Transgene mRNA Levels Research Articles

Prednisolone and rapamycin reduce the plasma cell gene signature and may improve AAV gene therapy in cynomolgus macaques

Adeno-associated virus (AAV) vector gene therapy is a promising approach to treat rare genetic diseases; however, an ongoing challenge is how to best modulate host immunity to improve transduction efficiency and therapeutic outcomes. This report presents two studies characterizing multiple prophylactic immunosuppression regimens in male cynomolgus macaques receiving an AAVrh10 gene therapy vector expressing human coagulation factor VIII (hFVIII). In study 1, no immunosuppression was compared with prednisolone, rapamycin (or sirolimus), rapamycin and cyclosporin A in combination, and cyclosporin A and azathioprine in combination. Prednisolone alone demonstrated higher mean peripheral blood hFVIII expression; however, this was not sustained upon taper. Anti-capsid and anti-hFVIII antibody responses were robust, and vector genomes and transgene mRNA levels were similar to no immunosuppression at necropsy. Study 2 compared no immunosuppression with prednisolone alone or in combination with rapamycin or methotrexate. The prednisolone/rapamycin group demonstrated an increase in mean hFVIII expression and a mean delay in anti-capsid IgG development until after rapamycin taper. Additionally, a significant reduction in the plasma cell gene signature was observed with prednisolone/rapamycin, suggesting that rapamycin’s tolerogenic effects may include plasma cell differentiation blockade. Immunosuppression with prednisolone and rapamycin in combination could improve therapeutic outcomes in AAV vector gene therapy.

Tyrosine-Mutant AAV8 Vector Mediated Efficient and Safe Gene Transfer of Pigment Epithelium-Derived Factor to Mouse Lungs.

Recombinant adeno-associated viruses (rAAV) are an important tool for lung targeted gene therapy. Substitution of tyrosine with phenylalanine residues (Y-F) in the capsid have been shown to protect the AAV vector from ubiquitin/proteasome degradation, increasing transduction efficiency. We tested the mutant Y733F-AAV8 vector for mucus diffusion, as well as the safety and efficacy of pigment epithelium-derived factor (PEDF) gene transfer to the lung. For this purpose, Y733F-AAV8-PEDF (1010 viral genome) was administered intratracheally to C57BL/6 mice. Lung mechanics, morphometry, and inflammation were evaluated 7, 14, 21, and 28 days after injection. The tyrosine-mutant AAV8 vector was efficient at penetrating mucus in ex vivo assays and at transferring the gene to lung cells after in vivo instillation. Increased levels of transgene mRNA were observed 28 days after vector administration. Overexpression of PEDF did not affect in vivo lung parameters. These findings provide a basis for further development of Y733F-AAV8-based gene therapies for safe and effective delivery of PEDF, which has anti-angiogenic, anti-inflammatory and anti-fibrotic activities and might be a promising therapy for lung inflammatory disorders.

Plp1 in the enteric nervous system is preferentially expressed during early postnatal development in mouse as DM20, whose expression appears reliant on an intronic enhancer.

Recently, the myelin proteolipid protein gene (Plp1) was shown to be expressed in the glia of the enteric nervous system (ENS) in mouse. However, beyond this, not much is known about its expression in the intestine. To address this matter, we investigated Plp1 expression at the mRNA and protein levels in the intestine of mice at different ages (postnatal days 2, 9, 21, and 88). In this study, we show that Plp1 expression preferentially occurs during early postnatal development, primarily as the DM20 isoform. Western blot analysis indicated that DM20 migrated according to its formula weight when isolated from the intestine. However, mobilities of both PLP and DM20 were faster than expected when procured from the brain. The 6.2hPLP(+)Z/FL transgene, which uses the first half of the human PLP1 gene to drive expression of a lacZ reporter gene, recapitulated the developmental pattern observed with the native gene in the intestine, indicating that it can be used as a proxy for Plp1 gene expression. As such, the relative levels of β-galactosidase (β-gal) activity emanating from the 6.2hPLP(+)Z/FL transgene suggest that Plp1 expression is highest in the duodenum, and decreases successively along the segments, toward the colon. Moreover, removal of the wmN1 enhancer region from the transgene (located within Plp1 intron 1) resulted in a dramatic reduction in both transgene mRNA levels and β-gal activity in the intestine, throughout development, suggesting that this region contains a regulatory element crucial for Plp1 expression. This is consistent with earlier studies in both the central and peripheral nervous systems, indicating that it may be a common (if not universal) means by which Plp1 gene expression is governed.

The CUL3 adaptor protein SPOP plays a role in tauopathy mechanisms

AbstractBackgroundThe pathological accumulation of the microtubule binding protein tau drives age‐related neurodegeneration in a variety of disorders, collectively called tauopathies. In the most common tauopathy, Alzheimer’s disease (AD), the accumulation of pathological tau strongly correlates with cognitive decline. The underlying molecular mechanisms that drive neurodegeneration in tauopathies remain unknown and no effective disease modifying pharmacological interventions for tauopathy currently exist.MethodsWe employed classical forward genetic approaches and identified multiple loss of function alleles in the C. elegans spop‐1 gene that ameliorate tauopathy, suggesting SPOP is required for tau mediated neurodegeneration. CRISPR based genome editing methodology enabled the generation of customized SPOP‐1 loss of function and null alleles. Molecular genetics, behavioral, neuronal reporter assays, and biochemical analyses were also employed to characterize the consequences of spop‐1 loss of function on tauopathy related phenotypes in model systems.ResultsKnockout of SPOP‐1 rescues tau mediated behavioral deficits caused by neuronal dysfunction in tau transgenic C. elegans. Biochemical analysis revealed that SPOP‐1 loss of function promotes clearance of phosphorylated and total tau species from C. elegans neurons, but no change in tau transgene mRNA levels. Tau transgenic animals exhibit obvious neurodegeneration of GABAergic neurons, but loss of spop‐1 rescues neurodegeneration. While SPOP functions as an CUL3 E3 ligase adaptor protein, CUL3 function is not required for SPOP loss of function rescue of tauopathy. Genetic epistasis analysis suggests the nuclear speckle resident poly(A) RNA binding protein sut‐2 and spop‐1 function in a parallel molecular pathway.ConclusionsSPOP is a novel modifier of tauopathy phenotypes. Combined with previous findings investigating ALYREF, PARN/TOE1, SUT‐1 and SUT‐2/MSUT2, this work suggests phase‐separated nuclear speckles are an important cellular site controlling susceptibility to pathological tau. Recent work showing SPOP modification of PR dipeptides derived from C9orf72 expansion suggests common pathways may be at work in the neurodegenerative molecular mechanisms of tauopathy and repeat dipeptides.

Oxamyl exerts developmental toxic effects in zebrafish by disrupting the mitochondrial electron transport chain and modulating PI3K/Akt and p38 Mapk signaling

Oxamyl, a carbamate insecticide, is mainly used to control nematodes in the agricultural field. Although oxamyl is a widely used insecticide that is associated with ecological concerns, limited studies have examined the toxic effects of oxamyl on the developmental stage and the underlying mechanisms. In this study, the developmental toxicity of oxamyl was demonstrated using zebrafish, which is a representative model as it is associated with rapid embryogenesis and a toxic response similar to that of other vertebrates. The morphological alteration of zebrafish larvae was analyzed to confirm the sub-lethal toxicity of oxamyl. Analysis of transgenic zebrafish (olig2:dsRED and flk1:eGFP line) and mRNA levels of genes associated with individual organ development revealed that oxamyl exerted toxic effects on the development of neuron, notochord, and vascular system. Next, the adverse effect of oxamyl on the mitochondrial electron transport chain was examined. Treatment with oxamyl altered the PI3K/Akt signaling and p38 Mapk signaling pathways in zebrafish. Thus, this study elucidated the mechanisms underlying the developmental toxicity of oxamyl and provided information on the parameters to assess the developmental toxicity of other environmental contaminants.

Congenic expression of poly-GA but not poly-PR in mice triggers selective neuron loss and interferon responses found in C9orf72 ALS

Expansion of a (G4C2)n repeat in C9orf72 causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the link of the five repeat-encoded dipeptide repeat (DPR) proteins to neuroinflammation, TDP-43 pathology, and neurodegeneration is unclear. Poly-PR is most toxic in vitro, but poly-GA is far more abundant in patients. To directly compare these in vivo, we created congenic poly-GA and poly-PR mice. 40% of poly-PR mice were affected with ataxia and seizures, requiring euthanasia by 6 weeks of age. The remaining poly-PR mice were asymptomatic at 14 months of age, likely due to an 80% reduction of the transgene mRNA in this subgroup. In contrast, all poly-GA mice showed selective neuron loss, inflammation, as well as muscle denervation and wasting requiring euthanasia before 7 weeks of age. In-depth analysis of peripheral organs and blood samples suggests that peripheral organ failure does not drive these phenotypes. Although transgene mRNA levels were similar between poly-GA and affected poly-PR mice, poly-GA aggregated far more abundantly than poly-PR in the CNS and was also found in skeletal muscle. In addition, TDP-43 and other disease-linked RNA-binding proteins co-aggregated in rare nuclear inclusions in the hippocampus and frontal cortex only in poly-GA mice. Transcriptome analysis revealed activation of an interferon-responsive pro-inflammatory microglial signature in end-stage poly-GA but not poly-PR mice. This signature was also found in all ALS patients and enriched in C9orf72 cases. In summary, our rigorous comparison of poly-GA and poly-PR toxicity in vivo indicates that poly-GA, but not poly-PR at the same mRNA expression level, promotes interferon responses in C9orf72 disease and contributes to TDP-43 abnormalities and neuron loss selectively in disease-relevant regions.

The Integration of Multiple Nuclear-Encoded Transgenes in the Green Alga Chlamydomonas reinhardtii Results in Higher Transcription Levels

The integration of genes into the nuclear genome of Chlamydomonas reinhardtii is mediated by Non-Homologous-End-Joining, thus resulting in unpredicted insertion locations. This phenomenon defines ‘the position-effect’, which is used to explain the variation of expression levels between different clones transformed with the same DNA fragment. Likewise, nuclear transgenes often undergo epigenetic silencing that reduces their expression; hence, nuclear transformations require high-throughput screening methods to isolate clones that express the foreign gene at a desirable level. Here, we show that the number of integration sites of heterologous genes results in higher mRNA levels. By transforming both a synthetic ferredoxin-hydrogenase fusion enzyme and a Gaussia-Luciferase reporter protein, we were able to obtain 33 positive clones that exhibit a wide range of synthetic expression. We then performed a droplet-digital polymerase-chain-reaction for these lines to measure their transgene DNA copy-number and mRNA levels. Surprisingly, most clones contain two integration sites of the synthetic gene (45.5%), whilst 33.3% contain one, 18.1% include three and 3.1% encompass four. Remarkably, we observed a positive correlation between the raw DNA copy-number values to the mRNA levels, suggesting a general effect of which transcription of transgenes is partially modulated by their number of copies in the genome. However, our data indicate that only clones harboring at least three copies of the target amplicon show a significant increment in mRNA levels of the reporter transgene. Lastly, we measured protein activity for each of the reporter genes to elucidate the effect of copy-number variation on heterologous expression.

<em>In Vivo</em> Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

Does the Mutant CAG Expansion in Huntingtin mRNA Interfere with Exonucleolytic Cleavage of its First Exon?

Silencing mutant huntingtin mRNA by RNA interference (RNAi) is a therapeutic strategy for Huntington's disease. RNAi induces specific endonucleolytic cleavage of the target HTT mRNA, followed by exonucleolytic processing of the cleaved mRNA fragments. We investigated the clearance of huntingtin mRNA cleavage products following RNAi, to find if particular huntingtin mRNA sequences persist. We especially wanted to find out if the expanded CAG increased production of a toxic mRNA species by impeding degradation of human mutant huntingtin exon 1 mRNA. Mice expressing the human mutant HTT transgene with 128 CAG repeats (YAC128 mice) were injected in the striatum with self-complementary AAV9 vectors carrying a miRNA targeting exon 48 of huntingtin mRNA (scAAV-U6-miRNA-HTT-GFP). Transgenic huntingtin mRNA levels were measured in striatal lysates after two weeks. For qPCR, we used species specific primer-probe combinations that together spanned 6 positions along the open reading frame and untranslated regions of the human huntingtin mRNA. Knockdown was also measured in the liver following tail vein injection. Two weeks after intrastriatal administration of scAAV9-U6-miRNA-HTT-GFP, we measured transgenic mutant huntingtin in striatum using probes targeting six different sites along the huntingtin mRNA. Real time PCR showed a reduction of 29% to 36% in human HTT. There was no significant difference in knockdown measured at any of the six sites, including exon 1. In liver, we observed a more pronounced HTT mRNA knockdown of 70% to 76% relative to the untreated mice, and there were also no significant differences among sites. Our results demonstrate that degradation is equally distributed across the human mutant huntingtin mRNA following RNAi-induced cleavage.

Transcriptional and post-transcriptional targeting for enhanced transient gene expression in CHO cells.

To develop a simple approach to increase titers of transient gene expression in CHO cells without relying on host cell line engineering as recent reports suggest that for PEI-mediated transfections, under optimized conditions, DNA delivery into cells and nuclei is not the limiting factor. N, N-Dimethyl acetamide (DMA) was utilized to enhance transcription. To target post-transcriptional events, we evaluated the co-expression of various genes involved in the unfolded protein response, namely XBP1S, ATF4, CHOP and HSPA5. XBP1S overexpression led to a 15-85% increase in titer for multiple therapeutic proteins. Mechanistic studies confirmed that addition of 0.125% DMA increased transgene mRNA levels as expected. However, overexpression of XBP1S had no effect on transgene mRNA levels, indicating that it influenced post-transcriptional events. Since DMA and XBP1S targeted different pathways, the combination of the two approaches led to an additive improvement in protein titer (150-250% titer increase). Transcriptional and post-transcriptional pathways of transient gene expression can be targeted to increase titers without resorting to host cell line engineering in a simple, short, 7day production process.

547. Untranslated Region Optimization Increases Transgene mRNA and Protein Levels, Resulting in Enhanced Therapeutic Efficacy of AAV Vector Gene Transfer In Vivo for Crigler-Najjar Syndrome

Optimization of the transgene expression cassette for adeno-associated virus (AAV) vector-mediated gene therapy has been mainly focused on the design of efficient promoter/enhancers and on the optimization of the transgene codon usage. Addition of intron sequences to expression cassette of a given transgene has also been shown to be beneficial in terms of expression levels.In an effort to develop a liver gene transfer approach to treat Crigler-Najjar (CN) syndrome, an autosomal recessive disorder caused by mutations in the UDP-glucuronosyltransferase 1 isotype A1 (UGT1A1) gene and associated with toxic accumulation of unconjugated bilirubin in serum, we designed several UGT1A1 transgene expression cassettes and we packaged them into serotype 8 AAV vectors.We first performed the codon optimization of the UGT1A1 wildtype (w.t.) cDNA following two algorithms (v1 and v2), which led to cDNAs with nucleotide sequences significantly different from each other (nucleotide sequence homology of 77.5% for v1 and 78.3% for v2 vs. w.t.). Surprisingly, the sequence with a codon adaptation index closer to that of w.t. (w.t.=0.76, v1=0.76, v2=0.96) thus predicted to be the least efficient in terms of expression, showed the best performance in vitro in human hepatocyte cell lines, both at the RNA and protein levels. These results were confirmed in vivo in Ugt1a1-/- mice, a model of CN syndrome, where gene transfer with the optimized UGT1A1 resulted in better reduction of total bilirubin in serum. Next, we analyzed the 5’ untranslated region (UTR) of the expression cassette for sequences that may affect the efficacy of the transgene expression levels. Aside from the introduction of an optimized Kozak consensus sequence, the human beta2-globin (hBB2) intron sequence associated with the promoter revealed the presence of out of frame and in frame ATG codons before the start site of the UGT1A1 transgene. Removal of these cryptic ATGs from the intron sequence enhanced transgene expression, which resulted in stable, long-term correction of the disease phenotype following in vivo gene transfer. This demonstrated that the intron sequence optimization increases mRNA and protein levels in vitro and enhance the efficacy of the vector in vivo. Similar analysis was conducted in other common promoter/intron combinations used in AAV vector gene transfer.In conclusion, our results provide evidence that the combination of a potent liver specific promoter, codon optimization, and optimized untranslated region elements allow for decreasing the therapeutic dose of AAV vectors.

108. Versican G1 Domain Stimulates Adenoviral Transgene Expression in a JAK/STAT Dependent Manner

Gene therapy using either adeno-associated (AAV) or adenoviral (AdV) vectors has been shown to be effective in the ocular environment. Understanding the mechanisms of this enhancement could benefit gene therapy protocols in general. Vitreous, the gelatinous material filling the posterior eye, enhances the expression of transgenes delivered by AdV and AAV. Vitreous has no effect on vector internalization but is associated with increased transgene mRNA levels and mediates enhancement in hyaluronan-dependent and independent-manners (Chaudhuri et al. Mol Therapy 2007). We hypothesize that versican, a hyaluronan-binding proteoglycan expressed in vitreous, has a role in both mechanisms. Furthermore, we define an intracellular pathway through which the increased gene expression occurs.Versican-containing supernatant (VCS) was produced by culturing versican-secreting ACHN cells or HepG2 cells transfected to transiently express the recombinant versican G1 domain either lacking (G1-) or containing (G1+) the hyaluronan-binding region. Y79 or Weri retinoblastoma cells, or SKNDZ neuroblastoma cells that lack a cellular hyaluronan binding receptor were transduced in the presence of bovine vitreous, VCS, or either G1 domain with AdV vectors delivering a luciferase reporter transgene.Incubation of target cells with vitreous enhanced AdV or AAV gene expression greater than 4-fold (p<0.001) when Y79 cells, Weri cells, or SKNDZ cells were used as targets. Boiling vitreous prevented this enhancement. Thus, heat-stable hyaluronan alone was not responsible. Incubation with dasatanib (a Src kinase inhibitor), VCS, G1+, or G1- mimicked the effects of vitreous. Small-molecule inhibition of JAK1/2 or STAT3/5 using ruxolitinib or C188-9 mitigated the enhancement of transgene expression mediated by vitreous, dasatanib, VCS or either G1 domain, while inhibition of the mTOR pathway using rapamycin or everolimus showed no effect on enhancement. Vitreous or VCS treatment enhanced STAT3 phosphorylation in comparison to untreated cells.These results support a model in which Src kinase activity inhibits AdV or AAV viral vector mediated transgene expression. This inhibition can be overcome by hyaluronan-independent and dependent mechanisms mediated by the G1 domain of versican through activation of the JAK/STAT signaling pathway. Therefore, the versican G1 domain could be used as an adjuvant to enhance gene therapy protocols.

Isoglutaminyl cyclase contributes to CCL2-driven neuroinflammation in Alzheimer's disease.

The brains of Alzheimer’s disease (AD) patients are characterized by deposits of Abeta peptides and by accompanying chronic inflammation. Here, we provide evidence that the enzyme isoglutaminyl cyclase (isoQC) is a novel factor contributing to both aspects of AD pathology. Two putative substrates of isoQC, N-truncated Abeta peptides and the monocyte chemoattractant chemokine CCL2, undergo isoQC-catalyzed pyroglutamate (pGlu) modification. This triggers Abeta aggregation and facilitates the biological activity of CCL2, which collectively results in the formation of high molecular weight Abeta aggregates, glial cell activation, neuroinflammation and neuronal cell death. In mouse brain, we found isoQC to be neuron-specifically expressed in neocortical, hippocampal and subcortical structures, localized to the endoplasmic reticulum and Golgi apparatus as well as co-expressed with its substrate CCL2. In aged APP transgenic Tg2576 mice, both isoQC and CCL2 mRNA levels are up-regulated and isoQC and CCL2 proteins were found to be co-induced in Abeta plaque-associated reactive astrocytes. Also, in mouse primary astrocyte culture, a simultaneous up-regulation of isoQC and CCL2 expression was revealed upon Abeta and pGlu-Abeta stimulation. In brains of AD patients, the expression of isoQC and CCL2 mRNA and protein is up-regulated compared to controls and correlates with pGlu-Abeta load and with the decline in mini-mental state examination. Our observations provide evidence for a dual involvement of isoQC in AD pathogenesis by catalysis of pGlu-Abeta and pGlu-CCL2 formation which mutually stimulate inflammatory events and affect cognition. We conclude that isoQC inhibition may target both major pathological events in the development of AD.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1395-2) contains supplementary material, which is available to authorized users.

Increased Mitochondrial Gene Deletions and ROS Production in Transgenic Mice Overexpressing a Human 8-Oxoguanine DNA-Glycosylase Gene: A Causal Association with the Development of Hematopoietic Neoplasms

Alterations of nuclear genes in human diseases including neoplasms have been well investigated in past several decades and unequivocally established their predominant role in the pathogenesis. However, the relationship of mitochondrial genome alteration with human diseases remains largely unknown. Mitochondria are dynamic organelles involved in oxidative phosphorylation and production of reactive oxygen species (ROS). Accumulated evidence supports that mitochondrial DNA damage and dysfunction play vital roles in the development of a wide array of mitochondria-related human diseases, such as obesity, diabetes, infertility, neurodegenerative disorders and malignant tumors. We previously described the development of a transgenic (TG) mouse model for mitochondrial DNA damage by overexpressing human mitochondrial isoform of 8-oxoguanine DNA Glycosylase 1 (hOGG1) gene. Over-expression of this gene produced a wide range of adverse biological phenotypes, manifesting early-onset obesity, metabolic disturbance, female infertility, high frequency of B-cell lymphoma and human essential thrombocythemia like myeloproliferative disorder, involving the lymph node, bone marrow, spleen, liver and other extranodal sites. Development of these hematopoietic neoplasms appeared to be age-dependent. In the current study, we focused on the pathogenesis of the hematopoietic neoplasms by characterization of the neoplasms via pathologic, biochemical and molecular approaches. While expression of mOGG1 was very similar in parallel organs from transgenic and wild-type control mice, the hOGG1 TG mice expressed very high levels of human OGG1 transgene mRNA, being 6.8- and 112-fold as high as the endogenous mouse OGG, in the spleen and bone marrow. By contrast, hOGG1 transgene mRNA were not detected at all in the above two organs from control mice, indicating that the transgene is highly expressed in the hematopoietic organs in TG mice. We then measured mitochondrial NADH dehydrogenase 1 (ND1) gene expression as an indirect measure of mitochondrial respiratory function. ND1 mRNA levels in the spleen (4) and lymphoma (4) of TG mice were 83% and 58% higher, respectively, than those in the spleen (4) of wild-type control mice (P < 0.01), indicative of increased mitochondrial respiration in the lymphoma and spleen of hOGG1 TG mice. We next measured the levels of intracellular H2O2 production in the lymphoma and spleen of hOGG1 transgenic (4) and the spleen from wild-type control (4) mice. The amount of H2O2 produced in the lymphoma and the spleen of hOGG1 transgenic mice was ~166% and ~66% higher, respectively, than that in the spleen from wild-type control mice (P < 0.001). The amount of H2O2 produced in the lymphoma was ~60% higher than that in the spleen from hOGG1 transgenic mice (P < 0.05). Finally, we examined mitochondrial DNA alterations in TG mice. Mitochondrial DNA samples were extracted from various organs and lymphoma tissues from hOGG1 TG and age-matched non-TG control animals and subjected to PCR analysis using specific primer sets franking the breakpoints of 7 major mitochondrial DNA deletions. Six deletions (3.7, 3.82, 3.86, 4.2, 4.9 and 5.2 kilobase in length) have been previously reported in the literatures. One novel deletion of 15.kilobase was identified in hOGG1 TG mouse in our laboratory. Among 7 major mitochondrial DNA deletion analyzed, Five (3.7, 3.86, 4.2, 5.2 and 15 kilobase in length) deletions were detected in higher frequency in various organs of hOGG1 TG but not in non-TG control mice, suggesting that those deletions might be resulted from overexpression of the transgene hOGG1. Notably, 3 deletions (del3729, del3868, and del15139) were present in significantly higher frequencies in spleen with myeloproliferative disorders or lymphoma from TG mice in comparison to the spleen of the age-matched wild type animals (P<0.01). While the precise mechanisms leading to the development of hematopoietic neoplasms remain elusive, we hypothesized that major mitochondrial gene deletions and increased mitochondrial respiration, as a result of overexpressed hOGG1 gene in the mitochondria, may contribute significantly to the increased intracellular ROS in hematopoietic progenitor cell populations, which, in turn, causes further genetic mutation and the development of lymphoma and myeloproliferative disorder seen in these TG mice. DisclosuresNo relevant conflicts of interest to declare.

Tissue-Specific Transgene Expression in Induced Pluripotent Stem (iPS) Cell-Derived Megakaryocytes: Correction of Glanzmann Thrombasthenia (GT)

Abstract 387Megakaryocyte-specific transgene expression in patient-derived iPS cells offers a new approach to study and potentially treat disorders that affect megakaryocytes and platelets. The feasibility of this method was demonstrated in GT, a disorder resulting from an absence of functional platelet integrin alphaIIb/beta3, leading to impaired platelet aggregation and clinically presenting with severe bleeding. While rare, GT can serve as a model of an inherited platelet disorder that would benefit from corrective cell therapy. For this study, iPS cells were generated from the peripheral blood of healthy controls and two patients with GT. Patient 1 is homozygous for a novel alphaIIb exon/intron 4 splice site mutation, leading to an alternatively spliced transcript and premature stop codon. Patient 2 is homozygous for a previously reported alphaIIb missense mutation in exon 8 (p.G273D), leading to impaired intracellular transport and surface expression of the heterodimer. Both patients express <5% alphaIIb/beta3 on the surface of their platelets, which was confirmed in IPS cell-derived megakaryocytes. To achieve megakaryocyte-specific expression, transgene constructs were created using the murine proximal GPIbalpha promoter driving either an eGFP reporter or wild type alphaIIb cDNA. These constructs were inserted into the AAVS1 safe harbor locus using zinc finger nuclease-mediated homologous recombination. When targeted to a single allele of the AAVS1 locus in human ES cells, the GPIbalpha promoter driving eGFP showed a high-level of expression in ES cell-derived megakaryocytes. Inserting the GPIbalpha promoter driving alphaIIb into a single allele of the AAVS1 locus in GT iPS cells restored ∼60% surface expression of integrin alphaIIb/beta3 when compared to control iPS cell-derived megakaryocytes. Transgenic alphaIIb mRNA levels were similar to endogenous alphaIIb mRNA levels in differentiated megakaryocytes. When stimulated with thrombin, corrected GT iPS cell-derived megakaryocytes demonstrated both PAC-1 antibody binding and fibrinogen binding. This study shows the GPIbalpha promoter construct targeted to the AAVS1 locus drives a high-level of expression in iPS cell-derived megakaryocytes, which offers a novel tool to study megakaryocyte and platelet biology in samples obtained from affected individuals. For GT, this study demonstrates a higher level of correction than seen with a lentiviral approach. To our knowledge, this is the first report of the generation and correction of iPS cell lines from patients with a disease affecting platelet function. Disclosures:No relevant conflicts of interest to declare.

Role of non‐specific DNA in reducing coding DNA requirement for transient gene expression with CHO and HEK‐293E cells

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the TGE volumetric productivity has improved significantly over the past decade, the amount of plasmid DNA (pDNA) needed for transfection remains very high. Here, we examined the use of non-specific (filler) DNA to partially replace the transgene-bearing plasmid DNA (coding pDNA) in transfections of Chinese hamster ovary (CHO) and human embryo kidney (HEK-293E) cells. When the optimal amount of coding pDNA for either host was reduced by 67% and replaced with filler DNA, the recombinant protein yield decreased by only 25% relative to the yield in control transfections. Filler DNA did not affect the cellular uptake or intracellular stability of coding pDNA, but its presence lead to increases of the percentage of transfected cells and the steady-state level of transgene mRNA compared to control transfections. Studies of the physicochemical properties of DNA-polyethyleneimine (PEI) complexes with or without filler DNA did not reveal any differences in their size or surface charge. The results suggest that filler DNA allows the coding pDNA to be distributed over a greater number of DNA-PEI complexes, leading to a higher percentage of transfected cells. The co-assembly of filler DNA and coding pDNA within complexes may also allow the latter to be more efficiently utilized by the cell's transcription machinery, resulting in a higher level of transgene mRNA.

Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct

Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2–R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3′ half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production.

149 PRODUCTION OF KNOCK-DOWN MICE WITH shRNA CYTOCHROME P-450 4F16 GENE AND REDUCTION OF THE IMMUNE SYSTEM

The putative mouse homologue of cytochrome P-450 4F16 (Cyp4f16) is induced by interleukin-1 (Il-1), interleukin-6 (Il-6) and tumour necrosis factor-α (Tnf-α) and repressed by interleukin-10 (Il-10) and lipopolysaccharide (LPS). The Cyp4F16 is a subfamily of Cyp4F and it is also related to eicosanoids that are important mediators in the inflammatory cascade (Cui et al. 2001). To investigate the role of Cyp4F16, in the present study, we report the production of Cyp4f16 gene knock-down mice in 2 strains of mice, namely A/J and C57BL/6. The A/J is susceptible to infection and it is associated with Cyp4F16, whereas C57BL/6 is relatively resistant to infection. An shRNA-Cyp4F16 expression vector was microinjected into pronuclei of fertilized mouse oocytes and the embryos were transferred into pseudopregnant recipients. As a result, 25 and 50 mice were produced in the A/J and C57BL, respectively. Two mice in the A/J strain and 6 in the C57BL strain were confirmed by PCR as transgenic. Organs were collected in each of the lines produced by inbreeding and screened with real-time PCR for Cyp4f16 transcripts. The Cyp4f16 gene was expressed in a tissue-specific manner with high expression in the pancreas, spleen and lung and a lower level of transcription in the heart, muscle, thymus, kidney, testis and liver. In the spleen of transgenic Cyp4f16 knock-down mice, Cyp4f16 mRNA and protein expression levels were significantly lower than those of wild-type mice. The A/J Cyp4f16 knock-down mice suffered an inflammatory skin disease and tumours, but wild-type A/J mice and knock-down C57BL mice did not. Taken together, these results suggest that Cyp4f16 may play a regulatory role in the immune system and point to the use of the Cyp4f16 knock-down mouse as an experimental animal model for the study of the inflammatory process. This work was supported by a grant PJ0070762010 from BioGreen 21 Program, Rural Development Administration, Republic of Korea.

Abstract B9: Myc represses Ebf1 and Pax5 to impair early B-cell differentiation and drive progenitor B lymphomagenesis

Abstract Activation of Myc oncogenic transcription factors is a hallmark of rapidly dividing malignancies. The Eμ-Myc transgenic mouse is a validated model of MYC activation due to MYC/Ig translocations seen in human Burkitt lymphoma (BL). Normally, these transgenics develop pre-B cell (fractions C-E) stage lymphoma with a mean latency of 3–4 months. Here we report that Myc represses the transcription of Pax5 and Ebf1, which are master transcriptional regulators of B cell fate commitment, and that Pax5 and Ebf1 function as tumor suppressors that impair the tumorigenic potential of Myc-driven lymphoma. Expression analyses of wild-type and premalignant Eμ-Myc fractions A0 through F bone marrow and splenic B cells showed that Pax5 and Ebf1 expression is normally induced in fraction A2 progenitors, but that this response is markedly suppressed in Myc-expressing B cells. In addition, activation of c-Myc in P493-6 B lymphoid cells, which harbor a Tet-regulated c-Myc transgene, repressed Pax5 and Ebf1 mRNA levels, and ChIP analyses demonstrated this was associated with direct binding of c-Myc to the promoter-regulatory regions of Pax5 and Ebf1. Notably, FACS analyses demonstrated marked increases in the numbers of Fraction A1 cells (AA4.1 +B220+CD19−CD4+) and corresponding reductions in Fraction A2 (AA4.1+B220+CD19-CD4-) progenitors in Eμ-Myc versus wild-type mice. Thus, Myc impairs the A1-to-A2 developmental transition of B cells and this is associated with Myc-mediated repression of Pax5 and Ebf1. Collectively these findings, and those of others showing that Pax5 loss promotes the development of progenitor B cell tumors and that inactivating mutations in PAX5 and EBF1 are found in human B-ALL, supported the hypothesis that Ebf1 and Pax5 function as tumor suppressors. To test this, we transduced Eμ-Myc lymphomas harboring inactivating mutations in either Arf or p53 with retroviruses expressing Ebf1 or Pax5 together with a GFP marker, or with control GFP-only virus. Mixtures of GFP+ (transduced) and GFP− (nontransduced) lymphomas were then either cultured ex vivo in medium with IL7, or were directly transplanted into either Nude or NOD/SCID mice. Notably, Ebf1-expressing lymphoma cells underwent rapid growth arrest when cultured ex vivo, indicating Ebf1 cancels Myc's proliferative response. More importantly, enforced expression of Pax5 and especially Ebf1 markedly impaired the tumorigenic potential of Myc-driven lymphoma, irrespective of mutations in Arf or p53, where there was a profound selection against Pax5+GFP+ or Ebf1+GFP+ lymphoma cells in tumor-bearing recipients compared to recipients transplanted with GFP-only expressing lymphoma cells. To assess the effects of Ebf1 and Pax5 in Eμ-expressing (stages A1-F) B cells, as well as their ability to impair the development of lymphomas, we have generated Eμ-Ebf1 and Eμ-Pax5 transgenic mice. These are being crossed to both Eμ-Myc and Eμ-Myc/?μ-Bcl2 transgenics to assess whether Pax5 or Ebf1 will also block lymphoma onset. We predict that Pax5 and Ebf1 will impair lymphomagenesis and override the A1-to-A2 developmental block manifest in Eμ-MycB cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B9.

Characterization of a Transgenic Short Hairpin RNA-Induced Murine Model of Tafazzin Deficiency

Barth's syndrome (BTHS) is an X-linked mitochondrial disease that is due to a mutation in the Tafazzin (TAZ) gene. Based on sequence homology, TAZ has been characterized as an acyltransferase involved in the metabolism of cardiolipin (CL), a unique phospholipid almost exclusively located in the mitochondrial inner membrane. Yeast, Drosophila, and zebrafish models have been invaluable in elucidating the role of TAZ in BTHS, but until recently a mammalian model to study the disease has been lacking. Based on in vitro evidence of RNA-mediated TAZ depletion, an inducible short hairpin RNA (shRNA)-mediated TAZ knockdown (TAZKD) mouse model has been developed (TaconicArtemis GmbH, Cologne, Germany), and herein we describe the assessment of this mouse line as a model of BTHS. Upon induction of the TAZ-specific shRNA in vivo, transgenic mouse TAZ mRNA levels were reduced by >89% in cardiac and skeletal muscle. TAZ deficiency led to the absence of tetralineoyl-CL and accumulation of monolyso-CL in cardiac muscle. Furthermore, mitochondrial morphology from cardiac and skeletal muscle was altered. Skeletal muscle mitochondria demonstrated disrupted cristae, and cardiac mitochondria were significantly enlarged and displace neighboring myofibrils. Physiological measurements demonstrated a reduction in isometric contractile strength of the soleus and a reduction in cardiac left ventricular ejection fraction of TAZKD mice compared with control animals. Therefore, the inducible TAZ-deficient model exhibits some of the molecular and clinical characteristics of BTHS patients and may ultimately help to improve our understanding of BTHS-related cardioskeletal myopathy as well as serve as an important tool in developing therapeutic strategies for BTHS.