No description available.

Human adenoviruses (HAdVs) are common viruses often associated withgastrointestinal, ocular and respiratory infections. They can infect a widevariety of cells, both dividing and non-dividing. HAdVs attach to and infecttarget cells through interactions with cellular receptors. It has also beenshown that HAdVs can use soluble host components in body fluids forindirect binding to target cells, a feature that enables the usage of new typesof receptors resulting in a more efficient HAdV infection. We thereforeevaluated the influence of soluble components from four different bodyfluids on HAdV infection of epithelial cells, representing the respiratory andocular tropism of most HAdVs. We found that plasma, saliva, and tear fluidpromote binding and infection of HAdV-5 (species C) and that plasmapromotes infection of HAdV-31 (species A). Further binding and infectionexperiments identified coagulation factor IX (FIX) and X (FX) as thecomponents of plasma responsible for increase of HAdV-5 infection whileFIX alone mediates increase of HAdV-31 infection. We found that as little as1% of the physiological concentration of these factors is required to facilitatemaximum binding. The effect of coagulation factors on HAdV infection was thereafterextended to include all species A HAdVs: HAdV-12, -18 and -31. Species AHAdVs normally cause infections involving the airways and/or the intestine.These infections are often mild but species A HAdVs in general, and HAdV-31 in particular, have been shown to cause severe and life-threateninginfections in immunocompromised patients. We show here that FIXefficiently increase HAdV-18 and -31 (but not HAdV-12) binding andinfection of human epithelial cells, representing the respiratory andgastrointestinal tropism. FIX was shown to interact with the hexon proteinof HAdV-31 and surface plasmon resonance analysis revealed that theHAdV-31:FIX interaction is slightly stronger than that of the HAdV-5:FIX/FX interactions, but more interestingly, the half-lives of theseinteractions are profoundly different. By performing binding and infectionexperiments using cells expressing specific glycosaminoglycans (GAGs) and ivGAG-cleaving enzymes we found that the HAdV-31:FIX and HAdV-5:FIX/FX complexes bind to heparan sulfate-containing GAGs on targetcells, but we could also see a difference in GAG dependence and specificitybetween these complexes.We conclude that the use of coagulation factors might be of moreimportance than previously recognized and that this may affect not only theliver tropism seen when administering adenovirus vectors into thecirculation but also regulate primary infections by wild-type viruses of theirnatural target cells. We also believe that our findings may contribute tobetter design of HAdV-based vectors for gene and cancer therapy and thatthe interaction between the HAdV-31 hexon and FIX may serve as a targetfor antiviral treatment. HAdV vectors are mainly based on HAdV-5 and several problems haverecently become evident when using these vectors. Major challenges withHAdV-5 based vectors include pre-existing neutralizing antibodies, pooraccess to the receptor CAR (coxsackie and adenovirus receptor), and offtarget effects to the liver due to interactions with coagulation factors. Theneed for new HAdV vectors devoid of these problems is evident.HAdV-52 is one of only three HAdVs that are equipped with two differentfiber proteins, one long and one short. We show here, by means of bindingand infection experiments, that HAdV-52 can use CAR as a cellular receptor,but that most of the binding is dependent on sialic acid-containingglycoproteins. Flow cytometry, ELISA and surface plasmon resonanceanalyses revealed that the terminal knob domain of the long fiber (52LFK)binds to CAR, and the knob domain of the short fiber (52SFK) binds tosialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complexwith sialic acid revealed a new sialic acid binding site compared to otherknown adenovirus:glycan interactions. Moreover, glycan array analysisidentified α2,8-linked oligosialic acid, mimicking the naturally occurringpolysialic acid (PSia), as a potential sialic acid-containing glycan receptor for52SFK. ELISA and surface plasmon resonance confirmed the ability of52SFK to interact with PSia. Flow cytometry analysis also showed a fivefold vincrease in binding of 52SFK to PSia-expressing cells compared to controlcells. X-ray crystallographic analysis of 52SFK in complex with oligo-PSiarevealed engagement at the non-reducing end of oligo-PSia to the canonicalsialic acid-binding site, but also suggested the presence of a 'steering rim'consisting of positively charged amino acids contributing to the contact bylong-range electrostatic interactions. PSia is nearly absent on cells in healthy adults but can be expressed inhigh amounts on several types of cancers including: glioma, neuroblastomaand lung cancer. We show here that the short fiber of HAdV-52 bindsspecifically to PSia. Taking into account that HAdV-52 has a supposedly lowseroprevalence and is incapable of interacting with coagulation factors webelieve that HAdV-52 based vectors can be useful for treatment of cancertypes with elevated PSia expression.

