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PubMed2005

Transduction of satellite cells after prenatal intramuscular administration of lentiviral vectors.

BACKGROUND: We have previously reported long-term expression of lacZ in myocytes after in utero intramuscular injection of Mokola and Ebola pseudotyped lentiviral vectors. In further experiments, we have noted that these vectors also transduce small cells at the periphery of the muscle fibers that have the morphology of satellite cells, or muscle stem cells. In this study we performed experiments to further define the morphology and function of these cells. METHODS: Balb/c mice at 14-15 days gestation were injected intramuscularly with Ebola or Mokola pseudotyped lentiviral vectors carrying CMV-lacZ. Animals were harvested at various time points, muscles were stained with X-gal, and processed for electron microscopy (EM) and immunofluorescence. To determine whether transduced satellite cells were functionally capable of regenerating injured muscles, animals were injected with notexin in the same area 8 weeks after the in utero injection of viral vector. RESULTS: Transmission EM of transduced cells confirmed the ultrastructural appearance of satellite cells. Double immunofluorescence for beta-galactosidase and satellite cell markers demonstrated co-localization of these markers in transduced cells. In the notexin-injured animals, small blue cells were seen at the areas of regeneration that co-localized beta-galactosidase with markers of regenerating satellite cells. Central nucleated blue fibers were seen at late time points, indicating regenerated muscle fibers arising from a transduced satellite cell. CONCLUSIONS: This study demonstrates transduction of muscle satellite cells following prenatal viral vector mediated gene transfer. These findings may have important implications for gene therapy strategies directed toward muscular dystrophy.

Animals↗
PubMed2005

Shuttle of lentiviral vectors via transplanted cells in vivo.

Lentiviral vectors have turned out to be an efficient method for stable gene transfer in vitro and in vivo. Not only do fields of application include cell marking and tracing following transplantation in vivo, but also the stable delivery of biological active proteins for gene therapy. A variety of cells, however, need immediate transplantation after preparation, for example, to prevent cell death, differentiation or de-differentiation. Although these cells are usually washed several times following lentiviral transduction, there may be the risk of viral vector shuttle via transplanted cells resulting in undesired in vivo transduction of recipient cells. We investigated whether infectious lentiviral particles are transmitted via ex vivo lentivirally transduced cells. To this end, we explored potential viral shuttle via ex vivo lentivirally transduced cardiomyocytes in vitro and following transplantation into the brain and peripheral muscle. We demonstrate that, even after extensive washing, infectious viral vector particles can be detected in cell suspensions. Those lentiviral vector particles were able to transduce target cells in transwell experiments. Moreover, transmitted vector particles stably transduced resident cells of the recipient central nervous system and muscle in vivo. Our results of lentiviral vector shuttle via transduced cardiomyocytes are significant for both ex vivo gene therapy and for lentiviral cell tracing, in particular for investigation of stem cell differentiation in transplantation models and co-cultivation systems.

Animals↗
PubMed2006

Identification of a major histocompatibility complex class I-restricted T-cell epitope in the tumour-associated antigen, 5T4.

5T4 is a surface glycoprotein expressed on placental trophoblasts and also on a wide range of human carcinomas. Its highly restricted expression on normal tissues and broad distribution on many carcinomas make 5T4 a promising target for cancer immunotherapy. In the current study, we set out to investigate whether a 5T4-specific cytotoxic T lymphocyte (CTL) repertoire exists in healthy individuals. CD4-depleted peripheral blood mononuclear cells (PBMCs) from blood donors were screened using an ex vivo interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay. A panel of overlapping peptides, spanning the full length of the 5T4 protein, was used as a source of antigen. In the process of screening, one out of 30 blood donors demonstrated a positive ex vivo IFN-gamma ELISPOT response to a single 5T4 peptide. A polyclonal T-cell line was derived from this donor by culturing PBMCs with autologous peptide-pulsed dendritic cells (DCs). The resulting polyclonal T-cell line and clones were tested in a 51Cr-release assay and by ELISPOT and were shown to be peptide specific. Furthermore, antigen-presenting cells (APCs), infected with a viral vector expressing 5T4, were able to stimulate IFN-gamma production by the peptide-specific T-cell clones. A minimal CD8 epitope, PLADLSPFA, has been identified and found to be restricted through human leucocyte antigen (HLA) Cw7. Subsequently, we have demonstrated that HLA-Cw7-positive colorectal cancer patients vaccinated with a recombinant vaccinia viral vector encoding 5T4 (TroVax) are capable of mounting a strong IFN-gamma ELISPOT response to this novel CTL epitope. These findings have potential application in cancer immunotherapy in terms of subunit vaccine design and the monitoring of immune responses induced in patients by 5T4-based therapies.

Cancer Vaccines↗
PubMed1995

Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA.

The carotenoid biosynthetic pathway in higher plants was manipulated by using an RNA viral vector. A cDNA encoding phytoene synthase and a partial cDNA encoding phytoene desaturase (PDS) were placed under the transcriptional control of a tobamovirus subgenomic promoter. One to two weeks after inoculation, systemically infected Nicotiana benthamiana plants were analyzed for phytoene. Leaves from transfected plants expressing phytoene synthase developed a bright orange phenotype and accumulated high levels of phytoene. Cytoplasmic inhibition of plant gene expression by viral RNA was demonstrated with an antisense RNA transcript to a partial PDS cDNA derived from tomato. The leaves of the plants transfected with the antisense PDS sequence developed a white phenotype and also accumulated high levels of phytoene. A partial cDNA to the corresponding N. benthamiana PDS gene was isolated and found to share significant homology with the tomato antisense PDS transcript. This work demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a major, eukaryotic biosynthetic pathway. In addition, our results indicate that an antisense transcript generated in the cytoplasm of a plant cell can turn off endogenous gene expression.

Amino Acid Sequence↗
PubMed1996

The basal lamina is a physical barrier to herpes simplex virus-mediated gene delivery to mature muscle fibers.

A major impediment to successful implementation of gene therapy for treatment of muscular dystrophy is the restricted infectivity of mature muscle fibers with viral vectors. This phenomenon has been observed with adenovirus vectors and more recently with herpes simplex virus type 1 (HSV-1)-based vectors. Here we report findings of morphological studies designed to experimentally determine the mechanism underlying the rapid reduction in vector-mediated gene delivery concomitant with the maturation of muscle fibers. Using immunohistochemistry and confocal microscopy, we have colocalized HSV-1 and collagen IV, a major component of the basal lamina, in HSV-1-injected muscles and determined that the virus penetrates and expresses a transgene (lacZ) in muscle fibers of newborn animals but cannot efficiently penetrate adult myofibers. This was observed in normal as well as in immunocompromised animals, suggesting that the lack of adult myofiber transduction is not a result of an immune response and clearance of the viral vector. Since heparan sulfate proteoglycan, the initial attachment receptor for HSV-1, was shown to be preserved during the maturation of the myofibers by immunofluorescence assay and HSV-1 was able to infect isolated, viable myofibers in vitro, we suggest that the low-level HSV-1 transduction of mature myofibers is not a consequence of the loss of viral attachment sites on the surfaces of mature muscle fibers. Rather, our results indicate that the mature basal lamina acts as a physical barrier to HSV-1 infection of adult myofibers. This conclusion was further supported by the finding that HSV-1 displayed an intermediate level of transduction in mature dy/dy muscle which is defective for normal basal lamina formation. Together, these experiments suggest that efficient HSV vector transduction in mature skeletal muscle requires methods to permeabilize the basal lamina.

Animals↗
PubMed2026

Lipid-nanoparticle-mediated base editing of the trabecular meshwork rescues glaucoma in vivo.

