Lucia Monaco

Polycystin-1, the gene product of PKD1, induces resistance to apoptosis and spontaneous tubulogenesis in MDCK cells.

The major form of autosomal dominant polycystic kidney disease (ADPKD) results from mutation of a gene (PKD1) of unknown function that is essential for the later stages of renal tubular differentiation. In this report, we describe a novel cell culture system for studying how PKD1 regulates this process. We show that expression of human PKD1 in MDCK cells slows their growth and protects them from programmed cell death. MDCK cells expressing PKD1 also spontaneously form branching tubules while control cells form simple cysts. Increased cell proliferation and apoptosis have been implicated in the pathogenesis of cystic diseases. Our study suggests that PKD1 may function to regulate both pathways, allowing cells to enter a differentiation pathway that results in tubule formation.

A new Chinese hamster ovary cell line expressing α2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins

Chinese hamster ovary (CHO) cells are widely employed to produce glycosylated recombinant proteins. Our group as well as others have demonstrated that the sialylation defect of CHO cells can be corrected by transfecting the alpha2,6-sialyltransferase (alpha2,6-ST) cDNA. Glycoproteins produced by such CHO cells display both alpha2,6- and alpha2,3-linked terminal sialic acid residues, similar to human glycoproteins. Here, we have established a CHO cell line stably expressing alpha2,6-ST, providing a universal host for further transfections of human genes. Several relevant parameters of the universal host cell line were studied, demonstrating that the alpha2,6-ST transgene was stably integrated into the CHO cell genome, that transgene expression was stable in the absence of selective pressure, that the recombinant sialyltransferase was correctly localized in the Golgi and, finally, that the bioreactor growth parameters of the universal host were comparable to those of the parental cell line. A second step consisted in the stable transfection into the universal host of cDNAs for human glycoproteins of therapeutic interest, i.e. interferon-gamma and the tissue inhibitor of metalloproteinases-1. Interferon-gamma purified from the universal host carried 40.4% alpha2,6- and 59.6% alpha2,3-sialic acid residues and showed improved pharmacokinetics in clearance studies when compared to interferon-gamma produced by normal CHO cells.

Inhibition of Tumor Growth by Intramuscular Injection of cDNA Encoding Tumor Necrosis Factor α Coupled to NGR and RGD Tumor-Homing Peptides

The antitumor properties of tumor necrosis factor alpha (TNF) and its efficacy in selective destruction of tumor-associated vessels are well known. Besides the TNF protein, the TNF gene has been used for gene therapy of cancer and shown to induce antitumor responses both in animal models and in patients. We show here that the therapeutic properties of the TNF gene are improved by fusing the TNF sequence with those of peptides able to target tumor vessels, such as CNGRCG or ACDCRGDCFCG. Intramuscular administration of plasmid DNA encoding CNGRCG-TNF and ACDCRGDCFCG-TNF (pNGR-TNF and pRGD-TNF, respectively), but not plasmids encoding TNF (pTNF) or empty vector (pMock), inhibited the growth of subcutaneous murine B16F1 melanomas and RMA-T lymphomas implanted at sites distant from the site of plasmid injection. The combination of pNGR-TNF or pRGD-TNF with doxorubicin or melphalan induced stronger effects than single agents. These treatments induced antitumor effects without activating toxic or negative feedback mechanisms. In addition, pRGD-TNF increased the uptake of an antibody directed to a tumor-associated antigen. These results suggest that the therapeutic properties of NGR-TNF and RGD-TNF cDNAs are greater than those of TNF cDNA and provide the rationale for developing new gene therapy approaches based on vascular targeting with TNF coupled to tumor-homing peptides.

Genetic engineering of α2,6-sialyltransferase in recombinant CHO cells and its effects on the sialylation of recombinant interferon-γ.

The CHO cell line has achieved considerable commercial importance as a vehicle for the production of human therapeutic proteins, but is known to lack a functional copy of the gene coding for α2,6-sialyltransferase (EC 2.4.99.1). The cDNA for rat α2,6-ST was expressed in a recombinant CHO cell line making interferon-γ, using a novel in vitro amplification vector. The enzyme was expressed efficiently, and resulted in up to 60% of the total sialic acids on interferon-γ being linked in the α2,6-conformation. This sialic acid linkage distribution was more akin to that seen in natural human glycoproteins. In the most successful cell clones, expression of α2,6-sialyltransferase improved the overall level of sialylation by up to 56%, and had no adverse effects on cell growth, IFN-γ productivity or other aspects of IFN-γ glycosylation. These experiments demonstrate how the glycosylation machinery of rodent cells can be genetically manipulated to replicate human tissues.

Na-butyrate increases the production and α2,6-sialylation of recombinant interferon-γ expressed by α2,6- sialyltransferase engineered CHO cells

A non-human like glycosylation pattern in human recombinant glycoproteins expressed by animal cells may compromise their use as therapeutic drugs. In order to correct the CHO glycosylation machinery, a CHO cell line producing recombinant human interferon- γ (IFN) was transformed to replace the endogenous pseudogene with a functional copy of the enzyme α2,6-sialyltransferase (α2,6-ST). Both the parental and the modified CHO cell line were propagated in serum-free batch culture with or without 1 mM sodium butyrate. Although Na-butyrate inhibited cell growth, IFN concentration was increased twofold. The IFN sialylation status was determined using linkage specific sialidases and HPLC. Under non- induced conditions, IFN expressed by α2,6-engineered cells contained 68% of the total sialic acids in the α2,6- conformation and the overall molar ratio of sialic acids to IFN was 2.3. Sodium butyrate addition increased twofold the molar ratio of total sialic acids to IFN and 82% of total sialic acids on IFN were in the α2,6-conformation. In contrast, no effect of the sodium butyrate was noticed on the sialylation of the IFN secreted by the α2,6-ST deficient parental cell line. This study deals for the first time with the effect of Na-butyrate on CHO cells engineered to produce human like sialylation.

