Gene Therapy Strategies Incorporating Large Transgenes

Johnston, Jennifer M.; Doering, Christopher B.; Spencer, H. Trent

doi:10.5772/22437

Cited by 4 publications

(6 citation statements)

References 85 publications

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“…As insert size increases the lentiviral particles recovered by ultracentrifugation have been shown to decrease. 5,6 A prime example of this can be observed when considering gene therapy applications for the treatment of hemophilia A (for review see Johnston et al 7 ). Even after significant reductions in the size of the cDNA-encoding fVIII, low viral titer has been observed.…”

Section: Introductionmentioning

confidence: 99%

High-throughput screening identifies compounds that enhance lentiviral transduction

et al. 2014

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A difficulty in the field of gene therapy is the need to increase the susceptibility of hematopoietic stem cells (HSCs) to ex vivo genetic manipulation. To overcome this obstacle a high-throughput screen was performed to identify compounds that could enhance the transduction of target cells by lentiviral vectors. Of the 1280 compounds initially screened using the myeloid-erythroid-leukemic K562 cell line, 30 were identified as possible enhancers of viral transduction. Among the positive hits were known enhancers of transduction (camptothecin, etoposide and taxol), as well as the previously unidentified phorbol 12-myristate 13-acetate (PMA). The percentage of green fluorescent protein (GFP)-positive-expressing K562 cells was increased more than fourfold in the presence of PMA. In addition, the transduction of K562 cells with a lentiviral vector encoding fVIII was four times greater in the presence of PMA as determined by an increase in the levels of provirus in genetically modified cells. PMA did not enhance viral transduction of all cell types (for example, sca-1(+) mouse hematopoietic cells) but did enhance viral transduction of human bone marrow-derived CD34(+) cells. Notably, the percentage of GFP-positive CD34(+) cells was increased from 7% in the absence of PMA to greater than 22% in the presence of 1 nM PMA. PMA did not affect colony formation of CD34(+) cells or the expression of the hematopoietic markers CD34 and CD45. These data demonstrate that high-throughput screening can be used to identify compounds that increase the transduction efficiency of lentiviral vectors, identifying PMA as a potential enhancer of lentiviral HSC transduction.

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Section: Introductionmentioning

confidence: 99%

High-throughput screening identifies compounds that enhance lentiviral transduction

et al. 2014

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“…A possible explanation for the lower expressing rolD:nosFLZDY plants is gene silencing. Since gene silencing increases with increasing transcript size (Johnston et al 2011;Melnyk et al 2011), the larger rolD:nosFLZDY construct may have been subject to gene silencing in the tobacco plant, whereas the rolD:nosZ construct was not. N 2 OR from rolD:nosZ and rolD:nosFLZDY plant root tissue was shown to be correctly assembled.…”

Section: Discussionmentioning

confidence: 99%

Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root‐specific rolD promoter from Agrobacterium rhizogenes

Wan

Johnson

Altosaar

2011

Ecology and Evolution

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The nitrous oxide (N2O) reduction pathway from a soil bacterium, Pseudomonas stutzeri, was engineered in plants to reduce N2O emissions. As a proof of principle, transgenic plants expressing nitrous oxide reductase (N2OR) from P. stutzeri, encoded by the nosZ gene, and other transgenic plants expressing N2OR along with the more complete operon from P. stutzeri, encoded by nosFLZDY, were generated. Gene constructs were engineered under the control of a root-specific promoter and with a secretion signal peptide. Expression and rhizosecretion of the transgene protein were achieved, and N2OR from transgenic Nicotiana tabacum proved functional using the methyl viologen assay. Transgenic plant line 1.10 showed the highest specific activity of 16.7 µmol N2O reduced min−1 g−1 root protein. Another event, plant line 1.9, also demonstrated high specific activity of N2OR, 13.2 µmol N2O reduced min−1 g−1 root protein. The availability now of these transgenic seed stocks may enable canopy studies in field test plots to monitor whole rhizosphere N flux. By incorporating one bacterial gene into genetically modified organism (GMO) crops (e.g., cotton, corn, and soybean) in this way, it may be possible to reduce the atmospheric concentration of N2O that has continued to increase linearly (about 0.26% year−1) over the past half-century.

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“…Vector size constraints are an issue for fVIII gene therapy since the B domain deleted fVIII cDNA is approximately 4.4kb (for review see Johnston et al ., 2011). 14 For these reasons, lentiviral vectors are reasonable for gene therapy applications aimed at the treatment of hemophilia A utilizing ex vivo modification of HSCs. Within lentiviral vectors, a woodchuck post-transcriptional regulatory element (WPRE) is routinely added to the 3’ end of the transgene.…”

Section: Introductionmentioning

confidence: 99%

Generation of an optimized lentiviral vector encoding a high-expression factor VIII transgene for gene therapy of hemophilia A

et al. 2012

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We previously compared the expression of several human factor VIII (fVIII) transgene variants and demonstrated the superior expression properties of B domain deleted porcine fVIII. Subsequently, a hybrid human/porcine fVIII molecule (HP-fVIII) comprising 91% human amino acid sequence was engineered to maintain the high-expression characteristics of porcine fVIII. The bioengineered construct then was used effectively to treat knockout mice with hemophilia A. In the current study, we focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components in terms of virus production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: 1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), 2) HIV versus SIV viral vector systems, and 3) various internal promoters. The inclusion of a WPRE sequence had negligible effects on viral production and HP-fVIII expression. HIV and SIV vectors were compared and found to be similar with respect to transduction efficiency in both K562s and HEK-293T cells. However, there was an enhanced expression of HP-fVIII by the SIV system, which was evident in both K562 and BHK-M cell lines. To further compare expression of HP-fVIII from an SIV-based lentiviral system, we constructed expression vectors containing the high expression transgene and a human elongation factor-1 alpha (EF1α), cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter. Expression was significantly greater from the CMV promoter, which also yielded therapeutic levels of HP-fVIII in hemophilia A mice. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE.

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Gene Therapy Strategies Incorporating Large Transgenes

Cited by 4 publications

References 85 publications

High-throughput screening identifies compounds that enhance lentiviral transduction

High-throughput screening identifies compounds that enhance lentiviral transduction

Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root‐specific rolD promoter from Agrobacterium rhizogenes

Generation of an optimized lentiviral vector encoding a high-expression factor VIII transgene for gene therapy of hemophilia A

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