Ho, Sui Fan Tong. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 68-71). / LIST OF TABLES / LIST OF FIGURES / ACKNOWLEDGEMENTS / ABSTRACT / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 2. --- OBJECTIVES --- p.12 / Chapter 3. --- MATERIALS AND METHODS --- p.13 / Chapter 3.1 --- Materials --- p.13 / Chapter 3.1.1 --- Enzymes --- p.13 / Chapter 3.1.2 --- DNA Markers --- p.13 / Chapter 3.1.3 --- General Reagents --- p.13 / Chapter 3.2 --- General Methods --- p.15 / Chapter 3.2.1 --- Phenol and Phenol/Chloroform (1:1) Preparation --- p.15 / Chapter 3.2.2 --- Buffer Preparation --- p.15 / Chapter 3.2.3 --- Agarose Gel Electrophoresis --- p.18 / Chapter 3.2.4 --- Polyacrylamide Gel Electrophoresis --- p.18 / Chapter 3.3 --- DNA Study --- p.19 / Chapter 3.3.1 --- Haemophilia B Patient --- p.19 / Chapter 3.3.2 --- Blood Collection --- p.20 / Chapter 3.3.3 --- DNA Extraction --- p.20 / Chapter 3.3.4 --- DNA Quantitation --- p.21 / Chapter 3.3.5 --- Polymerase Chain Reaction --- p.22 / Chapter 3.3.6 --- Purification of PCR Products --- p.28 / Chapter 3.3.7 --- Sequencing --- p.32 / Chapter 3.3.8 --- Cloning --- p.37 / Chapter 4. --- RESULTS --- p.40 / Chapter 4.1 --- DNA Extraction --- p.40 / Chapter 4.2 --- Calibration of the Coy TempCycler --- p.42 / Chapter 4.3 --- Optimization of PCR --- p.44 / Chapter 4.3.1 --- PCR-1 --- p.44 / Chapter 4.3.2 --- PCR-2 --- p.46 / Chapter 4.3.3 --- PCR-3 --- p.46 / Chapter 4.3.4 --- PCR-4 --- p.48 / Chapter 4.3.5 --- PCR-5 --- p.49 / Chapter 4.3.6 --- PCR-6 --- p.50 / Chapter 4.3.7 --- PCR-7 --- p.51 / Chapter 4.4 --- Purification of PCR Product --- p.52 / Chapter 4.4.1 --- GC-1 --- p.52 / Chapter 4.4.2 --- GC-2 --- p.52 / Chapter 4.4.3 --- GC-3 --- p.53 / Chapter 4.4.4 --- PAGE-1 --- p.54 / Chapter 4.4.5 --- PAGE-2 --- p.54 / Chapter 4.4.6 --- Agarose Gel Extraction with Glasswool Exclusion --- p.55 / Chapter 4.5 --- Direct Sequencing of PCR Products --- p.55 / Chapter 4.6 --- Cloning --- p.55 / Chapter 5. --- DISCUSSION --- p.57 / Chapter 5.1 --- DNA Extraction --- p.57 / Chapter 5.2 --- Polymerase Chain Reaction --- p.57 / Chapter 5.3 --- Purification of PCR Products --- p.58 / Chapter 5.4 --- Sequencing --- p.61 / Chapter 5.5 --- Cloning --- p.61 / Chapter 6. --- CONCLUSION --- p.67 / Chapter 7. --- PHOTOGRAPHS --- p.64 / Chapter 8. --- REFERENCES --- p.68

Haemophilia B is a bleeding disorder caused by various mutations of the coagulation Factor IX gene (F9) resulting in qualitative or quantitative Factor IX protein (FIX) deficiency. Factor replacement therapy is the current standard of care. Cure may be possible in the near future by gene therapy — the transfer of normal copies of F9 to patients with haemophilia, causing establishment of FIX production and correction of the bleeding phenotype. Mesenchymal stem cells (MSC) are potential vehicles for gene delivery through ex vivo gene transfer and subsequent transplantation to the patient. Lentiviral vectors can transduce MSC effectively and mediate long term gene expression. However, gene expression may decline with time due to transgene silencing. Ubiquitous Chromatin Opening Element (UCOE) is a set of genetic sequences cloned from housekeeping genes that can maintain a transcriptionally competent, open chromatin structure and was shown to prevent gene silencing by resisting DNA methylation. We tested human F9 expression and FIX protein secretion by transducing MSC with lentiviral vectors that carry the FIX gene under the control of A2UCOE (A2UCOE-hF9). A2UCOE is a 2.2 kb sequence cloned from the HNRPA2B1–CBX3 gene loci that harbour UCOE function. A2UCOE-eGFP, an enhanced Green Fluorescent Protein (eGFP) gene expression construct, was used to assist in vector titration of A2UCOE-hF9 by flow cytometry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). MSC were transduced at various Multiplicities of Infection (MOIs) by A2UCOE-hF9 lentiviral vector. Upon transduction, F9 mRNA expression and FIX secretion were measured by qRT-PCR and ELISA respectively. Osteogenic and adipogenic differentiation assay were performed to compare differentiation potential before and after transduction at an MOI of 1. F9 mRNA expression and FIX secretion were both undetectable in untransduced MSC. Upon transduction, vector dose-dependent increase in F9 mRNA expression and FIX secretion were detected at MOIs of 1, 2, 4 and 8. The level of secreted FIX ranges from 20 to 150 μIU in 72 hours. Osteogenic and adipogenic differentiation were not affected post-transduction at an MOI of 1. In conclusion, FIX secretion by MSC was detected upon A2UCOE-hF9 lentiviral transduction. However, the level of FIX appeared to be low compared to published studies. Further studies are required to determine the cause of low FIX expression, develop methods to maximize FIX expression and confirm whether A2UCOE can prevent gene silencing and maintain sustainable gene expression. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Research in Medicine

Permanent repair of the F9 gene is a significant goal to cure Hemophilia B disease. Advanced gene therapy using CRISPR/Cas9 system can increase circulation level of Factor IX proteins to a significant level without the need of demanding infusions of FIX concentrates. Induced pluripotent stem cells represent an ideal cell for gene therapy because patient-derived cells could be reprogrammed into iPSCs, genetically modified, selected, expanded and then induced to differentiate into fully functional hepatocytes in vitro. This study covered a portion of a 5-year project which ultimately aims at establishing therapeutic results in transgenic Hemophilia B mice by injecting genetically corrected iPSC-derived hepatocytes into the liver. The purpose of this thesis is to summarize what has been completed up to now: generation of the proper model of Hemophilia B human iPSCs using CRISPR/Cas9-mediated genome editing and differentiation of healthy and disease specific iPSCs into hepatocytes which will allow disease modelling to look for cell function, viability, homogeneity and drug screening. Further research will be done to effectively knock-in the F9 allele into liver safe harbor site of disease specific iPSCs, which will express FIX at a significant level to show therapeutic effects.

Hemophilia B is a debilitating and life-threatening disorder caused by a deficiency in or dysfunction of factor IX (FIX), a complex plasma glycoprotein required for the formation and maintenance of blood clots. Treatment of hemophilia B involves infusion of replacement FIX currently derived from two sources: FIX purified from pools of human plasma (pd-FIX) and a single recombinant FIX product generated in genetically engineered Chinese hamster ovary (CHO) cells. Both of these FIX products are prohibitively expensive, limiting of the treatment options of hemophiliacs worldwide. As a result, a more abundant and affordable FIX product would greatly improve the life prospects for hemophiliacs. The biological activity of FIX is dependent upon its numerous post-translational modifications (PTMs), including gamma-carboxylation, proteolytic maturation, phosphorylation, sulfation, and glycosylation. Of these PTMs, those known to be vital for activity are gamma-carboxylation of multiple glutamate residues near the N-terminus and proteolytic cleavage of the FIX propeptide. When expressed at a high rate in exogenous expression systems, however, the ability of current systems to effect the necessary PTMs is severely rate limited, restricting the production of active FIX. The transgenic pig bioreactor represents a promising source for the production of large quantities biologically active FIX due to its demonstrated ability to perform the required FIX PTMs. It was the goal of this study to characterize the PTM structure and the resulting function of recombinant FIX when expressed at 1-3 mg/ml in the transgenic pig mammary epithelium (tg-FIX). It was found that the expressed tg-FIX is comprised of a heterogeneous mixture of FIX PTM isoforms. This mixture represents a spectrum of tg-FIX molecules of varying gamma-carboxyglutamic acid (Gla) and propeptide content, indicating that rate limitations in effecting these PTMs are present. A purification process was developed utilizing heparin-affinity chromatography to purify the total population of tg-FIX from pig milk, a complex multi-phase feedstock. Subsequently, a process was developed to fractionate the total population of tg-FIX into subpopulations based upon the extent of post-translational