Mutations in MYOC, the most common genetic cause of glaucoma, cause misfolded myocilin to accumulate in the endoplasmic reticulum (ER), leading to trabecular meshwork (TM) dysfunction, elevated intraocular pressure, and progressive vision loss. While gene editing offers curative potential, current delivery methods rely on viral vectors, which are limited by inflammation, off-target effects, and poor translatability. Here, we report a nonviral lipid nanoparticle (LNP) platform that enables selective in vivo delivery of mRNA encoding an adenine base editor and single guide RNA (LNP-ABE) to TM cells. A direct comparison of LNP-mCherry with lentiviral GFP revealed that LNPs outperform viral vectors, achieving markedly higher efficiency and greater selectivity for the TM without inducing ocular inflammation. In a Cre-inducible Tg.CreMYOCY437H glaucoma mouse model, LNP-Cre mRNA selectively induced mutant MYOC expression in the TM, faithfully recapitulating key disease features. A single administration of LNP-ABE achieved efficient on-target editing of mutant MYOC, reducing mutant myocilin protein by approximately 46%, decreasing aggregates, alleviating ER stress, and fully rescuing the glaucomatous phenotype in Tg.CreMYOCY437H mice. Importantly, no off-target editing or ocular toxicity was detected. These findings establish LNP-based mRNA delivery as a safe, efficient, and clinically translatable approach for TM-targeted genome editing with broad therapeutic potential in glaucoma.

Animals↗
PubMed2004

Vaccines and vaccine strategies against HIV.

The HIV/AIDS pandemic is a global emergency and a preventive HIV vaccine is urgently needed. HIV has, however, proved a difficult pathogen to vaccinate against. This is largely because HIV has a very high mutation rate and can escape immune responses, it has a latent stage where it can rest silently integrated into host DNA, and neutralising antibodies that can neutralise diverse field strains have so far proved difficult to induce. There is however, considerable evidence now that HIV-specific CD4 and CD8 T cells can provide partial control of HIV replication and delay or prevent disease. Technologies to quantify and analyse HIV-specific T cells have advanced recently, and in particular ELISpot, intracellular cytokine staining and tetramer studies have provided clear analyses of the ability of HIV vaccines to induce T cell responses. The use of pools of overlapping HIV peptides as in vitro antigens has also provided a standardised reagent for accurate measurement of T cell responses. HIV protein vaccines have not induced broad neutralising antibodies or T cell responses and failed to protect humans in the only phase III efficacy trial yet completed. Viral vectors, such as canarypox, engineered to express HIV genes, have induced HIV-specific CD8 T cell responses in a minority of subjects in phase II trials and are proceeding to human efficacy trials. Currently, the most effective method of inducing CD8+ CTL immunity in non-human primates utilises priming with naked plasmid DNA and then boosting with recombinant viral vectors both encoding various parts of the HIV genome. Such vaccines have induced non-sterilising immunity to virulent Simian/Human immunodeficiency virus exposure in macaques and have entered phase I trials. Multiple other approaches are also being evaluated in what has become a global effort for a vaccine to prevent AIDS. Although an HIV vaccine is still a long way off, there is reason to be optimistic that a vaccine to prevent AIDS will eventually be developed.

AIDS Vaccines↗
PubMed2004

A phase I vaccination study with tyrosinase in patients with stage II melanoma using recombinant modified vaccinia virus Ankara (MVA-hTyr).

A significant percentage of patients with stage II melanomas suffer a relapse after surgery and therefore need the development of adjuvant therapies. In the study reported here, safety and immunological response were analyzed after vaccination in an adjuvant setting with recombinant modified vaccinia virus Ankara carrying the cDNA for human tyrosinase (MVA-hTyr). A total of 20 patients were included and vaccinated three times at 4-week intervals with 5x10(8) IU of MVA-hTyr each time. The responses to the viral vector, to known HLA class I-restricted tyrosinase peptides, and to dendritic cells transfected with tyrosinase mRNA, were investigated by ELISpot assay on both ex vivo T cells and on T cells stimulated in vitro prior to testing. The delivery of MVA-hTyr was safe and did not cause any side effects above grade 2. A strong response to the viral vector was achieved, indicated by an increase in the frequency of MVA-specific CD4+ and CD8+ T cells and an increase in virus-specific antibody titers. However, no tyrosinase-specific T-cell or antibody response was observed with MVA-hTyr in any of the vaccinated patients. Although MVA-hTyr provides a safe and effective antigen-delivery system, it does not elicit a measurable immune response to its transgene product in patients with stage II melanoma after repeated combined intradermal and subcutaneous vaccination. We presume that modification of the antigen and/or prime-boost vaccination applying different approaches to antigen delivery may be required to induce an effective tyrosinase-specific immune response.

