Transgene mRNA Levels Research Articles

Enhanced CHO Cell-Based Transient Gene Expression with the Epi-CHO Expression System

Transient gene expression systems in mammalian cells continue to grow in popularity due to their capacity to produce significant amounts of recombinant protein in a rapid and scalable manner, without the lengthy time periods and resources required for stable cell line development. Traditionally, production of recombinant monoclonal antibodies for pre-clinical assessment by transient expression in CHO cells has been hampered by low titers. In this report, we demonstrate transient monoclonal antibody titers of 140mg/l with CHO cells using the episomal-based transient expression system, Epi-CHO. Such titers were achieved by implementing an optimized transfection protocol incorporating mild-hypothermia and through screening of a variety of chemically defined and serum-free media for their ability to support elevated and prolonged viable cell densities post-transfection, and in turn, improve recombinant protein yields. Further evidence supporting Epi-CHO's capacity to enhance transgene expression is provided, where we demonstrate higher transgene mRNA and protein levels of two monoclonal antibodies and a destabilized enhanced green fluorescent protein with Epi-CHO compared to cell lines deficient in plasmid DNA replication and/or retention post-transfection. The results demonstrate the Epi-CHO system's capacity for the rapid production of CHO cell-derived recombinant monoclonal antibodies in serum-free conditions.

Valproic acid enhances recombinant mRNA and protein levels in transiently transfected Chinese hamster ovary cells

Valproic acid (VPA) is a small molecule that inhibits histone deacetylase activity. Here we report that VPA increases recombinant mRNA and protein levels in transiently transfected CHO DG44 cells. In the presence of VPA, transient recombinant antibody yields of up to 40 mg/L were achieved in simple batch cultures. The steady-state levels of the IgG light and heavy chain mRNAs were nearly 10 times higher than in the untreated control transfection even though the level of transfected plasmid DNA was the same in the presence or absence of VPA. The combination of VPA treatment and incubation of the transfected cells in mildly hypothermic conditions resulted in recombinant antibody yields of over 90 mg/L by 6 days post-transfection in batch cultures. The results demonstrated that the treatment of transfected CHO DG44 cells with VPA is a cost-effective strategy for enhancing transient gene expression by increasing the transgene mRNA levels.

Mice with podocyte-specific overexpression of wild type α-actinin-4 are healthy controls for K256E-α-actinin-4 mutant transgenic mice

Mutations in the gene ACTN4 encoding the actin bundling protein-alpha-actinin-4 underlie an inherited form of kidney lesions known as focal segmental glomerulosclerosis (FSGS). Previously, we developed a model for this condition by generating mice with podocyte-specific overexpression of a disease-causing mutant alpha-actinin-4 (K256E-ACTN4 (pod+)). However, whether alpha-actinin-4 overexpression artifacts and not the gain of affinity effects of the mutation accounted for the robust FSGS phenotype in these mice was unclear. To address this question, we developed a control line of mice with podocyte-specific overexpression of wildtype alpha-actinin-4 (wt-ACTN4 (pod+)). An 8.3 kb fragment of the mouse nephrin promoter (NPHS1) was used to drive expression of a hemagglutinin (HA)-tagged wildtype alpha-actinin-4 coding sequence in mice. Five founder lines expressing the HA-tagged alpha-actinin-4 protein in a podocyte-specific manner were obtained, as determined by co-immunofluorescence with HA and synaptopodin antibodies. Quantitative PCR revealed that renal transgene mRNA levels of wt-ACTN4 (pod+) mice are similar to K256E-ACTN4 (pod+) mice. In contrast to K256E-ACTN4 (pod+) mice which exhibit albuminuria, podocyte foot process effacement and glomerular scarring, wt-ACTN4 (pod+) mice are healthy and indistinguishable from non-transgenic littermates. These findings suggest that the K256E mutation itself and not overexpression of alpha-actinin-4 protein per se accounts for the FSGS phenotype in our transgenic model.

Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines

SUMMARYReplicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor (TLR) 3 is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DC). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid based DNA vaccines. We show that mouse CD8α+ DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent fashion by dsRNA present within those cells. However, we find that cytotoxic T cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

Generating stable, high-producing cell lines for recombinant protein production requires an understanding of the potential limitations in the cellular machinery for protein expression. In order to increase our understanding of what makes a stable high producer, we have generated a panel of 17 recombinant monoclonal antibody expressing Chinese hamster ovary subclones (CHO-mAb) with specific productivities ranging between 3 and 75 pg cell(-1) day(-1) using the dihydrofolate reductase (dhfr) expression system and compared the molecular features of these high- and low-producer clones. The relative heavy chain (HC) and light chain (LC) transgene copy numbers and mRNA levels were determined using real-time quantitative PCR (RT qPCR). We observed that not only higher transgene copy numbers and mRNA levels of both HC and LC were characteristic for the high-producer clones as compared to the low-producer clones but also a more favorable HC to LC transgene copy numbers ratio. By studying the long-term stability of the CHO-mAb subclones in the absence of methotrexate (MTX) selective pressure over 36 passages we observed a 35-92% decrease in volumetric productivity, primarily caused by a significant decrease in HC and LC mRNA levels with little change in the transgene copy numbers. Using Southern blot hybridization we analyzed the HC and LC transgene integration patterns in the host chromosome and their changes in course of gene amplification and long-term culturing. We observed that MTX-induced gene amplification caused chromosomal rearrangements resulting in clonal variability in regards to growth, productivity, and stability. No further obvious DNA rearrangements occurred during long-term culturing in the absence of MTX, indicating that other mechanisms were responsible for the decreased transcription efficiency. Our results implicate that the amplified transgene sequences were arranged in tandem repeats potentially triggering repeat-induced gene silencing. We hypothesize that the decline in transgene mRNA levels upon long-term culturing without MTX was mainly caused by transgene silencing consequently leading to a loss in mAb productivity. The exact molecular mechanisms causing production instability are not yet fully understood. The herein described extensive characterization studies could help understand the limitations to high-level, stable recombinant protein production and find ways to improving and accelerating the process for high-producer cell line generation and selection.

Mild Hypothermia Improves Transient Gene Expression Yields Several Fold in Chinese Hamster Ovary Cells

Large-scale transient gene expression (TGE) in mammalian cells is a rapid method to generate recombinant proteins, but the volumetric productivity for secreted proteins is still more than an order of magnitude lower than the yields typically achieved with recombinant cell lines. Here transient recombinant protein production in Chinese hamster ovary cells transfected with linear 25 kDa polyethylenimine was significantly enhanced by incubation of the cells at temperatures ranging from 29 to 33 degrees C after DNA delivery. With this approach, transient recombinant antibody yields of 60-80 mg/L were achieved within 6 days of transfection. The increase in TGE correlated with the accumulation of cells in the G1 phase of the cell cycle, increased cell size, higher cell viability, higher steady-state levels of transgene mRNA, reduced consumption of nutrients, and decreased accumulation of waste products. The enhancement of TGE was not vector-dependent, but the presence of the woodchuck hepatitis virus post-transcriptional regulatory element in the 3' untranslated region of the transgene mRNA increased transient recombinant antibody expression more than 3-fold at 31 degrees C as compared to expression at 37 degrees C. The yields achieved by the low-temperature enhancement of TGE in CHO cells makes this technology feasible for the rapid production of gram amounts of secreted recombinant proteins at large scale (up to 100 L).

Enhancement of post-transcriptional gene silencing by grafting

We showed previously that grafting transmitted silencing occurred when transgenic ACC oxidase 1 (ACO1) overexpressing tomato plants that also produced siRNAs were grafted onto transgenic stocks that already showed strong silencing. The presence of siRNAs in these overexpressing scions may indicate that silencing, though inefficient, may already occur at a low level before grafting. To test if a silencing state with a relatively high level of target mRNA can be shifted towards further more effective silencing, we grafted an ACO1 antisense (AS) line with a high level of antisense ACO1 transgene mRNA and low level of siRNAs to the ACO1 strong silencer stock. The AS mRNA level was reduced dramatically two weeks after grafting. More interestingly, self-grafting of ACO1 overexpressers and AS lines also induced strong silencing in the scions. We suggest that grafting transmitted silencing may involve the switching from an inefficient or weak silencing state to a stronger silencing by a systemic silencing signal, similar to the change of silencing states that sometimes occurs during development. Control experiments using non-transgenic stocks designed to test whether wounding alone is responsible for generating a signal that enhances silencing in transgenic scions gave negative results. We propose that the build-up of silencing signal and / or molecules at both sides of the grafting junction and their sudden release when the phloem is reconnected may be critical to grafting transmitted silencing.