Expression of recombinant human granulocyte colony-stimulating factor in CHO dhfr- cells: new insights into the in vitro amplification expression system.

The in vitro amplification method for heterologous gene expression in mammalian cells is based on the stable transfection of cells with long, linear DNA molecules having several copies of complete expression units, coding for the gene of interest, linked to one terminal unit, coding for the selectable marker. DNA concatenamers containing additional expression units can also be prepared: we exploited this feature by co-polymerizing expression units coding for granulocyte colony-stimulating factor (G-CSF) with cassettes for dihydrofolate reductase (DHFR) and for neomycin (Nm) resistance, as selectable markers. We were thus able to obtain high level production of G-CSF in chinese hamster ovary (CHO) dhfr- cells by combining in vitro amplification to just one step of in vivo amplification. This approach required a considerably shorter time than the classical, stepwise amplification by methotrexate.

Glomerular filtration is required for transfection of proximal tubular cells in the rat kidney following injection of DNA complexes into the renal artery

Gene transfer to the kidney can be achieved with various DNA vectors, resulting in transgene expression in glomerular or tubular districts. Controlling transgene destination is desirable for targeting defined renal cells for specific therapeutic purposes. We previously showed that injection of polyplexes into the rat renal artery resulted in transfection of proximal tubular cells. To investigate whether this process involves glomerular filtration of the DNA-containing particles, fluorescent polyethylenimine polyplexes were prepared, containing fluoresceinated poly-L-lysine. This allowed visualization of the route of the particles into the kidney. Our polyplexes were filtered through the glomerulus, since fluorescent proximal tubuli were observed. Conversely, fluorescent lipopolyplexes containing the cationic lipid DOTAP were never observed in tubular cells. Size measurements by laser light scattering showed that the mean diameter of polyplexes (93 nm) was smaller than that of lipopolyplexes (160 nm). The size of the transfecting particles is therefore a key parameter in this process, as expected by the constraints imposed by the glomerular filtration barrier. This information is relevant, in view of modulating the physico-chemical properties of DNA complexes for optimal transgene expression in tubular cells.

Development and characterization of pituitary GH3 cell clones stably transfected with a human proinsulin cDNA

Successful β-cell replacement therapy in insulin-dependent (type I) diabetes is hindered by the scarcity of human donor tissue and by the recurrence of autoimmune destruction of transplanted β cells. Availability of non-β cells, capable of releasing insulin and escaping autoimmune recognition, would therefore be important for diabetes cell therapy. We developed rat pituitary GH3 cells stably transfected with a furin-cleavable human proinsulin cDNA linked to the rat PRL promoter. Two clones (InsGH3/clone 1 and 7) were characterized in vitro with regard to basal and stimulated insulin release and proinsulin transgene expression. Mature insulin secretion was obtained in both clones, accounting for about 40% of total released (pro)insulin-like products. Immunocytochemistry of InsGH3 cells showed a cytoplasmic granular insulin staining that colocalized with secretogranin II (SGII) immunoreactivity. InsGH3 cells/clone 7 contained and released in vitro significantly more insulin than clone 1. Secretagogue-stimulated insulin secretion was observed in both InsGH3 clones either under static or dynamic conditions, indicating that insulin was targeted also to the regulated secretory pathway. Proinsulin mRNA levels were elevated in InsGH3 cells, being significantly higher than in PTC3 cells. Moreover, proinsulin gene expression increased in response to various stimuli, thereby showing the regulation of the transfected gene at the transcriptional level. In conclusion, these data point to InsGH3 cells as a potential β-cell surrogate even though additional engineering is required to instruct them to release insulin in response to physiologic stimulations.

Controlling the Glycosylation of Recombinant Proteins Expressed in Animal Cells by Genetic and Physiological Engineering

The cDNA coding for rat alpha-2,6-sialyltransferase (α2,6-ST) was expressed in a recombinant CHO cell line producing interferon-gamma, using a novel in vitro amplification vector. The α2,6-ST was expressed efficiently, 20–30% of the sialic acid pool on interferon-gamma being linked in the α2,6-conformation. Despite the overall level of sialylation was not significantly increased, growth and interferon-gamma production in bioreactor were not compromised by the transfection and expression of the gene encoding for the α2,6-ST.

Generation of high-recombinant- protein-producing Chinese hamster ovary (CHO) cells.

The purpose of this chapter is to propose a practical procedure for the generation and selection of Chinese hamster ovary cells producing high levels of recombinant protein by combining in vitro and in vivo amplification of the foreign gene. A detailed description of the expression and amplification plasmids utilized for the in vitro generation of long DNA concatenamers, as well as the cell transfection and selection protocols are given. The procedure required for in vivo gene amplification using the dihydrofolate reductase/methotrexate system is also described.