Long-term Transgene Research Articles

Long-term T-DNA insert stability and transgene expression consistency in field propagated sugarcane

This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. T-DNA inserts are stable; no transgene rearrangements were observed. AmCYAN1 and PMI protein accumulation levels were maintained. There was no evidence that production of either protein declined across generations and no transgene silencing was observed in three commercial sugarcane varieties through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes over 4years of field testing. Long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized. This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. These data are critical supporting information needed for successful commercialization of GM sugarcane. Here seventeen transgenic events, containing the AmCYAN1 gene driven by a CMP promoter and the E. coli PMI gene driven by either a CMP or Ubi promoter, were used to monitor T-DNA insert stability and consistency of transgene encoded protein accumulation through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes. The experiments were conducted in three commercial sugarcane varieties over 4years of field testing. DNA gel blot analysis showed that the T-DNA inserts are stable; no transgene rearrangements were observed. Quantitative ELISA showed no evidence of decreasing AmCYAN1 and PMI protein levels across generations and no transgene silencing was observed. These results indicate that long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized.

Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells

Objectives:Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors.Methods: Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p'HR.cppt.3'1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(β-globin intron)-eGFP containing CMV promoter and β-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution.Results:GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (β-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction. Surprisingly, neither the HS4 insulator nor the UCOE sequence improved the GFP expression level or stability. Clonal cell lines with HCV E1 gene were generated from LV-CMV-E1 vector-infected 293T cells. A representative recombinant cell line maintained stable E1expression for at least 9 weeks without significant difference in morphology compared with untreated 293T cells.Conclusion: The results suggest that all five vectors can stably transduce 293T cells, producing long term transgene expression with different efficiencies. However, neither the insulator nor the UCOE improved the GFP expression. The vectors containing the promoter CMV or CMV (β-globin intron) generated the highest gene expressions, manifesting as more favorable candidates for recombinant protein production in HEK293T cells.

Genetic Modification of Cancer Cells Using Non-Viral, Episomal S/MAR Vectors for In Vivo Tumour Modelling

The development of genetically marked animal tumour xenografts is an area of ongoing research to enable easier and more reliable testing of cancer therapies. Genetically marked tumour models have a number of advantages over conventional tumour models, including the easy longitudinal monitoring of therapies and the reduced number of animals needed for trials. Several different methods have been used in previous studies to mark tumours genetically, however all have limitations, such as genotoxicity and other artifacts related to the usage of integrating viral vectors. Recently, we have generated an episomally maintained plasmid DNA (pDNA) expression system based on Scaffold/Matrix Attachment Region (S/MAR), which permits long-term luciferase transgene expression in the mouse liver. Here we describe a further usage of this pDNA vector with the human Ubiquitin C promoter to create stably transfected human hepatoma (Huh7) and human Pancreatic Carcinoma (MIA-PaCa2) cell lines, which were delivered into “immune deficient” mice and monitored longitudinally over time using a bioluminometer. Both cell lines revealed sustained episomal long-term luciferase expression and formation of a tumour showing the pathological characteristics of hepatocellular carcinoma (HCC) and pancreatic carcinoma (PaCa), respectively. This is the first demonstration that a pDNA vector can confer sustained episomal luciferase transgene expression in various mouse tumour models and can thus be readily utilised to follow tumour formation without interfering with the cellular genome.

Sustained Long-Term Engraftment and Transgene Expression of Peripheral Blood CD34+ Cells Transduced with Third-Generation Lentiviral Vectors

As mobilized peripheral blood (MPB) represents an attractive cell source for gene therapy, we investigated the ability of third-generation lentiviral vectors (LVs) to transfer the enhanced green fluorescent protein gene into MPB CD34(+) cells in culture conditions allowing expansion of transplantable human hematopoietic stem cells. To date, few studies have reported transduction of MPB cells with vesicular stomatitis virus G pseudotyped LVs. The critical issue remains whether primitive, hematopoietic repopulating cells have, indeed, been transduced. In vitro (5 weeks' culture in FLT3 ligand + thrombopoietin + stem cell factor + interleukin 6) and in vivo (serial transplantation in NOD/SCID mice) experiments show that MPB CD34(+) cells can be effectively long-term transduced by LV and maintain their proliferation, self-renewal, and multilineage differentiation potentials. We show that expansion following transduction improves the engraftment of transduced MPB CD34(+) (4.6-fold expansion of SCID repopulating cells by limiting dilution studies). We propose ex vivo expansion after transduction as an effective tool to improve gene therapy protocols with MPB. Disclosure of potential conflicts of interest is found at the end of this article.