Comparison of lentiviral vector titration methods

BackgroundLentiviral vectors are efficient vehicles for stable gene transfer in dividing and non-dividing cells. Several improvements in vector design to increase biosafety and transgene expression, have led to the approval of these vectors for use in clinical studies. Methods are required to analyze the quality of lentiviral vector production, the efficiency of gene transfer and the extent of therapeutic gene expression.ResultsWe compared lentiviral vector titration methods that measure pg p24/ml, RNA equivalents/ml, transducing units (TU/ml) or mRNA equivalents. The amount of genomic RNA in vector particles proves to be reliable to assess the production quality of vectors encoding non-fluorescent proteins. However, the RNA and p24 titers of concentrated vectors are rather poor in predicting transduction efficiency, due to the high variability of vector production based on transient transfection. Moreover, we demonstrate that transgenic mRNA levels correlate well with TU and can be used for functional titration of non-fluorescent transgenes.ConclusionThe different titration methods have specific advantages and disadvantages. Depending on the experimental set-up one titration method should be preferred over the others.

Inflammatory cytokine regulation of transgene expression in human fibroblast‐like synoviocytes infected with adeno‐associated virus

An ideal gene transfer vector for chronic inflammatory diseases such as rheumatoid arthritis (RA) would provide local transgene expression only when the disease is active. To determine whether adeno-associated virus (AAV) possesses this ability, the effects of inflammatory cytokines on transgene expression were evaluated in human RA fibroblast-like synoviocytes (FLS). Human FLS were infected with AAV in the presence or absence of inflammatory cytokines or synovial fluid obtained from patients with RA. Transgene expression was monitored by either enzyme-linked immunosorbent assay or flow cytometry. Transgene messenger RNA (mRNA) was measured by real-time quantitative reverse transcription-polymerase chain reaction. Inflammatory cytokines increased transgene expression in FLS by up to 60-fold. Synovial fluid from patients with RA, but not from patients without arthritis, was also able to increase expression in synoviocytes. Protein expression correlated with transgene mRNA levels. The enhanced expression required the continued presence of cytokines because, upon removal, transgene expression returned to baseline levels. Expression could be repeatedly reinduced by reexposure to cytokines. The effect was not promoter specific and was demonstrated to be phosphatidylinositol 3-kinase-dependent. These results suggest that expression of a therapeutic transgene can be controlled by the presence of inflammation following AAV gene transfer, making it an attractive vector for chronic inflammatory diseases such as RA.

Transgenic mice with osteoblast-targeted insulin-like growth factor-I show increased bone remodeling

To determine the effects of locally-expressed insulin-like growth factor (IGF-I) on bone remodeling, a transgene was produced in which murine IGF-I cDNA was cloned downstream of a gene fragment comprising 3.6 kb of 5′ upstream regulatory sequence and most of the first intron of the rat Col1a1 gene. The construct was expressed at the mRNA and protein level in transfected osteoblasts. Five lines of transgenic mice were generated by embryo microinjection. Transgene mRNA levels were highest in calvaria, long bone and tendon, and lower in skin. Serum IGF-I and body weight were increased in males and females only in the highest expressing line. Histomorphometry showed that transgenic calvaria were wider and had greater marrow area and bone area. Transgenic calvaria had increased osteoclast number per bone surface. Percent collagen synthesis and cell replication were increased in transgenic calvaria. Femur length, cortical width and cross-sectional area were increased in transgenic femurs of the highest expressing line, while femoral trabecular bone volume was little affected. Thus, broad overexpression of IGF-I in cells of the osteoblast lineage increased indices of bone formation and resorption.

