this study we demonstrate that by using a ketone isotere of biotin as a cofactor we can ligate this probe to BAP-modified AAV capsids. Because ketones are absent from AAV, BAP-modified AAV particles can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage modification methodology in the context of a mammalian cell lysate for the labeling of AAV vector particles with various fluorophores, and for the attachment of a synthetic cyclic arginine-glycine-aspartate (RGD) peptide (c(RGDfC)) to target integrin receptors that are present on neovasculature. Fluorophore labeling allowed the straightforward determination of intracellular particle distribution. Ligand conjugation mediated a significant increase in the transduction of endothelial cells in vitro, and permitted the intravascular targeting of AAV vectors to tumor-associated vasculature in vivo. These results suggest that this approach holds significant promise for future studies aimed at understanding and modifying AAV vector-cellular interactions.
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Ligasas de Carbono-Nitrógeno/química , Dependovirus/química , Proteínas de Escherichia coli/química , Proteínas Represoras/química , Animales , Biotina/química , Biotina/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Línea Celular , Dependovirus/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Femenino , Colorantes Fluorescentes/química , Técnicas de Transferencia de Gen , Vectores Genéticos/química , Vectores Genéticos/genética , Células HeLa , Humanos , Ligandos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Desnudos , Técnicas de Sonda Molecular , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Péptidos Cíclicos/química , Péptidos Cíclicos/genética , Péptidos Cíclicos/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
This study evaluated healing of equine metatarsal osteotomies and ostectomies in response to percutaneous injection of adenoviral (Ad) bone morphogenetic protein (BMP)-2, Ad-BMP-6, or beta-galactosidase protein vector control (Ad-LacZ) administered 14 days after surgery. Radiographic and quantitative computed tomographic assessment of bone formation indicated greater and earlier mineralized callus in both the osteotomies and ostectomies of the metatarsi injected with Ad-BMP-2 or Ad-BMP-6. Peak torque to failure and torsional stiffness were greater in osteotomies treated with Ad-BMP-2 than Ad-BMP-6, and both Ad-BMP-2- and Ad-BMP-6-treated osteotomies were greater than Ad-LacZ or untreated osteotomies. Gene expression of ostectomy mineralized callus 8 weeks after surgery indicated upregulation of genes related to osteogenesis compared to intact metatarsal bone. Expression of transforming growth factor beta-1, cathepsin H, and gelsolin-like capping protein were greater in Ad-BMP-2- and Ad-BMP-6-treated callus compared to Ad-LacZ-treated or untreated callus. Evidence of tissue biodistribution of adenovirus in distant organs was not identified by quantitative PCR, despite increased serum antiadenoviral vector antibody. This study demonstrated a greater relative potency of Ad-BMP-2 over Ad-BMP-6 in accelerating osteotomy healing when administered in this regimen, although both genes were effective at increasing bone at both osteotomy and ostectomy sites.
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Proteínas Morfogenéticas Óseas/genética , Curación de Fractura/genética , Fracturas Óseas/terapia , Terapia Genética/métodos , Osteogénesis/genética , Osteotomía , Factor de Crecimiento Transformador beta/genética , Adenoviridae/genética , Animales , Fenómenos Biomecánicos , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 6 , Callo Óseo/diagnóstico por imagen , Callo Óseo/fisiopatología , Modelos Animales de Enfermedad , Fracturas Óseas/diagnóstico por imagen , Fracturas Óseas/fisiopatología , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Caballos , Humanos , Operón Lac , Huesos Metatarsianos/diagnóstico por imagen , Huesos Metatarsianos/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Tomografía Computarizada por Rayos X , Torque , Torsión MecánicaRESUMEN
Adeno-associated virus (AAV) vectors are associated with relatively mild host immune responses in vivo. Although AAV induces very weak innate immune responses, neutralizing antibodies against the vector capsid and transgene still occur. To understand further the basis of the antiviral immune response to AAV vectors, studies were performed to characterize AAV interactions with macrophages. Primary mouse macrophages and human THP-1 cells transduced in vitro using an AAV serotype 2 (AAV2) vector encoding green fluorescent protein did not result in measurable transgene expression. An assessment of internalized vector genomes showed that AAV2 vector uptake was enhanced in the presence of normal but not heat-inactivated or C3-depleted mouse/human serum. Enhanced uptake in the presence of serum coincided with increased macrophage activation as determined by the expression of NF-kappaB-dependent genes such as macrophage inflammatory protein 2 (MIP-2), interleukin-1beta (IL-1beta), IL-8, and MIP-1beta. AAV vector serotypes 1 and 8 also activated human and mouse macrophages in a serum-dependent manner. Immunoprecipitation studies demonstrated the binding of iC3b complement protein to the AAV2 capsid in human serum. AAV2 did not activate the alternative pathway of the complement cascade and lacked cofactor activity for factor I-mediated degradation of C3b to iC3b. Instead, our results suggest that the AAV capsid also binds complement regulatory protein factor H. In vivo, complement receptor 1/2- and C3-deficient mice displayed impaired humoral immunity against AAV2 vectors, with a delay in antibody development and significantly lower neutralizing antibody titers. These results show that the complement system is an essential component of the host immune response to AAV.