modification. Q ion-exchange chromatography was utilized to fractionate tg-FIX based upon molecular acidity which was found to be correlated to both biological activity and Gla content. The resulting biologically active tg-FIX population contained an average of 7 of the 12 Gla residues found in pd-FIX. Immuno-affinity chromatography was subsequently utilized to further fractionate tg-FIX into mature tg-FIX and propeptide-containing tg-FIX populations. The isolated FIX PTM populations were subjected to functional analysis by investigating in vitro clotting activity, activation by factor XIa, and in vivo pharmacokinetics. From this analysis it was found that mature tg-FIX with an average 7 Gla residues, representing approximately 9% of the total tg-FIX produced, exhibits wild-type in vitro clotting activity and normal activation by factor XIa. The remainder of the tg-FIX produced, characterized by either a lower Gla content or the presence of the propeptide, was found to be inactive and displayed less efficient activation by factor IXa. In an in vivo pharmacokinetic study in the hemophilia B mouse model, biologically active tg-FIX was found to possess altered circulating properties. Tg-FIX was characterized by a lower recovery, approximately one-sixth that of pd-FIX, but an extended circulation half-life. From this study it was found that the mean residence time of tg-FIX after injections is approximately twice that observed for pd-FIX. These altered pharmacokinetic properties are likely linked to the unique tg-FIX PTM structure, perhaps through altered endothelial cell binding characteristics caused by the reduced Gla content. / Ph. D.

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser. Även tryckt utgåva.

The adenovirus (Ad) family consists of 52 different human types, which are divided into seven species (A-G). Human Ads cause disease in the respiratory tract, lymphoid tissue, intestine, urinary tract, and/or in the eye. Most, but not all Ads have been demonstrated to use the coxsackie-adenovirus receptor (CAR) as an efficient receptor in vitro, but CAR has been questioned as an in vivo-receptor for various reasons. Thus, there are reasons to believe that Ads use other mechanisms for binding to target cells. In an attempt to investigate the impact of tear fluid during in vitro infection of ocular Ads (i.e. Ad37), using corneal cells, we found that human tear fluid promoted infection of an Ad with pronounced respiratory tropism (i.e. Ad5) used here as a control, but surprisingly not of Ad37. Furthermore using a virus overlay protein blotting assay we found that Ad5 bound to several tear fluid proteins. One of these, human lactoferrin (hLf) which is a component that belongs to the innate immune system in various body fluids, was alone able to promote both binding and infection of all species C Ads (Ad1, Ad2, Ad5, Ad6) in epithelial cells. hLf was also found to promote gene delivery (GFP) from an Ad5-based vector. Further we have identified lactoferricin (Lfcin), the N-terminal part of hLf, as to be responsible for this effect. We also show that plasma, saliva, and tear fluid promote infection of Ad5 in respiratory and ocular epithelial cells, and that plasma promotes infection of Ad31. The component in plasma that is responsible for this effect is likely to be coagulation factor IX (FIX) and X (FX), since both these factors were able to promote binding and infection of Ad5 and/or Ad31 in epithelial cells. Finally, we show that the excess of fiber production from initial Ad infection and the release of fibers before the particle itself is released caused masking of the tropism-specific receptors in both infected and non-infected surrounding cells. This means that the overproduction of fibers affects the ability of Ad to spread within tissues. We conclude that soluble components in body fluids, such as hLf, FIX, and FX have the ability to mediate binding and infection of selected human Ads (species C and Ad31) in epithelial cells that represent the tropism of these Ads. We suggest that these components may serve as bridges between the virion and the cell surface. This is contributes to the knowledge about Ad lifecycle, and might help to improve the de-/retargeting of gene therapy based on Ad vectors.

<p>Hemophilia A and B are X-linked recessive bleeding disorders caused by the deficiency of coagulation factor VIII (FVIII) and Factor IX (FIX), respectively. Current treatment involves life-long protein replacement therapy which is invasive, expensive and inaccessible to the majority of hemophiliacs worldwide. Treatment is further compromised by the development of neutralizing antibodies. Thus, the development of an alternative treatment that is safer, cost effective and non-invasive that circumvents immune response induction is desirable.</p> <p>To this end, a chitosan-mediated gene therapy strategy delivered orally was developed to provide clinically relevant plasma expression of FVIII or FIX. Hemophilia A mice that ingested chitosan nanoparticles containing FVIII DNA transiently expressed canine FVIII reaching >100 mU one day post treatment, together with partial phenotypic correction. Residual FVIII activity was detected for several days. Repeated administration of nanoparticles restored FVIII expression for 4 weeks and reduced clotting time in treated mice. Interestingly, inhibitors and non-neutralizing antibodies were not detectable throughout the experiment.</p> <p>The immunomodulatory effects of chitosan-mediated oral gene delivery was investigated in naive hemophilia A mice and mice with pre-existing inhibitors. Administration of nanoparticles containing human FVIII DNA in naive mice suppressed systemic antibody responses and provided long-term tolerance to rhFVIII protein immunizations for at least 8 weeks. This tolerance was transferable to naive mice, suggesting development of regulatory T cells. In contrast, repeated oral nanoparticle administration was unable to suppress FVIII-specific antibody responses in hemophilia A mice with pre-existing inhibitors.</p> <p>Treatment of hemophilia B is challenged by a 25-50 fold higher therapeutic threshold. Nevertheless, hemophilia B mice fed chitosan nanoparticles containing CpG-FIXi plasmid transiently expressed therapeutically relevant human FIX >14mU/mL plasma.</p> <p>Chitosan nanoparticle formulation was optimized <em>in vitro</em> for improved transfection efficiency. Nanoparticles formulated at a chitosan:DNA charge ratio of >2:1 (N:P) provided DNA protection against proton and enzymatic degradation that mimic conditions of the stomach and intestine, respectively. The inclusion of 25 mM sodium acetate-acetic acid decreased transfection of HEK 293 cells 4-fold, while 50 mM sodium sulphate increased uptake by ~40%. Optimal transfection was achieved with chitosan chloride (CL 213) formulated at a charge ratio of 3:1 in 50 mM sodium sulphate.</p> <p>These findings suggest chitosan nanoparticles can provide clinically relevant FVIII and FIX transgene expression, which is amenable to a one-tablet-a-day dosing strategy. Taken together, chitosan-mediate gene therapy delivered orally is proposed as a potential non-invasive alternative strategy for hemophilia treatment and without inducing neutralizing and non-neutralizing antibody production.</p> / Doctor of Philosophy (PhD)

O fator IX (FIX) da coagulação sanguínea é uma proteína dependente de vitamina K de grande valor farmacêutico no tratamento da Hemofilia B, o qual é baseado na administração do fator de coagulação derivado de plasma humano ou da proteína recombinante produzida em células murinas. A terapia baseada nestas abordagens apresenta alto custo e está associada às contaminações com vírus e príons, além do desenvolvimento de inibidores de FIX. Esses efeitos aumentam o risco de morbidade e mortalidade relacionadas às hemorragias. Neste trabalho, clonamos o cDNA do FIX em um vetor lentiviral e avaliamos a expressão da proteína recombinante em duas linhagens celulares humanas. A clonagem do cDNA do FIXh no vetor de expressão lentiviral 1054 foi confirmada através da análise com enzimas de restrição específicas obtendo-se as bandas esperadas de 1407 pb e 10054 pb visualizadas em gel de agarose. As linhagens celulares 293T e SK-Hep-1 foram transduzidas com o vetor lentiviral 1054-FIX gerado em nosso laboratório e as células que apresentaram maior expressão de EGFP foram selecionadas e separadas por citometria de fluxo. A quantificação da expressão de FIXrh foi realizada por ensaios de ELISA e cromogênico. A quantificação de FIXrh total foi de 500 ng/106 células para a linhagem 293T e 803 ng/106 células para a linhagem SK-Hep-1. A atividade biológica específica de FIXh nas células 293T e SK-Hep-1 foi 0,047 UI/106 células e 0,186 UI/106 células, respectivamente. Com o intuito de avaliar o perfil de produção de FIXrh ativo ao longo do tempo, foi realizado um acompanhamento de 180 dias, no qual foi observado que a linhagem SK-Hep-1 cessou a expressão de FIX, enquanto as células 293T mantiveram a expressão durante o período. O FIXrh foi caracterizado por western blot confirmando a presença de uma banda imunoreativa esperada de 57 kDa. As linhagens 293T e SK-Hep-1 apresentaram 7,67 e 17 cópias do vetor inserido/célula, respectivamente. Considerando a importância do processo de ?