Adult↗
PubMed2006

Role of integrin cross-regulation in parvovirus B19 targeting.

Most viral vectors used for gene therapy lack the ability to target a defined cell population. Parvovirus B19 has a restricted tropism for human erythroid progenitor cells and uses activated alpha5beta1 integrins as coreceptors for entry [Weigel-Kelley, K.A., Yoder, M.C., and Srivastava, A. (2003). Blood 102, 3927-3933]. In this study we examined the role of coexpressed integrins in alpha5beta1 integrin coreceptor function. Antibody-mediated cross-linking of beta1, beta2, and beta3 integrins and the integrin-associated protein (IAP) increased parvovirus B19 entry into nontarget K562 cells. Functional silencing of one integrin group, however, reduced the virus uptake- promoting function of a subsequently activated integrin group, indicating that the three integrins did not operate in isolation but through shared signaling pathways. This was further corroborated by direct competition between simultaneously clustered beta2 and beta1 integrins that could be overcome by stabilizing clustered beta1 integrins in a high-affinity conformation. In contrast, parvovirus B19 entry into primary erythroid progenitor cells was characterized by strong clustering-induced beta1 integrin coreceptor activity that was not abolished by subsequent beta2 and beta3 integrin activation and was, in fact, substantially increased in the presence of preclustered beta2 and beta3 integrins. Thus, integrin function is regulated in a cell type-specific manner through coexpressed integrins and preferential parvovirus B19 entry into erythroid progenitor cells is promoted by a robust beta1 integrin response that is enhanced through stable preclustering of coexpressed integrins. These results have implications for other viral vectors that use integrins as receptors/coreceptors and for gene therapy of hematopoietic progenitor cells using parvovirus B19 vectors.

Antibodies, Viral↗
PubMed1998

Immune responses limit adenovirally mediated gene expression in the adult mouse eye.

In order to investigate the immunological consequences of gene transfer to the eye using viral vectors, adenovirus carrying a lacZ reporter gene (AV.LacZ) was injected either subretinally, subconjunctivally or into the anterior chamber of three groups of adult mice: immunocompetent or transiently immunosuppressed BALB/c mice and congenic immunodeficient nude mice. Adenovirally mediated lacZ expression persisted for approximately 3 weeks following injection of the vector into the anterior chamber, retina or extra ocular tissues of the conjuctiva of BALB/c mice. It appears that T cell-mediated immune responses limit the duration of AV-mediated ocular gene expression in adult mice since lacZ gene expression was detected for at least 15 weeks in T cell-deficient BALB/c nude mice, although the level of transgene expression decreased with time. Since intra-ocular AV-mediated gene expression was not significantly longer than extra-ocular expression, it appears that the eye is not normally immune-privileged with respect to viral vectors. Inflammatory cells were detected in the vitreous after anterior chamber injection and in the retina after subretinal injection of adenovirus. The presence of both CD4+ and CD8+ T cells was established by immunophenotyping. Reinjection of BALB/c mice resulted in rapid decline in reporter gene expression, but successful readministration was possible in the case of immunodeficient nude mice. However, after transient depletion of T cells, achieved by intraperitoneal injection of both CD8- and CD4-specific antibodies, the duration of expression in BALB/c mice was longer in the eye (at least 12 weeks, again with decrease in level over time), than in extra-ocular tissues (8 weeks) provided the animal was not reinjected with virus raising the possibility of partial ocular immune-privilege after transient immunosuppression.

Adenoviridae↗
PubMed2000

Transient modulation of gene expression in the neurulation staged mouse embryo.