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Proteínas del Sistema Complemento/fisiología , Dependovirus/inmunología , Vectores Genéticos/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Línea Celular , Dependovirus/genética , Expresión Génica , Humanos , Inmunoprecipitación , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/virología , Ratones , Ratones Endogámicos C57BLRESUMEN
In articular fractures, both bone and cartilage are injured. We tested whether stem cells transduced with bone morphogenetic protein 2 (BMP2) can promote bone and cartilage repair. Distal femoral articular osteotomies in nude rats were treated with stem cells, either wild-type or transduced with an adenoviral (Ad) BMP2. Cells were delivered in alginate (ALG) carrier or by direct injection in saline solution. Gene expression of these cells at the osteotomy site was confirmed by in vivo imaging. At day 14, only the group that received AdBMP2 stem cells by direct injection showed completely healed osteotomies, while other groups remained unhealed (P < 0.0003). In ALG groups, bone healing was impeded by the development of a chondroid mass, most pronounced in the AdBMP2 ALG group (P < 0.002). We were successful in achieving repair of both bone and cartilage in vivousing direct stem cell injection. Our data suggests that BMP2 augmentation might be critically important in achieving this effect.
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Proteínas Morfogenéticas Óseas/metabolismo , Fracturas Óseas/metabolismo , Fracturas Óseas/terapia , Terapia Genética , Células Madre Mesenquimatosas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adenoviridae/genética , Alginatos/metabolismo , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/genética , Modelos Animales de Enfermedad , Femenino , Fracturas Óseas/genética , Técnicas de Transferencia de Gen , Genes Reporteros/genética , Ácido Glucurónico/genética , Ácido Glucurónico/metabolismo , Ácidos Hexurónicos/metabolismo , Humanos , Masculino , Osteotomía , Ratas , Tomógrafos Computarizados por Rayos X , Factor de Crecimiento Transformador beta/genéticaRESUMEN
OBJECTIVE: To evaluate host cell permissiveness and cytotoxic effects of recombinant and modified adenoviral vectors in equine chondrocytes, synovial cells, and bone marrow-derived mesenchymal stem cells (BMD-MSCs). SAMPLE POPULATION: Articular cartilage, synovium, and bone marrow from 15 adult horses. PROCEDURES: Equine chondrocytes, synovial cells, and BMD-MSCs and human carcinoma (HeLa) cells were cultured and infected with an E-1-deficient adenovirus vector encoding the beta-galactosidase gene or the green fluorescent protein gene (Ad-GFP) and with a modified E-1-deficient vector with the arg-gly-asp capsid peptide insertion and containing the GFP gene (Ad-RGD-GFP). Percentages of transduced cells, total and transduced cell counts, and cell viability were assessed 2 and 7 days after infection. RESULTS: -Permissiveness to adenoviral vector infection was significantly different among cell types and was ranked in decreasing order as follows: HeLa cells > BMD-MSCs > chondrocytes > synovial cells. Morphologic signs of cytotoxicity were evident in HeLa cells but not in equine cells. Numbers of transduced cells decreased by day 7 in all cell types except equine BMD-MSCs. Transduction efficiency was not significantly different between the Ad-GFP and Ad-RGD-GFP vectors. CONCLUSION AND CLINICAL RELEVANCE: Sufficient gene transfer may be achieved by use of an adenovirus vector in equine cells. High vector doses can be used in equine cells because of relative resistance to cytotoxic effects in those cells. Greater permissiveness and sustained expression of transgenes in BMD-MSCs make them a preferential cell target for gene therapy in horses.