Long-term Gene Expression Using the Lentiviral Vector in Rat Chondrocytes

The optimal approach to a long-term stable transgene expression in chondrocytes has not been established. Recently, lentiviral vectors have been used for transfection of some cultured cell lines. Our study tests the hypothesis that lentiviral vectors lead to longer gene expression in primary chondrocytes. We transfected lentiviral and adenoviral vectors carrying the green fluorescence protein gene to chondrocytes at different infection rates and cultured them in collagen Type I gel for up to 6 weeks. We also transplanted the cells of gel-suspended chondrocytes into the backs of nude mice. The mRNA expression of collagen Type II and aggrecan core protein was tested by real time polymerase chain reaction. The morphologic features and proliferation of chondrocytes were observed. Lentiviral vectors could transfect the green fluorescence protein gene to chondrocytes and the adenoviral vector, and there was no influence on the proliferation and phenotype of the chondrocytes. The percentage of lentiviral green fluorescence protein positive cells was much greater than the adenoviral green fluorescence protein at the end of 6 weeks. Stable green fluorescence protein expression was observed only in the lentivirus-transfected implants. The gene transfected by the lentiviral vector can be expressed efficiently for a long time and may be useful for gene transfer in cartilage defect repair.

742. Human Artificial Chromosome (HAC) Vector Provides Long-Term Therapeutic Transgene Expression in Normal Human Primary Fibroblasts

Top of pageAbstract Human artificial chromosomes (HACs) are segregating freely from host chromosomes through a set of cell divisions. Accordingly, the host genome is not disrupted and the expression of the transgene could be sustained for a prolonged period without suffering the effects of its surrounding sequence on the host genome. These are the ideal properties required for vectors in gene therapy, and therefore HACs are potentially useful to ensure both safety and duration of gene expression in therapeutic gene delivery. We constructed a novel human chromosome 21-derived HAC (21|[Delta]|pqHAC) vector, which devoid of most expressed genes by telomere truncation at the genomic regions proximal to the centromere in both p and q arms, and inserted human erythropoietin (EPO) gene, which is a growth factor for erythroid cells and widely used for the clinical treatment of anemia in renal insufficiency, into the HAC (EPO-21|[Delta]|pqHAC) as a model of therapeutic transgenes. Although low transfer efficiency of intact HACs to the cells has hampered the studies using normal human primary cells, the major targets for ex vivo gene therapy, we successfully demonstrated the introduction of EPO-21|[Delta]|pqHAC vector into normal primary human fibroblasts (hPFs) with micro cell-mediated chromosome transfer (MMCT). However, the mitotic stability of EPO-21|[Delta]|pqHAC vector in normal hPFs and the growth ability of the resultant hPF clones to obtain graft cells for transplantation remained to be elucidated. Here, we studied the hPF clones harboring the EPO-21|[Delta]|pqHAC vector cytogenetically and demonstrated that the structurally defined, extra artificial chromosome was highly maintained in normal hPFs under non-selective condition at a constant copy number mostly in a single copy per cell without accompanying aberration of host chromosomes. Mitotic-loss rates were determined, and the level of stability of EPO-21|[Delta]|pqHAC vector was comparable with those reported for the other top-down and bottom-up artificial chromosomes in human fibro-sarcoma cell line HT1080 cells. These hPF clones proliferated from a single colony up to 3.8x107 cells and then their growth stopped, suggesting that cell senescence occurred and the introduction of the EPO-21|[Delta]|pqHAC vector did not cause immortalization. We also showed the long-term expression of human epo gene in the hPFs carrying the EPO-21|[Delta]|pqHAC vector. In conclusion, this study shows the potential application of the 21|[Delta]|pqHAC vector in hormone-replacement gene therapy, which might ensure safety with no lesion of host chromosomes and provides long-term therapeutic transgene expression. This also implies that utilization of the HAC vector offers novel options for ex vivo gene therapy.