Transient modulation of gene expression in the embryo during early organogenesis will allow studies to be conducted that determine tissue- and stage-specific function(s) of genes. To achieve this goal, viral vectors and antisense oligodeoxynucleotides have been used to produce gain-of-function and loss-of-function models. Adenoviral transduction of whole embryos, embryonic heart and vasculature, and primary neural crest cell culture has been reported. The morphological consequences of overexpression or decreasing expression of selected genes have been evaluated using these tools. Gene-teratogen interaction studies have also been performed. The viral vectors appear to be important tools for modulating gene expression and hold great promise for future research.

Adenoviridae↗
PubMedDate not supplied

Towards gene therapy in the nervous system.

The integration of gene transfer techniques with neuroscience provides new opportunities to study the cellular and molecular aspects of neuron survival, dysfunction, and degeneration. A variety of viral vectors have been used to probe the effects of new gene expression in neurons, glial cells, and progenitor cells. These vectors provide an avenue to deliver genes with potential therapeutic value as shown by neuroprotective effects in animal models subsequent to the transduction of selected cell types. In addition, basic research in this field provides insight into the signalling mechanisms that dictate cell survival and programmed cell death and helps shape future strategies for gene therapy in the central nervous system. New generations of viral vectors will offer improved gene expression and move the field of gene delivery/therapy closer to clinical reality for the prevalent neurodegenerative conditions that include Alzheimer's and Parkinson's.

Gene Expression Regulation↗
PubMed2004

Functional expression of ATM gene carried by HSV amplicon vector in vitro and in vivo.

Ataxia-telangiectasia (AT) is a human autosomal recessive disease with a pleiotropic phenotype characterized by cerebellar degeneration, immunodeficiency, premature aging, cancer predisposition, and radiation sensitivity. The gene mutated in AT, ATM (for AT-mutated), had been cloned and found to have ionizing radiation and oxidative stress-inducible kinase activity. No treatment can stop the progression of the disease. In this study, the complete open-reading frame of ATM cDNA was cloned into a Herpes simplex virus type-1 (HSV-1) amplicon vector (pTO-ATM), and the transduction of cultured AT cells was demonstrated by immunohistochemistry and Western blot analysis. Functional gene expression was evaluated by cell colony-forming assays following exposure to oxidative stress. The survival of AT cells with ATM gene transduction was about 100% higher compared to nontransduced cells after t-butyl hydroperoxide treatments. Next, the normal ATM gene expression in different regions of the rat brain was studied. Immunohistochemistry staining demonstrated weak endogenous ATM protein expression in neurons of the caudate-putamen, with significantly higher levels of expression detected in neurons in other brain regions. Exogenous ATM gene expression from pTO-ATM after viral transduction in the caudate-putamen of the adult rat was examined. At 3 days after injection of the pTO-ATM viral vector, abundant positive ATM staining of the neurons was found at the injection sites, in comparison to the controls. These data demonstrate that the relatively large ATM cDNA can be transduced and expressed in vitro and in vivo from an HSV amplicon viral vector. These data provide initial evidence that the replacement of the ATM gene into the cells of AT patients might be possible some day.

Animals↗
PubMed1999

Preclinical safety evaluation of human gene therapy products.

Human gene therapy products include naked DNA and viral as well as non-viral vectors containing nucleic acids. There is limited experience on the preclinical toxicity studies necessary for the safety evaluation of these products, which have been outlined in several recently released guidelines. Requirements for the preclinical safety evaluation of human gene therapy products are both specific and non-specific. All key preclinical studies should be performed in compliance with Good Laboratory Practices. Non-specific requirements are in fact common to all pharmaceutical products. Critical specific issues to be addressed are: the safety evaluation of the vector and the toxicity of the expressed protein(s), which are the two components of gene therapy products, the quality of the test article, the selection of animal species, and the verification that the administration method successfully transports the gene of interest, with the vector, to the target site(s). The treatment schedule should mimic the intended human therapeutic design. The host's immune response against the gene therapy product has to be evaluated to detect possible adverse effects and immune neutralization by antibodies. The biodistribution of the gene of interest is also essential and can be evaluated by molecular biology techniques, such as PCR. Specific confinement is required for the safe manipulation of viral vectors.

Animals↗
PubMed2006

Fibrin microbeads (FMB) as a 3D platform for kidney gene and cell therapy.

Cell and gene therapy may alter the outcome of renal diseases, such as hereditary nephropathies, acute and chronic glomerulonephritis and allograft nephropathy. However, owing to blockade of many viral and cellular vehicles by the complex glomerular architecture, the exact nature of gene and cell delivery into specific renal compartments remains currently unknown. To study the interaction of viral vectors with a variety of renal cells and mesenchymal stem cells (MSCs), we employed a novel biological three-dimensional (3D) matrix comprised of fibrin microbeads (FMB) in comparison to monolayer cell culture. Our studies showed that renal cells of both established and primary lines can grow efficiently on FMB and differentiate into epithelial structures, as shown by electron microscopy. Gene delivery into renal cells in 3D was observed for several viral vectors and growth in 3D on FMB conferred resistance to renal cancer cells in the context of oncolytic adenoviruses. Finally, MSCs from various rodent species attached to FMB, grew robustly, survived for several weeks and could efficiently be transduced on FMB. Thus, on the basis of growth, differentiation and transduction of renal cells in 3D, FMB emerge as a novel 3D cellular microenvironment that differs substantially from monolayer cell cultures.

Cell Differentiation↗
PubMed2000

Generation of a ribozyme-adenoviral vector against K-ras mutant human lung cancer cells.

ras mutations represent one of the most common oncogenetic lesions in human non-small cell lung cancer (NSCLC) and adversely affect the survival of patients afflicted with this disease. ras-directed gene therapy in the past employed primarily antisense oligonucleotides (AS-ODN) or expression vectors (such as a viral vector construct) that deliver the antisense sequence to inactivate the mutant oncogene message. These approaches produced minimal toxicity, and yet were limited in efficacy. Ribozymes present a viable alternative in antisense therapy by virtue of their renewable catalytic capability for site-specific RNA cleavage. We recently produced an adenoviral vector with a hammerhead ribozyme transgene (KRbz) that is specific for the K-ras codon 12 mutant sequence GUU, given the considerations that (a) in the United States, approx 30% of human NSCLCs express K-ras oncogene mutations, nearly all of which reside in codon 12; (b) anti-K-ras, anti-H, as well as anti-N-ras hammerhead ribozymes are potent growth inhibitors in various human cancers tested; and (c) in vitro and animal model studies suggest that ribozymes directed at oncogene (K- and H-ras C-fos, BCR-ABL) or human immunodeficiency viral gene messages are more effective than their antisense counterpart. This article describes the techniques involved in the production of the KRbz-adenoviral vector that is specific for the K-ras mutation GTT, and summarizes its in vivo antitumor effect against NSCLC xenografts expressing the relevant K-ras mutation in athymic mice.

Carcinoma, Non-Small-Cell Lung↗
PubMed2006

Effective gene therapy in an authentic model of Tay-Sachs-related diseases.

Tay-Sachs disease is a prototypic neurodegenerative disease. Lysosomal storage of GM2 ganglioside in Tay-Sachs and the related disorder, Sandhoff disease, is caused by deficiency of beta-hexosaminidase A, a heterodimeric protein. Tay-Sachs-related diseases (GM2 gangliosidoses) are incurable, but gene therapy has the potential for widespread correction of the underlying lysosomal defect by means of the secretion-recapture cellular pathway for enzymatic complementation. Sandhoff mice, lacking the beta-subunit of hexosaminidase, manifest many signs of classical human Tay-Sachs disease and, with an acute course, die before 20 weeks of age. We treated Sandhoff mice by stereotaxic intracranial inoculation of recombinant adeno-associated viral vectors encoding the complementing human beta-hexosaminidase alpha and beta subunit genes and elements, including an HIV tat sequence, to enhance protein expression and distribution. Animals survived for >1 year with sustained, widespread, and abundant enzyme delivery in the nervous system. Onset of the disease was delayed with preservation of motor function; inflammation and GM2 ganglioside storage in the brain and spinal cord was reduced. Gene delivery of beta-hexosaminidase A by using adeno-associated viral vectors has realistic potential for treating the human Tay-Sachs-related diseases.

Animals↗