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Adenoviridae/fisiología , Adenoviridae/patogenicidad , Condrocitos/citología , Terapia Genética/veterinaria , Caballos , Células Madre/citología , Membrana Sinovial/citología , Adenoviridae/genética , Animales , Células de la Médula Ósea , Condrocitos/virología , Expresión Génica , Terapia Genética/efectos adversos , Vectores Genéticos/efectos adversos , Células HeLa , Caballos/virología , Humanos , Células Madre/virología , Membrana Sinovial/virologíaRESUMEN
Gene-eluting stents are being evaluated in animals as an alternative approach to inhibiting in-stent restenosis. Adeno-associated virus type 2 (AAV2) and adenovirus are commonly used for gene transfer applications. We tested the hypothesis that these vectors can achieve prolonged and localized gene delivery to the vessel wall, using stents as delivery platforms. AdbetaGal (5 x 10(9) plaque-forming units) and AAV2betaGal (5.3 x 10(9) DNase-resistant particles) were used to coat BiodivYsio stents with matrix HI coating (Abbott Vascular Devices, Galway, Ireland). After balloon injury, external iliac arteries of New Zealand White rabbits were stented. The reverse transcription-polymerase chain reaction was used to assess viral spread. Expression of LacZ was demonstrated with both vectors at five time points (3, 7, 14, 21, and 28 days). In the adenovirus group the median percentage of cells expressing the transgene on day 3 was 2.73%, which increased to a median expression of 7.31% at 28 days (p > 0.05). Expression was localized to medial cells on day 3, but was observed predominantly in neointimal cells on day 28. In the AAV group, day 3 expression was 5.78%, which decreased to 2.12% on day 28 (p = 0.05). No systemic dissemination of virus was seen in any group. Adenovirus- and AAV2-coated stents can be used to deliver genes to the blood vessel wall for up to 28 days.
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Adenoviridae/genética , Vasos Sanguíneos/metabolismo , Dependovirus/genética , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Stents , Animales , Vasos Sanguíneos/citología , Vasos Sanguíneos/enzimología , Expresión Génica , Vectores Genéticos/genética , Conejos , Transgenes , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
OBJECTIVES: Capsid-modified AAV vectors can mediate enhanced gene transfer to neoplasms characterized by low AAV receptor expression. Here we sought to determine the therapeutic potential of a capsid-modified AAV vector for gene therapy of ovarian carcinoma (OvCa). METHODS: We tested a panel of OvCa cell lines for AAV2-mediated gene transduction and for sensitivity to ganciclovir (GCV) following AAVHSVtk administration. Levels of AAV internalization and attachment receptor were assessed by flow cytometry and immunohistochemistry. The role of receptors in AAV-mediated gene transfer was assessed by competition assays. Finally, we examined the ability of a modified vector with an integrin-binding RGD motif inserted into the AAV capsid to improve gene delivery to OvCa and enhance AAVHSVtk/GCV-mediated killing by cytotoxicity assay. RESULTS: All OvCa cell lines were poorly transduced with AAV2 vectors and showed variably sensitive to AAVHSVtk/GCV. While OvCa cell lines expressed AAV2 internalization receptors (alphav integrins), expression of the AAV2 attachment receptor, HSPG, was variable and not detected on many lines. Analysis of archived clinical specimens showed no detectable HSPG expression on approximately 45% of primary human tumors. Gene transfer to OvCa was increased several fold using the RGD-modified vector. Gene transfer was independent of HSPG and specific to the targeted receptor. Importantly, the RGD-modified capsid markedly increased the ability of the AAVHSVtk to kill OvCa cells in the presence of GCV. CONCLUSIONS: The development of AAV vectors targeted to cell surface receptors other than HSPG will be critical to the advancement of AAV-mediated gene therapy for treating OvCa.
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Vectores Genéticos , Proteoglicanos de Heparán Sulfato/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Adenoviridae/metabolismo , Cápside/química , Línea Celular Tumoral , Femenino , Citometría de Flujo , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Humanos , Inmunohistoquímica , Neoplasias Ováricas/patología , Plásmidos/metabolismoRESUMEN
The development of rationally designed targeted gene delivery vectors is an important focus for gene therapy. While genetic modification of AAV can produce vectors with modified tropism, incorporation of targeting peptides into the structural context of the AAV virion often results in loss of function or loss of virion integrity. To address this issue, we have developed a targeting system using metabolically biotinylated AAV. We generated serotype 1, 2, 3, 4, and 5 AAV capsids with small peptide insertions that are metabolically biotinylated in packaging cells during vector production by coexpression of the Escherichia coli BirA, biotin ligase, gene. Biotin moieties are exposed on the surface of assembled AAV particles and can interact with avidin. Metabolically biotinylated AAV vectors produced in this manner maintained endogenous titer and tissue tropism, could be purified on monomeric avidin resin, and could be retargeted to cells engineered to express an artificial avidin-biotin receptor. This technology provides not only a single platform for the purification of multiple AAV vector serotypes, but also a means for the development of multiple targeted AAV vectors utilizing a single capsid modification via straightforward avidin-biotin ligand coupling.