Single Gene Vector Research Articles

Development of a 10 g/L process for a difficult-to-express multispecific antibody format using a holistic process development approach

Ichnos has developed a multi-specific antibody platform based on the BEAT® (Bispecific engagement by antibodies based on the T-cell receptor) interface. The increased complexity of the bi- and multi-specific formats generated with this platform makes these molecules difficult-to-express proteins compared to standard monoclonal antibodies (mAbs). This report describes how expression limitations of a bi-specific bi-paratopic BEAT antibody were improved in a holistic approach. An initial investigation allowed identification of a misbalance in the subunits composing the BEAT antibody as the potential root cause. This misbalance was then addressed by a signal peptide optimization, and the overall expression level was increased by the combination of two vector design elements on a single gene vector. Further improvements were made in the selection of cell populations and an upstream (USP) platform process was applied in combination with a cell culture temperature shift. This allowed titer levels of up to 6 g/L to be reached with these difficult-to-express proteins. Furthermore, a high-density seeding process was developed that allowed titers of around 11 g/L for the BEAT antibody, increasing the initial titer by a factor of 10. The approach was successfully applied to a tri-specific antibody with titer levels reaching 10 g/L. In summary, a platform process for difficult-to-express proteins was developed using molecular biology tools, cell line development, upstream process optimization and process intensification.

Modulating Oncolytic Adenovirus Immunotherapy by Driving Two Axes of the Immune System by Expressing 4-1BBL and CD40L.

Oncolytic viruses (OVs) can have utility for direct killing of cancer cells, but may also serve to activate the immune system against cancer cells. While viruses alone can serve as immune stimulators, there is great interest in arming OVs with genes encoding immune stimulatory proteins to amplify their effects. In this work, we have tested the efficacy of conditionally-replicating adenoviruses (CRAds) with and without selected immunostimulatory payloads, murine CD40L (mCD40L) or 4-1BBL (m4-1BBL), in an immune competent mouse model of melanoma. When CRAd657-m4-1BBL and CRAd657-mCD40L were injected into B16-hCAR murine melanoma tumors, both single vectors delayed tumor growth and prolong survival compared to empty CRAd657. However, combined injection of both CRAd-m4-1BBL and CRAd-mCD40L mediated significantly better control of tumor growth. All of the payloads increased immune cell infiltration into tumors and notably reduced expression of PD-1 exhaustion marker on T cells. Tumor volumes were negatively associated with total infiltrating T cell population. We found that the payloads increased immune cell infiltration into tumors with some specificities: recruitment of CD8+ T cells was higher with m4-1BBL expression, while mCD40L expression induced more CD4+ T cell infiltration. Importantly, the combination of CRAd657-m4-1BBL and CRAd657-mCD40L induced the highest immune cells/T cell infiltration and the highest anti-TRP-2 tumor-associated antigen T cell responses than empty or single gene vector. This combination also caused depigmentation in areas adjacent to the tumor sites in more animals. These data indicate that driving two axes of the immune system with combined immune stimulatory payloads can lead to improved anticancer immune responses and better tumor control in an immune competent model of cancer.

Construction and Functional Analysis of CRISPR/Cas9 Vector of FAD2 Gene Family in Soybean

Soybean oleic acid content is one of the important indexes to evaluate the quality of soybean oil. In the synthesis pathway of soybean fatty acids, the FAD2 gene family is the key gene that regulates the production of linoleic acid from soybean oleic acid. In this study, CRISPR/Cas9 gene editing technology was used to regulate FAD2 gene expression. Firstly, the CRISPR/Cas9 single knockout vectors GmFAD2-1B and GmFAD2-2C and double knockout vectors GmFAD2-2A-3 were constructed. Then, the three vectors were transferred into the recipient soybean variety Jinong 38 by Agrobacterium-mediated cotyledon node transformation, and the mutant plants were obtained. Functional analysis and comparison of the mutant plants of the T2 and T3 generations were carried out. The results showed that there was no significant difference in agronomic traits between the CRISPR/Cas9 single and double knockout vectors and the untransformed CRISPR/Cas9 receptor varieties. The oleic acid content of the plants that knocked out the CRISPR/Cas9 double gene vector was significantly higher than that of the single gene vector.