P1- and VPg-transgenic plants show similar resistance to Potato virus A and may compromise long distance movement of the virus in plant sections expressing RNA silencing-based resistance

Nicotiana benthamiana was transformed with P1 or VPg cistron of Potato virus A (PVA, genus Potyvirus). For both transgenes, T1 progeny displayed (i) resistance to PVA infection, (ii) susceptibility, or (iii) systemic infection followed by recovery of new leaves from PVA infection (RC), regardless of the transgene. In RC plants, fully recovered leaves contained no detectable PVA RNA, were highly resistant to challenge inoculation with PVA, and had barely detectable steady-state levels of transgene mRNA; transgene-homologous siRNA was not detected, in contrast to leaves undergoing recovery. Tops in RC plants and PVA-susceptible transgenic plants were replaced with scions from wild-type plants; only scions on the latter became PVA-infected. These findings suggest that vascular movement of PVA from lower, infected parts of RC plants was compromised in the recovered section expressing RNA silencing-based resistance, which adds a novel dimension to the current models for potyvirus movement.

Bean Yellow Dwarf Virus replicons for high‐level transgene expression in transgenic plants and cell cultures

A novel stable transgenic plant expression system was developed using elements of the replication machinery of Bean Yellow Dwarf Virus (BeYDV). The system contains two transgenes: 1) The BeYDV replicon vector with an expression cassette flanked by cis-acting DNA elements of BeYDV, and 2) The viral replication initiator protein (Rep) controlled by an alcohol-inducible promoter. When Rep expression was triggered by treatment with ethanol, it induced release of the BeYDV replicon from stably integrated T-DNA and episomal replication to high copy number. Replicon amplification resulted in substantially increased transgene mRNA levels (up to 80-fold) and translation products (up to 10-fold) after induction of Rep expression by ethanol treatment in tobacco NT1 cells and leaves of whole potato plants. Thus, the BeYDV stable transformant replicon system is a powerful tool for plant-based production of recombinant proteins.

Characterization of resistance in transgenic Nicotiana benthamiana encoding N-terminal deletion and assembly mutants of the tobacco etch potyvirus coat protein

The resistance of transgenic Nicotiana benthamiana plants encoding wild type, truncated and point mutants of the tobacco etch virus (TEV) coat protein (CP) was analyzed. After R1 plants from 45 transgenic lines were challenged with TEV, six percent of the lines exhibited high resistance, 38% exhibited low resistance, and the remainder were susceptible. The phenomenon of recovery and delay in symptom development was observed in 65% and 56% of the resistant and susceptible lines, respectively. Plants containing genes that encode sequences of two assembly-deficient mutants of TEV-CPDelta1-63 exhibited resistance to infection, suggesting that self-assembly of the CP is not responsible for resistance. Highly resistant lines accumulated low levels of transgene mRNA and non-detectable amounts of protein, and tissues accumulated lower amounts of transgene mRNA following recovery than before infection. In addition, co-suppression of replication of a recombinant tobamovirus containing the TEV-CPDelta1-63 sequence was observed in several lines, suggesting homology-dependent degradation of RNA, most likely through induction of post-transcriptional gene silencing. Plants not exhibiting high resistance via gene silencing exhibited moderate levels of resistance that is attributed to and/or affected by the CP molecule.

946. CD8+ T Cells Are Partially Mediate the Immune-Mediated Silencing of Adenoviral Vector-Encoded Transgene Expression in the Rat Brain

First generation adenoviral vectors (RAd) transfer genes into the brain with high efficiency, and provide high levels, long term expression, and therapeutic efficacy in models of brain tumors and neurodegeneration, in the absence of priming of an anti-adenoviral immune response. Systemic immunization against RAd, however, induces a cellular adaptive immune response which completely eliminates the expression of the transgene from the brain, and causes a long-term inflammatory response. To elucidate the mechanisms by which the immune response clears transgene expression from the brain, we examined, following the delivery of RAds into the striatum, and systemic immune priming, whether the immune mediated inhibition of transgene expression causes brain neurotoxicity, reduces the levels of transgene transcripts, reduces vector genome copy numbers, and is mediated by CD8+ T cells. Our experiments provided the following results: (1) at 7 and 14 days post-immunization there was an infiltration of lymphocytes and macrophages surrounding transduced cells; (2) macrophages containing remains of transduced cells could be identified indicating the existence of cytotoxicity of transduced cells; (3) there was neuronal loss in the substantia nigra, but not in the striatum; (4) transgene mRNA levels were reduced by more than 90%, but genome copy numbers were only reduced by 10-20%; (5) the depletion of CD8+ T cells using the monoclonal antibody OX8, reduced circulating CD8+ T cells by >90% and partially protected against the immunization-induced silencing of transgene expression. In summary, our data indicate that immune-mediated silencing of RAd-encoded transgene expression in the brain occurs through a combination of non-cytolytic, and, also cytotoxic mechanisms. The percentage contribution of each mechanism is currently being evaluated.

Proteasome Inhibition Enhances AAV-Mediated Transgene Expression in Human Synoviocytes in Vitro and in Vivo

To explore the potential applicability of recombinant adeno-associated virus (rAAV) vectors in the treatment of rheumatoid arthritis (RA), primary human fibroblast-like synoviocytes (FLS) derived from patients with RA were infected with rAAV encoding mouse IL-10 under the control of the CMV promoter. Addition of the proteasome inhibitor carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (zLLL) to the cultures dramatically enhanced expression of the IL-10 transgene, in a dose-dependent manner. The increased expression was transient, peaking at 3 days and returning to near baseline by 7 days. The enhancement was observed even when zLLL was added 13 days after infection with rAAV. The effect of zLLL was not specific to either the mIL-10 transgene or the CMV promoter, as similar findings were observed using an rAAV construct encoding alpha1-anti-trypsin under the control of the chick beta-actin promoter or GFP, driven by the CMV promoter. Transgene expression could be repeatedly induced by reexposure to zLLL. Transgene mRNA levels increased in parallel with protein levels. Transgene expression could also be repeatedly induced in vivo by administering zLLL to SCID mice previously injected with rAAV-infected FLS. These data demonstrate that proteasome inhibition can dramatically enhance transgene expression in human RA FLS following infection with rAAV and suggest a possible approach to regulating synovial transgene expression in vivo.

Factors limiting autogene‐based cytoplasmic expression systems

The relatively low levels of transfection that can be achieved by current gene delivery systems have limited the therapeutic utility of gene transfer. This is especially true for non-viral gene delivery systems, where the levels of gene expression achieved are usually well below the levels achieved by viral gene transfer systems. Previous work from our laboratory describes an enhanced dual promoter autogene-based cytoplasmic expression system that gives rise to levels of gene expression 20-fold higher than that of a CMV nuclear expression plasmid control. Here various strategies are described to increase the levels of autogene-based gene expression by changing variables such as the type of nuclear promoter, phage RNAP gene, and IRES element. Although insights into the function of various IRES elements were gained, none of these changes demonstrated a significant increase in gene expression. However, determination of the mRNA levels achieved using quantitative RNase protection assays and immunofluorescence experiments revealed that transgene mRNA levels were saturated at up to 10 times higher than all other mRNA in the transfected cell combined. It follows that mRNA production, as well as translation, are important factors limiting autogene-based cytoplasmic expression.

Lentivirus-mediated gene transfer to human epidermis.

For long-term cutaneous gene therapy, the therapeutic gene must be targeted to stem cells and be stably transmitted to and expressed in descendant cells. Retroviral vectors are highly efficient in gene transfer to human keratinocyte stem cells in culture; however, they cannot transduce quiescent stem cells in vivo. As lentiviral vectors (LVV) transduce non-proliferating cells, their ability to target human epidermal stem cells was evaluated. LVV were highly efficient in gene transfer to clonogenic keratinocytes in vitro. Despite higher transgene DNA content and comparable levels of transgene mRNA, levels of transgene product directed by lentivectors were 3-folds lower than that of retrovectors. When transduced keratinocytes were grafted onto mice, transgene expression persisted for at least 20 wk; however, transgene product was detected primarily in the uppermost layers of epidermis. Inclusion of an element that is known to facilitate nuclear export of intron-less transcripts, resulted in enhanced transgene expression in keratinocytes. In vivo transduction of xenografted human skin with these vectors resulted in efficient gene transfer to epidermal progenitor cells. These results demonstrate stem cell transduction by LVV and point out the utility of using these vectors for direct gene transfer to and sustained expression in human epidermis.

A high-throughput method for quantifying transgene expression in transformed plants with real-time PCR analysis

When analysing plant transformation in plant transgenic lines, determining the level of transgene expression is essential. Northern blot analysis and reverse-transcription polymerase chain reaction (RT-PCR) are currently used for this purpose and enable qualitative and semiquantitative estimation of transgene mRNA levels. We have introduced a real-time PCR method for quantitative determination of transgene expression level in transgenic potato plants containing the gene for coat protein (CP) of potato virus Y strain NTN (PVYNTN) in order to provide a reliable, high-throughput method that could successfully replace the Northern blot analysis. The method has been compared with other available methods for gene expression analysis with respect to accuracy, sensitivity, specificity, and throughput. The effectiveness of the real-time PCR assay was confirmed on transgenic tobacco plants. It proved to be accurate, sensitive rapid, and sufficiently reproducible for further application in high-throughput molecular characterisation of transgenic lines. In addition, the described assay enables detection of the virus at increased sensitivity and reproducibility and is therefore appropriate for use in routine PVYNTN detection.

314. mRNA Production Is the Major Factor Limiting Autogene-Based Cytoplasmic Expression

The relatively low levels of transfection that can be achieved by current gene delivery systems have limited the therapeutic utility of gene transfer. This is especially true for non-viral gene delivery systems, where the levels of gene expression achieved are usually well below the levels achieved by viral gene transfer systems. Previous work from our laboratory describes an enhanced dual promoter autogene-based cytoplasmic expression system that gives rise to levels of gene expression 20 fold higher than that of a CMV nuclear expression plasmid control. Here we describe various strategies to increase the levels of autogene based gene expression by changing variables such as the type of nuclear promoter, phage RNAP gene, and IRES sequence. It was found that none of these changes demonstrated a significant increase in gene expression. However determination of the mRNA levels achieved using quantitative RNase protection assays and immunofluorescence experiments, revealed transgene mRNA levels up to 10 times higher than all other mRNA in the transfected cell combined. It follows that mRNA production is a major factor limiting autogene based cytoplasmic expression.

Factors Binding a Non-classical Cis-element Prevent Heterochromatin Effects on Locus Control Region Activity

A locus control region (LCR) is a cis-acting gene-regulatory element capable of transferring the expression characteristics of its gene locus of origin to a linked transgene. Furthermore, it can do this independently of the site of integration in the genome of transgenic mice. Although most LCRs contain subelements with classical transcriptional enhancer function, key aspects of LCR activity are supported by cis-acting sequences devoid of the ability to act as direct transcriptional enhancers. Very few of these "non-enhancer" LCR components have been characterized. Consequently, the sequence requirements and molecular bases for their functions, as well as their roles in LCR activity, are poorly understood. We have investigated these questions using the LCR from the mouse T cell receptor (TCR) alpha/Dad1 gene locus. Here we focus on DNase hypersensitive site (HS) 6 of the TCRalpha LCR. HS6 does not support classical enhancer activity, yet has gene regulatory activity in an in vivo chromatin context. We have identified three in vivo occupied factor-binding sites within HS6, two of which interact with Runx1 and Elf-1 factors. Deletion of these sites from the LCR impairs its activity in vivo. This mutation renders the transgene locus abnormally inaccessible in chromatin, preventing the normal function of other LCR subelements and reducing transgene mRNA levels. These data show these factor-binding sites are required for preventing heterochromatin formation and indicate that they function to maintain an active TCRalpha LCR assembly in vivo.