Immune-mediated loss of transgene expression from virally transduced brain cells is irreversible, mediated by IFNγ, perforin, and TNFα, and due to the elimination of transduced cells

The adaptive immune response to viral vectors reduces vector-mediated transgene expression from the brain. It is unknown, however, whether this loss is caused by functional downregulation of transgene expression or death of transduced cells. Herein, we demonstrate that during the elimination of transgene expression, the brain becomes infiltrated with CD4 and CD8 T cells and that these T cells are necessary for transgene elimination. Further, the loss of transgene-expressing brain cells fails to occur in the absence of IFNγ, perforin, and TNFα receptor. Two methods to induce severe immune suppression in immunized animals also fail to restitute transgene expression, demonstrating the irreversibility of this process. The need for cytotoxic molecules and the irreversibility of the reduction in transgene expression suggested to us that elimination of transduced cells is responsible for the loss of transgene expression. A new experimental paradigm that discriminates between downregulation of transgene expression and the elimination of transduced cells demonstrates that transduced cells are lost from the brain upon the induction of a specific antiviral immune response. We conclude that the anti-adenoviral immune response reduces transgene expression in the brain through loss of transduced cellsFil: Zirger, Jeffrey M.. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados UnidosFil: Puntel, Mariana. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bergeron, Josee. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados UnidosFil: Wibowo, Mia. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados UnidosFil: Moridzadeh, Rameen. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados UnidosFil: Bondale, Niyati. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados UnidosFil: Barcia, Carlos. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados UnidosFil: Kroeger, Kurt M.. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados UnidosFil: Liu, Chunyan. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados UnidosFil: Castro, Maria Graciela. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados Unidos. University of Michigan; Estados UnidosFil: Lowenstein, Pedro R.. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados Unidos. University of Michigan; Estados Unido

The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells.

Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies

The changing role of cell culture in the generation of transgenic livestock

Transgenesis may allow the generation of farm animals with altered phenotype, animal models for research and animal bioreactors. Although such animals have been produced, the time and expense involved in generating transgenic livestock and then evaluating the transgene expression pattern is very restrictive. If questions about the ability and efficiency of expression could be asked solely in vitro rapid progress could be achieved. Unfortunately, experiments addressing transcriptional control in vitro have proved unreliable in their ability to indicate whether a transgene will be transcribed or not. However, initial studies suggest that cell culture may be able to predict in vivo post-transcriptional events. We review these issues and propose that strategies which engineer the transgene integration site could enhance the probability for efficient expression. This approach has now become feasible with the development of techniques allowing animals to be generated from somatic cells by nuclear transfer. The important step in this procedure is the use of cells grown in culture as the source of genetic information, allowing the selection of specific transgene integration events. This technology which has dramatically increased the potential use of transgenic livestock for both agricultural and biotechnological applications, is based on standard cell culture methodology. We are now at the start of a new era in large animal transgenics

A Myelin Proteolipid Protein-LacZ Fusion Protein Is Developmentally Regulated and Targeted to the Myelin Membrane in Transgenic Mice

Transgenic mice were generated with a fusion gene carrying a portion of the murine myelin proteolipid protein (PLP) gene, including the first intron, fused to the E. coli LacZ gene. Three transgenic lines were derived and all lines expressed the transgene in central nervous system white matter as measured by a histochemical assay for the detection of β-galactosidase activity. PLP-LacZ transgene expression was regulated in both a spatial and temporal manner, consistent with endogenous PLP expression. Moreover, the transgene was expressed specifically in oligodendrocytes from primary mixed glial cultures prepared from transgenic mouse brains and appeared to be developmentally regulated in vitro as well. Transgene expression occurred in embryos, presumably in pre- or nonmyelinating cells, rather extensively throughout the peripheral nervous system and within very discrete regions of the central nervous system. Surprisingly, beta-galactosidase activity was localized predominantly in the myelin in these transgenic animals, suggesting that the NH_2-terminal 13 amino acids of PLP, which were present in the PLP-LacZ gene product, were sufficient to target the protein to the myelin membrane. Thus, the first half of the PLP gene contains sequences sufficient to direct both spatial and temporal gene regulation and to encode amino acids important in targeting the protein to the myelin membrane