518. Long-Term Lentiviral Vector-Mediated Transgene Expression in Neural Progenitor Cells Following Implantation into the Injured Rat Spinal Cord

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons (DAN) in the substantia nigra (SNi). The ultimate treatment of PD is to prevent DAN from the cell death. We previously showed that hepatocyte growth factor (HGF) resulted in effective prevention of DAN from cell death and remarkable behavioral recovery in 6-hydroxy-dopamine-lesioned parkinsonian rats. In this study, we have extend the preclinical exploration to primate model of PD. Seven days after stereotaxic transfection of human HGF plasmid or pVAX1 control plasmid into the unilateral striatum, infusion of MPTP into the right internal carotid artery of animals produces toxin-induced injury to the right nigro-striatal pathway with sparing of other dopaminergic neurons on the infused side and with negligible or little injury to the opposite, untreated side. After 2 to 3 weeks period, animals exhibited stable moderate parkinsonian features. But animals transfecterd with human HGF plasmid showed normal states. Interestingly, animals transfected with pVAX1 plasmid demonstrated the apomorphine and amphetamine-induced rotational asymmetry. However, the transfection of human HGF plasmid resulted in a significant inhibition of abnormal rotation over 3 months. In immunohistochemistry, more than 90% doperminergic neurons of PD model animals transfecred with pVAX1 plasmid were lost, while more than 70% those of animals transfected with human HGF plasmid were survived. Microdialysis demonstrated that concentrations of dopamine in striatum and SNi in animals transfected with human HGF plasmid were higher compared with PD model. Overall, the present study demonstrated that over-expression of human HGF prevented neuronal death in primate PD model, providing a potential of novel gene therapy for PD using human HGF plasmid.

Expression of therapeutic levels of factor VIII in hemophilia A mice using a novel adeno/adeno-associated hybrid virus.

We have generated an E1a/E1b/E3-deleted adeno/adeno-associated (Ad/AAV) hybrid virus driven by a small nuclear RNA (pHU1-1) promoter for expression of a B domain-deleted (Thr761-Asn1639) factor VIII transgene (FVIIIDelta761-1639). Productive replication of Ad/AAV/FVIIIDelta761-1639 in AAV rep-expressing cells resulted in generation of monomeric and dimeric mini-adenoviral (mAd) replicative forms that retained the AAV integration elements (mAd/FVIIIDelta761-1639). In vitro studies using Ad/AAV/FVIIIDelta761-1639 generated approximately 2-logs greater FVIII activity than mAd/FVIIIDelta761-1639. To determine its capacity for in vivo excision and/or genomic integration, Ad/AAV/FVIIIDelta761-1639 was injected by tail vein into three groups of hemophilia A mice (2 x 10(11) vp [n = 3]; 4 x 10(11) vp [n = 3]; 8 x 10(11) vp [n = 3]), with clear concentration-dependent increase in FVIII activity (range 160-510 mU/ml; plasma activity 16%-51% of normal). Peak activity was seen by Day (D) 5, with slow return to baseline by D28 (0.1-0.9% activity); in only 3/9 mice was loss of FVIII activity associated with development of anti-FVIII antibodies. Quantitative-PCR using genomic DNA isolated from D28 liver, spleen, heart, lungs, and kidney demonstrated the highest concentration in liver (approximately 10 genomes/cell), with little to no organ toxicity at early (D5 or 6) or late (D28) post-infusion time points. There was no evidence for spontaneous transgene excision or genomic integration in vivo as evaluated by quantitative PCR and genomic blotting. These data establish (i) the feasibility and applicability of developing high-titer Ad/AAV hybrid viruses for FVIII delivery using a small cellular promoter, (ii) the potential utility of this virus for generation of "gutted" monomeric and dimeric mAD/FVIII retaining AAV integration elements, and (iii) that the development of strategies for regulated Rep68/78 co-expression may provide a novel approach for excision, integration, and long-term FVIII transgene expression.

80. Lack of Immune Response Against rtTA Correlates with Long-Term Doxycycline-Regulated Transgene Expression in Nonhuman Primate after Intramuscular Injection of Recombinant Adeno-Associated Virus

80. Lack of Immune Response Against rtTA Correlates with Long-Term Doxycycline-Regulated Transgene Expression in Nonhuman Primate after Intramuscular Injection of Recombinant Adeno-Associated Virus

Long-term localized transgene expression in the pancreas achieved by intra-arterial adenoassociated virus-mediated gene transfer

Long-term localized transgene expression in the pancreas achieved by intra-arterial adenoassociated virus-mediated gene transfer