-carboxilação, foi realizada uma análise da expressão gênica dos genes envolvidos neste processo, tais como o VKORC1, ?-carboxilase e o inibidor calumenina, nas linhagens celulares. Os resultados demonstraram razões elevadas entre os genes VKORC1 e calumenina e VKORC1 e ?-carboxilase nas duas linhagens. A cinética de crescimento das células foi realizada por um período de 7 dias apresentando diferenças significativas entre as células SK-Hep-1 transduzidas e não transduzidas, enquanto que as células 293T não presentaram diferenças estatísticas no crescimento celular. A suplementação do meio de cultura com íons Ca+2 e Mg+2 foi testada para avaliar sua influência na expressão de FIXrh ativo. As células 293T apresentaram melhor desempenho nas concentrações de 0,5 mmol/L de Ca+2 e 1,0 mmol/L de Mg+2 e as células SK-Hep-1 no meio de cultura não suplementado. Nossos dados indicam que a linhagem hepática SK-Hep-1 é a melhor produtora de FIXrh funcional e as comparações realizadas entre os dois tipos celulares são importantes na caracterização do comportamento de linhagens geneticamente modificadas voltadas para a expressão de proteínas recombinantes heterólogas e abre novos caminhos para futuros estudos que visam o melhoramento da produção desse tipo de proteína. / Blood coagulation factor IX is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the treatment of Hemophilia B which is based on the plasma-derived coagulation factors or recombinant protein produced in murine cells. Coagulation therapy based on these approaches has high costs and is closely associated with prion and virus contamination besides the FIX inhibitors development. These effects increase the risk for bleeding-related morbidity and mortality. The purpose of this study was to clone hFIX into a lentiviral vector and evaluate the expression of the recombinant protein in two human cell lines. The cloning of the hFIX cDNA into 1054 lentiviral expression vector was confirmed by enzymatic restriction obtaining the expected 1407 bp and 10054 bp bands in agarose gel. The 293T and SK-Hep-1 cell lines have been stable transduced with 1054-FIX lentiviral vector generated in our laboratory and the cells with higher expression of EGFP were selected and separated by flow cytometry. The quantification of the expression of rhFIX was performed by ELISA and chromogenic assays. The concentration of total rhFIX was 500 ng/106 cells in 293T cell line and 803 ng/106 cells in SK-Hep-1 cell line. The biological activity of FIX secreted by 293T and SK-Hep-1 was 0,047 UI/106 cells and 0,186 UI/106 cells, respectively. In order to evaluate the active rhFIX production profile over time, we conducted a monitoring of 180 days, which was noted that the SK-Hep-1 cell line ceased FIX expression, while 293T cells maintained the expression during this period. rhFIX was characterized by western blot analysis confirming the presence of a expected 57 kDa immunereactive band. The 293T and SK-Hep-1 cell lines showed 7.67 and 17 integrated vector copies/cell, respectively. Considering the importance of the ?-carboxylation process, we performed a gene expression analysis of genes involved in this process, such as VKORC1, ?-carboxylase and calumenin, in cell lines. The results showed high ratios among the genes VKORC1 and calumenin and among VKORC1 and ?-carboxylase in both cell lines. The cell growth kinetics was performed by a 7-day period, showed significant differences between SK-Hep-1 transduced cells and non-transduced cells, whereas 293T cells showed no difference in cell growth. Enrichment of culture medium with Ca +2 and Mg +2 ions was tested to evaluate its influence on the expression of active FIX. 293T cells showed better performance in 0.5 mmol/L Ca+2 and 1.0 mmol/L Mg +2 concentrations and SK-Hep-1 cells in culture medium control. Our data indicate that transduced SK-Hep-1 cells are the best producer of functional rhFIX, and comparisons between these two cell lines are important in characterizing the behavior of genetically modified cell lines focused on the heterologous expression of recombinant proteins and opens new avenues for future studies aimed at improving the production of this type of protein.