Sustained gene expression in the retina by improved episomal vectors

Gene and cellular therapies are nowadays part of therapeutic strategies for the treatment of diverse pathologies. The drawbacks associated with gene therapy-low levels of transgene expression, vector loss during mitosis, and gene silencing-need to be addressed. The pEPI-1 and pEPito family of vectors was developed to overcome these limitations. It contains a scaffold/matrix attachment region, which anchors its replication to cell division in eukaryotic cells while in an extrachromosomal state and is less prone to silencing, due to a lower number of CpG motifs. Recent success showed that ocular gene therapy is an important tool for the treatment of several diseases, pending the overcome of the aforementioned limitations. To achieve sustained gene delivery in the retina, we evaluated several vectors based on pEPito and pEPI-1 for their ability to sustain transgene expression in retinal cells. These vectors stably transfected and replicated in retinal pigment epithelial (RPE) cells. Expression levels were promoter dependent with constitutive promoters cytomegalovirus immediate early promoter (CMV) and human CMV enhancer/human elongation factor 1 alpha promoter yielding the highest levels of transgene expression compared with the retina-specific RPE65 promoter. When injected in C57Bl6 mice, transgene expression was sustained for at least 32 days. Furthermore, the retina-specific RPE65 promoter showed higher efficiency in vivo compared to in vitro. In this study, we demonstrate that by combining tissue-specific promoters with a mitotic stable system, less susceptible to epigenetic silencing such as pEPito-based plasmids, we can achieve prolonged gene expression and a sustained therapeutic effect.Fundacao para a Ciencia e Tecnologia, Portugal [PEst/OE/EQB-LA 0023/2013, SFRH/BD/76873/2011, SFRH/BD/70318/2010, PTDC/SAU/BEB/098475/2008]; European Union [PIRG-GA-2009-249314

Isolated sequences from the linked Myf-5 and MRF4 genes drive distinct patterns of muscle-specific expression in transgenic mice

In developing mouse embryos, MyoD family regulatory genes are expressed specifically in muscle precursors and mature myofibers. This pattern, taken together with the well-established ability of MyoD family members to convert a variety of cell types to skeletal muscle, suggests a significant role for these genes in regulating skeletal myogenesis. The possibility that expression of these genes may be causally associated with segregation of the myogenic lineage from other mesodermal derivatives, or with the subsequent maintenance of muscle phenotypes at later times, raises the issue of how MyoD family genes are themselves regulated during development. In this work, we have initiated studies to identify DNA sequences that govern Myf-5 and MRF4 (herculin, myf-6) transcription. Myf-5 is the first of the MyoD family to be expressed in the developing mouse embryo, while MRF4 is the most abundantly expressed myogenic factor in postnatal animals. In spite of their strikingly divergent patterns of expression, Myf-5 and MRF4 are tightly linked in the mouse genome; their translational start codons are only 8.5 kilobases apart. Here, the 5' flanking regions of the mouse Myf-5 and MRF4 genes were separately linked to a bacterial β-galactosidase (lacZ) gene, and these constructs were each used to produce several lines of transgenic mice. Transgene expression was monitored by X-gal staining of whole embryos and by in situ hybridization of embryo sections. For the Myf-5/lacZ lines, the most intense transgene expression was in the visceral arches and their craniofacial muscle derivatives, beginning at day 8.75 post coitum (p.c.). This correlates with endogenous Myf-5 expression in visceral arches. However, while Myf-5 is also expressed in somites starting at day 8 p.c., transgene expression in the trunk is not observed until day 12 p.c. Thus, the Myf-5/lacZ construct responds to early Myf-5 activators in the visceral arches but not in the somites, suggesting that myogenic determination in the nonsomitic head mesoderm may be under separate control from that of the somitic trunk mesoderm. MRF4/lacZ lines displayed an entirely different pattern from Myf-5. Transgene expression appeared in muscles starting at day 16.5 p.c. and became increasingly prominent at later times. However, an early wave of myotomal expression that is characteristic of the endogenous MRF4 was not recapitulated by the transgene

Transgenic Overexpression of LARGE Induces alpha-Dystroglycan Hyperglycosylation in Skeletal and Cardiac Muscle