Control of Multigene Expression Stoichiometry in Mammalian Cells Using Synthetic Promoters.

To successfully engineer mammalian cells for a desired purpose, multiple recombinant genes are required to be coexpressed at a specific and optimal ratio. In this study, we hypothesized that synthetic promoters varying in transcriptional activity could be used to create single multigene expression vectors coexpressing recombinant genes at a predictable relative stoichiometry. A library of 27 multigene constructs was created comprising three discrete fluorescent reporter gene transcriptional units in fixed series, each under the control of either a relatively low, medium, or high transcriptional strength synthetic promoter in every possible combination. Expression of each reporter gene was determined by absolute quantitation qRT-PCR in CHO cells. The synthetic promoters did generally function as designed within a multigene vector context; however, significant divergences from predicted promoter-mediated transcriptional activity were observed. First, expression of all three genes within a multigene vector was repressed at varying levels relative to coexpression of identical reporter genes on separate single gene vectors at equivalent gene copies. Second, gene positional effects were evident across all constructs where expression of the reporter genes in positions 2 and 3 was generally reduced relative to position 1. Finally, after accounting for general repression, synthetic promoter transcriptional activity within a local multigene vector format deviated from that expected. Taken together, our data reveal that mammalian synthetic promoters can be employed in vectors to mediate expression of multiple genes at predictable relative stoichiometries. However, empirical validation of functional performance is a necessary prerequisite, as vector and promoter design features can significantly impact performance.

Gene therapy for Cystic Fibrosis: Improved delivery techniques and conditioning with lysophosphatidylcholine enhance lentiviral gene transfer in mouse lung airways

ABSTRACT Purpose/Aim: Cystic fibrosis (CF) is the most common, fatal recessive genetic disease among the Caucasian population. Gene therapy has the potential to treat CF long term, however physiological barriers can prevent VSV-G pseudotyped lentiviral (LV) vectors from efficiently accessing the relevant receptors on the basolateral membrane of airway epithelial cells. The aims of this experiment were to use our new dose delivery techniques to determine whether conditioning the mouse lung conducting airways with lysophosphatidylcholine (LPC) improves the level of airway gene expression. Materials and Methods: Anaesthetised normal C57Bl/6 mice were intubated with an endotracheal cannula to non-invasively facilitate airway access. The airways were conditioned with 0.1% LPC, 0.3% LPC, or PBS (control) instilled via the ET tube. One hour later a VSV-G pseudotyped LV vector carrying the LacZ transgene was delivered. LacZ expression was measured by X-gal staining of the excised lungs 3 months after gene delivery. Results: Endotracheal intubation enabled precise dose delivery to the trachea and conducting airways. The cartilaginous airways of the groups conditioned with 0.1% and 0.3% LPC contained significantly larger numbers of LacZ positive cells compared to the PBS control group. In the LPC conditioned groups the majority of cell transduction was in ciliated epithelial cells. Conclusion: LPC conditioning prior to LV vector delivery, substantially enhanced the level of conducting airway gene expression after a single gene vector delivery. These results extend the previously established effectiveness of this protocol for producing gene expression in the nasal airways to the lung airways, the primary site of deleterious pathophysiology in CF individuals.

PS 01-23] SYNERGISTIC EFFECT OF A TISSUE KALLIKREIN 1 AND TISSUE INHIBITOR OF MATRIX METALLOPROTEINASE 1 CO—EXPRESSION VECTOR ON THE PROLIFERATION OF RAT VASCULAR SMOOTH MUSCLE CELLS

Objective: Tissue kallikrein 1 (TK1) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1) are important in inhibiting vascular smooth muscle cell (VSMC) proliferation and improving vascular remodeling, respectively. It was hypothesized that a combination of TK1 and TIMP1 genes, mediated by an adenovirus vector could augment or act in synergy to enhance the inhibitory effects. Design and Method: The promoter, mCMV carrying hTIMP1 cDNA was subcloned into pDC316—hTK1 to construct a recombinant plasmid carrying hTK1 and hTIMP1 genes. Subsequently, the double gene plasmid and adenovirus backbone plasmid were packaged into HEK293A cells. Gene transcription and protein expression were examined, respectively using reverse transcription—quantitative polymerase chain reaction (PCR) and western blotting assays. VSMC proliferation was assessed using cell counting and methyl—thiazolyl—tetrazoliuin methods. Results: The constructed plasmid containing hTK1 and hTIMP1 genes was correctly identified by means of PCR, double digestion and sequencing analysis. The co—expression vector, Ad—hTK1—hTIMP1 was successfully constructed and packaged into HEK293A cells. When VSMCs were transfected with the co—expression vector, the mRNA transcription and protein expression of hTK1 and hTIMP1 exhibited abundant expression in a concentration—dependent and time—dependent manner, independently. Conclusions: The co—expression vector synergistically inhibited the cell growth and proliferation induced by platelet—derived growth factor—BB compared with the single gene vector.

Synergistic effect of a tissue kallikrein 1 and tissue inhibitor of matrix metalloproteinase 1 co‑expression vector on the proliferation of rat vascular smooth muscle cells.

Tissue kallikrein 1 (TK1) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1) are important in inhibiting vascular smooth muscle cell (VSMC) proliferation and improving vascular remodeling, respectively. It was hypothesized that a combination of TK1 and TIMP1 genes, mediated by an adenovirus vector could augment or act in synergy to enhance the inhibitory effects. The promoter, mCMV carrying hTIMP1 cDNA was subcloned into pDC316-hTK1 to construct a recombinant plasmid carrying hTK1 and hTIMP1 genes. Subsequently, the double gene plasmid and adenovirus backbone plasmid were packaged into HEK293A cells. Gene transcription and protein expression were examined, respectively using reverse transcription-quantitative polymerase chain reaction (PCR) and western blotting assays. VSMC proliferation was assessed using cell counting and methyl-thiazolyl-tetrazoliuin methods. The constructed plasmid containing hTK1 and hTIMP1 genes was correctly identified by means of PCR, double digestion and sequencing analysis. The co-expression vector, Ad-hTK1-hTIMP1 was successfully constructed and packaged into HEK293A cells. When VSMCs were transfected with the co-expression vector, the mRNA transcription and protein expression of hTK1 and hTIMP1 exhibited abundant expression in a concentration-dependent and time-dependent manner, independently. In conclusion, the co-expression vector synergistically inhibited the cell growth and proliferation induced by platelet-derived growth factor-BB compared with the single gene vector.

MGMT enrichment and second gene co-expression in hematopoietic progenitor cells using separate or dual-gene lentiviral vectors

The DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) allows efficient in vivo enrichment of transduced hematopoietic stem cells (HSC). Thus, linking this selection strategy to therapeutic gene expression offers the potential to reconstitute diseased hematopoietic tissue with gene-corrected cells. However, different dual-gene expression vector strategies are limited by poor expression of one or both transgenes. To evaluate different co-expression strategies in the context of MGMT-mediated HSC enrichment, we compared selection and expression efficacies in cells cotransduced with separate single-gene MGMT and GFP lentivectors to those obtained with dual-gene vectors employing either encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or foot and mouth disease virus (FMDV) 2A elements for co-expression strategies. Each strategy was evaluated in vitro and in vivo using equivalent multiplicities of infection (MOI) to transduce 5-fluorouracil (5-FU) or Lin−Sca-1+c-kit+ (LSK)-enriched murine bone marrow cells (BMCs). The highest dual-gene expression (MGMT+GFP+) percentages were obtained with the FMDV-2A dual-gene vector, but half of the resulting gene products existed as fusion proteins. Following selection, dual-gene expression percentages in single-gene vector cotransduced and dual-gene vector transduced populations were similar. Equivalent MGMT expression levels were obtained with each strategy, but GFP expression levels derived from the IRES dual-gene vector were significantly lower. In mice, vector-insertion averages were similar among cells enriched after dual-gene vectors and those cotransduced with single-gene vectors. These data demonstrate the limitations and advantages of each strategy in the context of MGMT-mediated selection, and may provide insights into vector design with respect to a particular therapeutic gene or hematologic defect.

Suicide gene approach using a dual-expression lentiviral vector to enhance the safety of ex vivo gene therapy for bone repair

'Ex vivo' gene therapy using viral vectors to overexpress BMP-2 is shown to heal critical-sized bone defects in experimental animals. To increase its safety, we constructed a dual-expression lentiviral vector to overexpress BMP-2 or luciferase and an HSV1-tk analog, Δtk (LV-Δtk-T2A-BMP-2/Luc). We hypothesized that administering ganciclovir (GCV) will eliminate the transduced cells at the site of implantation. The vector-induced expression of BMP-2 and luciferase in a mouse stromal cell line (W-20-17 cells) and mouse bone marrow cells (MBMCs) was reduced by 50% compared with the single-gene vector. W-20-17 cells were more sensitive to GCV compared with MBMCs (90-95% cell death at 12 days with GCV at 1 μg ml(-1) in MBMCs vs 90-95% cell death at 5 days by 0.1 μg ml(-1) of GCV in W-20-17 cells). Implantation of LV-Δtk-T2A-BMP-2 transduced MBMCs healed a 2 mm femoral defect at 4 weeks. Early GCV treatment (days 0-14) postoperatively blocked bone formation confirming a biologic response. Delayed GCV treatment starting at day 14 for 2 or 4 weeks reduced the luciferase signal from LV-Δtk-T2A-Luc-transduced MBMCs, but the signal was not completely eliminated. These data suggest that this suicide gene strategy has potential for clinical use in the future, but will need to be optimized for increased efficiency.

Co-transduction of ribosomal protein L23 enhances the therapeutic efficacy of adenoviral-mediated p53 gene transfer in human gastric cancer

Induction of murine double minute 2 (MDM2) expression is thought to be a determinant of resistance to p53 gene therapy for cancer. Previous studies have revealed that ribosomal protein L23 (RPL23) inhibits MDM2-mediated p53 degradation through direct binding to MDM2. In addition, ectopically expressed RPL23 was reported to interact with MDM2 in both the nucleus and cytoplasm, by which RPL23 indirectly inhibited MDM2-p53 binding. Based on the known molecular properties of the RPL23 protein, it was speculated that co-transduction of RPL23 may protect wild‑type p53 protein from MDM2-mediated inactivation and, thus, improve the effect of delivering therapeutic exogenous p53. To test this hypothesis, we constructed a bicistronic adenoviral vector expressing both the RPL23 and p53 genes (Ad-RPL23/p53) and compared its tumor-suppressor activity in human gastric cancer with that of a single gene vector for p53 (Ad-p53). In the in vivo and in vitro experiments, we observed that treatment with Ad-RPL23/p53 resulted in a stronger antitumor response compared to that obtained using Ad-p53. Moreover, the antitumor response of the bicistronic adenovirus was obtained not only in MGC803 cells (endogenous mutant p53) but also in MKN45 cells (endogenous wild‑type p53) which were initially resistant to p53 gene transfer, indicating that co-transduction of RPL23 also expanded the utility of p53 gene therapy. Furthermore, in an orthotopic nude mouse model of human gastric cancer, we found that the survival benefit was greater after Ad-RPL23/p53 treatment than after Ad-p53. Taken together, the data presented here demonstrate that co-transduction of RPL23 enhances the therapeutic efficacy of adenoviral-mediated p53 gene transfer in models of human gastric cancer and support the use of this strategy for cancer treatment.

Acquiring transgenic tobacco plants with insect resistance and glyphosate tolerance by fusion gene transformation

The advantages of gene 'stacking' or 'pyramiding' are obvious in genetically modified (GM) crops, and several different multi-transgene-stacking methods are available. Using linker peptides for multiple gene transformation is considered to be a good method to meet a variety of needs. In our experiment, the Bt cry1Ah gene, which encodes the insect-resistance protein, and the mG ( 2 ) -epsps gene, which encodes the glyphosate-tolerance protein, were connected by a 2A or LP4/2A linker. Linker 2A is a peptide from the foot-and-mouth disease virus (FMDV) that has self-cleavage activity. LP4 is a peptide from Raphanus sativus seeds that has a recognition site and is cleaved by a protease. LP4/2A is a hybrid peptide that contains the first 9 amino acids of LP4 and 20 amino acids from 2A. We used the linker peptide to construct four coordinated expression vectors: pHAG, pHLAG, pGAH and pGLAH. Two single gene expression vectors, pSAh and pSmG(2), were used as controls. The six expression vectors and the pCAMBIA2301 vector were transferred into tobacco by Agrobacterium tumefaciens-mediated transformation, and 529 transformants were obtained. Molecular detection and bioassay detection data demonstrated that the transgenic tobaccos possessed good pest resistance and glyphosate tolerance. The two genes in the fusion vector were expressed simultaneously. The plants with the genes linked by the LP4/2A peptide showed better pest resistance and glyphosate tolerance than the plants with the genes linked by 2A. The expression level of the two genes linked by LP4/2A was not significantly different from the single gene vector. Key message The expression level of the two genes linked by LP4/2A was higher than those linked by 2A and was not significantly different from the single gene vector.

Enhanced apoptosis of glioma cell lines is achieved by co-delivering FasL-GFP and TRAIL with a complex Ad5 vector.

Brain tumors (BTs) are among the most malignant forms of human cancer. Unfortunately, current treatments are often ineffective and produce severe side effects. Cytotoxic gene therapy is an alternative treatment strategy, with the potential advantages of reduced toxicity to normal brain tissue. Apoptosis-inducing "death ligands" Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) are genes with substantial cytotoxic activity in susceptible tumor cells. Here, we compared the effectiveness of Ad vector-mediated delivery of Fas ligand-green fluorescent protein (FasL-GFP) fusion protein, human TRAIL, and both genes simultaneously. We examined a panel of 13 cell lines (eight derived from primary isolates) for susceptibility to Ad5-based vector infection and for sensitivity to FasL- and TRAIL-mediated apoptosis. All cell lines were efficiently transduced, but, as expected, varied in their sensitivity to ligand-induced apoptosis. Generally, sensitivity to FasL-GFP correlated with cell surface FasR levels, but no such correlation was seen for TRAIL and its functional receptors, DR4 and DR5. The vector expressing both FasL-GFP and TRAIL was more effective than either of the single-gene vectors at comparable transduction levels, and it was effective against a broader range of cell lines. In five cell lines, coexpression resulted in apoptosis levels greater than those predicted for strictly additive activity of the two death ligands. We believe that Ad vector-mediated delivery of multiple death ligands may be developed as a potential BT therapy, either alone or in conjunction with surgical resection of the primary tumor.

Development of multigene and regulated lentivirus vectors.

Previously we described safe and efficient three-component human immunodeficiency virus type 1 (HIV-1)-based gene transfer systems for delivery of genes into nondividing cells (H. Mochizuki, J. P. Schwartz, K. Tanaka, R. O. Brady, and J. Reiser, J. Virol. 72:8873-8883, 1998). To apply such vectors in anti-HIV gene therapy strategies and to express multiple proteins in single target cells, we have engineered HIV-1 vectors for the concurrent expression of multiple transgenes. Single-gene vectors, bicistronic vectors, and multigene vectors expressing up to three exogenous genes under the control of two or three different transcriptional units, placed within the viral gag-pol coding region and/or the viral nef and env genes, were designed. The genes encoding the enhanced version of green fluorescent protein (EGFP), mouse heat-stable antigen (HSA), and bacterial neomycin phosphotransferase were used as models whose expression was detected by fluorescence-activated cell sorting, fluorescence microscopy, and G418 selection. Coexpression of these reporter genes in contact-inhibited primary human skin fibroblasts (HSFs) persisted for at least 6 weeks in culture. Coexpression of the HSA and EGFP reporter genes was also achieved following cotransduction of target cells using two separate lentivirus vectors encoding HSA and EGFP, respectively. For the regulated expression of transgenes, tetracycline (Tet)-regulatable lentivirus vectors encoding the reverse Tet transactivator (rtTA) and EGFP controlled by a Tet-responsive element (TRE) were constructed. A binary HIV-1-based vector system consisting of a lentivirus encoding rtTA and a second lentivirus harboring a TRE driving the EGFP reporter gene was also designed. Doxycycline-modulated expression of the EGFP transgene was confirmed in transduced primary HSFs. These versatile vectors can potentially be used in a wide range of gene therapy applications.

The beta-galactosidase gene as a marker for hematopoietic reconstitution: individual cell analysis in a murine bone marrow transplantation model.

We have used a simple, single-gene retrovirus carrying the Escherichia coli beta-galactosidase reporter gene (lacZ), termed LlacZ. This virus was found to infect immortalized myeloid and lymphoid precursor/leukemic cell lines efficiently as well as primary murine bone marrow clonogenic progenitors, without apparent modulation of growth or phenotype. Following infection of bone marrow cells, a significant proportion of progenitors--36% of lineage-negative cells with low levels of c-kit expression (lin-/c-kit(lo)) known to be enriched with pluripotent hemopoietic stem cells, and 19% of Sca1-positive cells known to be enriched with transplantable cells with lymphomyeloid-reconstituting ability--were shown to express lacZ. Use of an LlacZ-infected population of post 5-fluorouracil bone marrow cells to reconstitute lethally irradiated mice demonstrated the presence of lacZ-expressing cells in the spleen at day 12 post-transplantation with provirus detected in individual spleen colonies (CFU-S). In the long term (3-6 months following transplantation), lacZ expression was detected in hematopoietic tissues of all recipient mice. The use of two-color in situ and flow cytometry analysis combined with lineage-specific antibodies showed lacZ expression in both myeloid and lymphoid cells in spleen and bone marrow. In addition, lacZ-expressing cells were detected in secondary recipient mice injected with bone marrow cells derived from primary LlacZ recipients. Overall, these data show the efficacy of a single gene vector for stem cell transduction, the utility of beta-galactosidase as a single cell marker for stem cell transduction and reconstitution ability, and the need for protocol optimization to see high-level multilineage gene expression.

Coexpression of cytidine deaminase and mutant dihydrofolate reductase by a bicistronic retroviral vector confers resistance to cytosine arabinoside and methotrexate.

The transfer of a drug resistance gene into hematopoietic cells is an approach being investigated to overcome the problem of myelosuppression produced by anticancer drugs. Chemotherapeutic agents are often given in combination in order to increase their effectiveness. Consequently, there is an advantage in designing vectors for gene transfer that are capable of expressing two drug resistance genes. We have constructed a bicistronic retroviral vector, MFG-DHFR-IRES/CD, which contains the mutated human dihydrofolate reductase (DHFR) cDNA with a phenylalanine-to-serine substitution at codon 31 (F31S) and the human cytidine deaminase (CD) cDNA. Murine fibroblast and hematopoietic cells were transduced with this vector and evaluated for their resistance to methotrexate (MTX) and cytosine arabinoside (ARA-C). The transduced fibroblast cells showed high levels of resistance to MTX and to ARA-C as determined by a clonogenic assay. Using enzymatic assays, we observed a coordinate increase in resistance to MTX and DHFR enzyme activity following an ARA-C selection. In addition, MTX selection produced an increase in CD enzyme activity and ARA-C resistance. Murine hematopoietic cells transduced with the bicistronic vector also showed drug resistance to both MTX and ARA-C. Interestingly, the double-gene construct conferred an equivalent level of drug resistance compared with single-gene vectors bearing only CD or DHFR genes in the hematopoietic cells. These results demonstrate the potential of the MFG-DHFR-IRES/CD vector to confer drug resistance to both MTX and ARA-C and may have future application in chemoprotection of normal hematopoietic cells in patients with cancer.