Development of Genetically Modified Chinese Hamster Ovary Host Cells for the Enhancement of Recombinant Tissue Plasminogen Activator Expression

Background: Chinese hamster ovary (CHO) cells are the most commonly used host system for the expression of high quality recombinant proteins. However, the development of stable, high-yielding CHO cell lines is a major bottleneck in the industrial manufacturing of therapeutic proteins. Therefore, different strategies such as the generation of more efficient expression vectors and establishment of genetically engineered host cells have been employed to increase the efficiency of cell line development. In order to examine the possibility of generating improved CHO host cells, cell line engineering approaches were developed based on ceramide transfer protein (CERT), and X-box binding protein 1s (XBP1s). Methods: CHO cells were transfected with CERT S132A, a mutant variant of CERT which is resistant to phosphorylation, or XBP1s expression plasmids, and then stable cell pools were generated. Transient expression of t-PA was examined in engineered cell pools in comparison to un-modified CHO host cells. Results: Overexpression of CERT S132A led to the enhancement of recombinant tissue plasminogen activator (t-PA) expression in transient expression by 50%. On the other hand, it was observed that the ectopic expression of the XBP1s, did not improve the t-PA expression level. Conclusion: The results obtained in this study indicate successful development of the improved CHO host cells through CERT S132A overexpression.

Effects of peptone supplementation in different culture media on growth, metabolic pathway and productivity of CHO DG44 cells; a new insight into amino acid profiles

The optimization of bioprocess conditions towards improved growth profile and productivity yield is considered of great importance in biopharmaceutical manufacturing. Peptones as efficient sources of nutrients have been studied for their effect on media development; however, their role on metabolic pathway is not well understood. In the present study, the effect of different concentration of peptones on a recombinant Chinese hamster ovary [CHO] cell line grown in three serum-free suspension cultures was determined. Six peptones of different origins and available amino acid profiles were investigated regarding their impact on cell growth, productivity, and metabolic pathways changes. In optimized feeding strategies, increases of 136% and 159% in volumetric productivity [for a low-nutrient culture media] and 55% [for a high-nutrient culture media] were achieved. Furthermore, particular sources of peptones with specific amino acid profile developed preferential results for each different culture medium. Two peptones, SoyA2SC and SoyE-110, were the only hydrolysates that showed production improvement in all three media. Casein Peptone plus Tryptone N1 and SoyA3SC showed different improved results based on their implemented concentration for each individual basal medium. The amino acid profile of peptones may provide clues to identify the most effective feeding strategies for recombinant CHO cells

Effect of peptone feeding on transient gene expression process in CHO DG44

Transient Gene Expression [TGE] gained popularity over the last decade as a rapid method for the production of milligram to gram quantities of recombinant proteins for preclinical studies in biophama industry. Thereby, the optimization of the TGE technique for Chinese hamster ovary [CHO] as the dominant host for the production of bio therapeutics is of great interest to reach the values for Human Embryo Kidney-293 [HEK-293] cells in terms of transfection efficiencies and production titers. TGE efficiencies are cell line and vector dependent. In transfection efficiency optimization experiments, different starting cell densities, different amounts of plasmid DNA and PEI transfection reagent were investigated to achieve the best conditions leading to maximum transfection efficiencies. Furthermore, in order to investigate the effect of peptone feeding on transfection efficiency, three different sources of peptones with the greatest effect in the CD DG44 basal media were selected; Casein Tryptone N1, Soy petone A2SC and Soy peptone E110. The transfection strategy performed here was able to make an outstanding increase in transfection efficiency of CHO DG44 cell line transfected with pTracer-SV40-mutated t-PA plasmid from 3.6% in our starting nonoptimized condition to 66.93% in finally optimized situation. Moreover, peptone feeding strategy used here was successful to increase volumetric productivities up to 37%. In addition, the amounts of both PEI and plasmid DNA were reduced up to 66% and 25%, respectively compared to our previous protocol. Here we described an optimization process for TGE in suspensionadapted CHO cells based on Polyethylenimine [PEI]/DNA concentration, DNA: PEI ratio, starting cell densities and peptone feeding strategy

[Enhancement of recombinant human tissue plasminogen activator expression in CHO cells using matrix attachment region containing vectors in combination with promoter activation strategy]

Development of high producing mammalian cell lines is a major bottleneck in manufacturing of recombinant therapeutic proteins. This study examines the effect of using the matrix attachment region from the human interferon beta gene in combination with promoter activation strategy with E1A 13S protein on human tissue plasminogen activator [t-PA] expression in Chinese hamster ovary [CHO] cells. The matrix attachment region was cloned in 3' and 5' flanking sides of the t-PA expression cassette in pTPA vector to generate pMTPA. After transfection of the cells with pTPA and pMTPA vectors, stable cell pools were developed and the t-PA expression level determined for each stable cell line. In the next step, E1A 13S expression plasmid was transfected to stable cell pools and t-PA titers were measured after 72 hours. Integration of pTPA and pMTPA vectors in the CHO genome was confirmed by PCR analysis on genomic DNA of stable cell pools. Analysis of the t-PA expression level showed a three-fold enhancement in pMTPA transfected cells compared to pTPAcontaining cells. t-PA expression was further enhanced up to 1771 U/ml by transient expression of E1A 13S in pMTPA stable cell pools. These results have shown that incorporation of matrix attachment region in an expression vector in combination with promoter activation can effectively enhance recombinant protein expression levels in CHO cells.

Immune responses against a new HIV-1 p24-gp41/pCAGGS-IL-12 DNA vaccine in balb/c mice

Development of an effective vaccine is highly needed in order to restrict the AIDS pandemic. DNA vaccines initiate both arms of immunity without the potential of causing disease. HIV-1 p24 and gp41 [gag and env] proteins play important roles in viral pathogenesis and are effective candidates for immune induction and vaccine design. In this study, new DNA vaccine candidates constructed from HIV-1 fused p24- gp41 or gp41 alone were evaluated in Balb/c mice for induction of cellular and humoral immune responses. Recombinant plasmids, pcDNA3.1/Hygro expression vector containing immunogenic sequences of fused p24-gp41 or gp41alone were produced. Dendrosome used as a system for carrying vectors in laboratory animals, and an IL-12 containing vector [pCAGGS-IL-12] was co-immunized with the p24-gp41 vector as a genetic adjuvant. Induction of effective immune responses against the designed vectors as DNA vaccine candidates in Balb/c mice was evaluated. Levels of total antibodies, IgG isotypes [IgG2a and IgG1]? IFN-alpha and IL-4 were measured by ELISA. MTT assay was used to evaluate lymphoproliferation. The results confirmed that the immunogenic epitopes of both p24 and gp41 genes are highly effective inducers of immune responses, and administration of fused p24-gp41 alone or along with IL-12 resulted in further enhancement of immune responses. Group 4 that received fused fragments [p24-gp41] along with an IL-12 expressing vector demonstrated a significantly higher Stimulation Index [SI] and IFN-alpha production [p<0.0001] with a significant increase in IgG2a/IgG1 ratio, indicating the stimulation of CMI towards Th1. Although gp41 containing vector [group 6] also showed significant increases in both proliferation and IFN-alpha production, the responses were persistently lower than that of p24-gp41 containing vectors. Total antibody production was highest in group 6 as expected. Dendrosome proved to be an efficient carrier of recombinant plasmids constructed in this study. Further studies are necessary to evaluate these constructs as HIV vaccine candidates

[ comparison of bone morphogenetic protein-7 mutant expression in prokaryotic and eukaryotic hosts]

Bone morphogenetic protein-7 [BMP-7] is a multifunctional growth factor predominantly recognized for its osteoinductive properties. Due to the high cost of this protein, the availability of BMP-7 for treatment is limited. The heterologous production of recombinant hBMP-7 has been performed in a number of expression systems. In this study a novel form of BMP-7 was expressed in eukaryotic and prokaryotic hosts. For expression in the prokaryotic system, the novel protein was secreted to the periplasmic space of Escherichia coli using a pelB signal sequence followed by singlestep purification by Ni2+-charged column chromatography. In the mammalian cell expression system, we transferred a full-length cDNA encoding precursor of the novel protein to CHO cells then selected stable clones by using the appropriate antibiotic concentration. Expressions in both systems were confirmed by Western blot analysis. The novel recombinant protein was produced as a 36-38 kDa dimer in the CHO cell line and a 16 kDa monomer in the Escherichia coli system. Quantitative analysis according to ELISA showed that the expression levels of the mutant protein in the eukaryotic and prokaryotic expression systems were 40 ng/ml and 135 ng/ml of the culture media, respectively. In this study, the expression level in Escherichia coli was at least three times more than observed in the CHO cells. However, further optimization is required to obtain a dimer protein in Escherichia coli. The results show that periplasmic expression may be suitable for the production of complex proteins such as BMPs

Cloning and expression of Leishmania major superoxide dismutase B1: a potential target antigen for serodiagnosis of Leishmaniasis

Leishmaniasis- a neglected public health problem- is a group of diseases affecting an estimated 12 million people worldwide. In the present study, recombinant Leishmania major superoxide dismutase B1 [rLmSODB1] has been utilized as a potential antigen for the serodiagnosis of human cutaneous [CL] and visceral leishmaniasis [VL] in the endemic regions of southern part of Iran. Additionally, the sensitivity and specificity of ELISA-based serodiagnosis using rLmSODB1 and the soluble Leishmania antigen [SLA] were compared. For the first time, rLmSODB1 has been cloned successfully and used for ELISA-based serodiagnosis. Sera from 30 CL and 24 VL cases were included in this study. Additional studies were also done for the evaluation of cross-reactivity using sera from 41 endemic controls including normal endemic donors [n= 20], systemic lupus erythematosus patients [n=5], rheumatoid arthritis patients [n= 5], and patients with tuberculosis [n=11]. Analysis indicated that rLmSODB1 was recognized by62.5% and 13.3% of sera from patients with VL and CL, showing a sensitivity of 72.7% and 53.6%, respectively. However 95.8% of VL and 30% of CL sera reacted with SLA, revealing sensitivities of 96% and 58.8%, respectively. Additionally, from 41 sera collected either from healthy subjects or patients affected with other diseases, 97.5% were negative with SLA or rLmSODB1 [specificity 97.6%]. These results show that rLmSODB1 almost does not react with sera from patients with tuberculosis and autoimmune diseases and may be considered as a candidate antigen for the specific immunodiagnosis of visceral leishmaniasis

Application of SYBR-Green Real Time RT-PCR for quantification of human immunodeficiency virus type-1 [HIV-1] and comparison with COBAS AMPLICOR test

In this study, a SYBR Green real-time RT-PCR assay for quantification of HIV-1 viral RNA was developed. This assay was performed based on amplification of the pol region of HIV-1 and product analysis by an ABI 7500 system. We quantified HIV-1 viral load in 26_seropositive patients by this system and the data were subsequently compared with results obtained with a reference technique represented by COBAS AMPLICOR HIB-1 Monitor test. The results demonstrated that this technique could detect up to 500 HIV-1 RNA copies/ml of plasma. The linearity of this approach was conserved over a wide range of HIV-1 copy numbers [5x10[2]- 5x10[9]]. Since no positive signal was observed in seronegative volunteers, the specificity of the test was calculated as 100%. Comparison of the results with those obtained by the reference quantification method, revealed a significant correlation between the results [R[2] =0.95]. On the basis of the most recent recorded cases for HIV-1 infection and AIDS in Iran, the prevalence of this disease is rising rapidly and the situation has been called to be alarming by national health representatives. Determination of HIV-1 viral load in plasma has been considered as the most effective single prediction tool for monitoring HIV-1 patients treated with antiviral drugs. In this study, we have developed a SYBR-Green Real Time RT-PCR assay for quantitative analysis of HIV-1 in infected patients. Since a synthetic RNA standard was used in this assay, the upper limit of detection was detected to be higher than the standard test [5x10[9] versus 7.5x10[5]]. This can be important in patients with acute high viral load infections. Reproducibility was assessed by Intra assay and Inter assay analysis, Coefficient of variations Ct, in reproducibility tests for Intra assay and Inter assay variability were less than 3% and 4.5% accordingly. The above results, indicates that the new developed test can be a used in substitution of the commercial assay for quantitative analysis of HIV-1

Immune response analysis of immunized Balb/C mice with HIV-1 fusion p24 - gp41 genes as DNA vaccine candidate

The global HIV epidemic continues to expand and exceeding previous predictions. An effective vaccine represents the best hope to curtail the HIV epidemic. DNA vaccines induce humoral and cellular responses and mimic live vaccines without their pathogenic potential. The importance of CD8[+]CTL responses in controlling HIV and SIV viremia has led to production of a series of vaccines candidates that effectively induce these responses. It is now widely believed that an HIV vaccine strategy must stimulate both a strong humoral [antibody] as well as cell-mediated [CTL] immune response. The p24 and gp41 play many important roles in host-virus interaction and pathogenesis. These proteins are considered as attractive vaccine candidate in which their immunogenecity and immunomodulatory effects have been confirmed. In this study, a construct, pcDNA3.1Hygro- [p24-gp41], was evaluated as a DNA vaccine candidate in Balb/C mice for generation of effective cellular immune responses. For immunizing, we used dendrosome, a novel family of vehicles for transfection and therapy. IFN-gamma cytokine production and total antibody were detected by ELISA. Lymphoprolifration assay was performed by MTT test. ELISA and MTT assays confirmed that the cited p24-gp41 fusion gene is able to enhance immune responses in mice. The construct that was used in this research can be a good candidate for DNA vaccine against HIV-1, if the future complementary tests demonstrate the same trends of immunogenic responses shown in this study