Background: LARGE is one of seven putative or demonstrated glycosyltransferase enzymes defective in a common group of muscular dystrophies with reduced glycosylation of alpha-dystroglycan. Overexpression of LARGE induces hyperglycosylation of alpha-dystroglycan in both wild type and in cells from dystroglycanopathy patients, irrespective of their primary gene defect, restoring functional glycosylation. Viral delivery of LARGE to skeletal muscle in animal models of dystroglycanopathy has identical effects in vivo, suggesting that the restoration of functional glycosylation could have therapeutic applications in these disorders. Pharmacological strategies to upregulate Large expression are also being explored.Methodology/Principal Findings: In order to asses the safety and efficacy of long term LARGE over-expression in vivo, we have generated four mouse lines expressing a human LARGE transgene. On observation, LARGE transgenic mice were indistinguishable from the wild type littermates. Tissue analysis from young mice of all four lines showed a variable pattern of transgene expression: highest in skeletal and cardiac muscles, and lower in brain, kidney and liver. Transgene expression in striated muscles correlated with alpha-dystroglycan hyperglycosylation, as determined by immunoreactivity to antibody IIH6 and increased laminin binding on an overlay assay. Other components of the dystroglycan complex and extracellular matrix ligands were normally expressed, and general muscle histology was indistinguishable from wild type controls. Further detailed muscle physiological analysis demonstrated a loss of force in response to eccentric exercise in the older, but not in the younger mice, suggesting this deficit developed over time. However this remained a subclinical feature as no pathology was observed in older mice in any muscles including the diaphragm, which is sensitive to mechanical load-induced damage.Conclusions/Significance: This work shows that potential therapies in the dystroglycanopathies based on LARGE upregulation and alpha-dystroglycan hyperglycosylation in muscle should be safe

Deletion of the Virion Host Shut-off Gene Enhances Neuronal-Selective Transgene Expression from an HSV Vector Lacking Functional IE Genes

The ability of herpes simplex virus (HSV) to establish lifelong latency in neurons suggests that HSV-derived vectors hold promise for gene delivery to the nervous system. However, vector toxicity and transgene silencing have created significant barriers to vector applications to the brain. Recently, we described a vector defective for all immediate-early gene expression and deleted for the joint region between the two unique genome segments that proved capable of extended transgene expression in non-neuronal cells. Sustained expression required the proximity of boundary elements from the latency locus. As confirmed here, we have also found that a transgene cassette introduced into the ICP4 locus is highly active in neurons but silent in primary fibroblasts. Remarkably, we observed that removal of the virion host shutoff (vhs) gene further improved transgene expression in neurons without inducing expression of viral genes. In rat hippocampus, the vhs-deleted vector showed robust transgene expression exclusively in neurons for at least 1 month without evidence of toxicity or inflammation. This HSV vector design holds promise for gene delivery to the brain, including durable expression of large or complex transgene cassettes

Green Fluorescent Protein (GFP) in Vector Systems Played Sense Role of Epigenetic in Plants

The green fluorescent protein (GFP) of jellyfish (_Aequorea victoria_) has significant advantages over other reporter genes, because expression can be detected in living cells without any substrates. Recently, epigenetic phenomena are important to consider in plant biotechnology experiments for elucidate unknown mechanism. Therefore, soybean immature cotyledons were generated embryogenesis cells and engineered with two different gene constructs (pHV and pHVS) using gene gun method. Both constructs contain a gene conferring resistance to hygromycin (_hpt_) as a selective marker and a modified glycinin (11S globulin) gene (_V3-1_) as a target. However, sGFP(_S65T_) as a reporter gene was used only in pHVS as a reporter gene for study the relation between using sGFP(_S65T_) and gene silencing phenomena. Fluorescence microscopic was used for screening after the selection of hygromycin, identified clearly the expression of sGFP(_S65T_) in the transformed soybean embryos bombarded with the pHVS construct. Protein analysis was used to detect gene expression overall seeds using SDS-PAGE. Percentage of gene down regulation was highly in pHV construct compared with pHVS. Thus, sGFP(_S65T_) as a reporter gene in vector system may be play useful role for transgenic evaluation and avoid gene silencing in plants for the benefit of plant transformation system

Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.

Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients