Fernando Revert

Stability of PEI–DNA and DOTAP–DNA complexes: effect of alkaline pH, heparin and serum

DNA complexes formed with nonviral vectors such as polyethylenimine (PEI) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) are widely used in gene therapy. These complexes prevent the interaction of DNA with the fluorescent probes usually employed to quantify DNA. We thus studied the procedures for DNA quantification from DNA complexes as well as their stability in the presence of DNase or mouse, rat and human sera. Release of the DNA from its complexes was accomplished by increasing the pH of the medium (from 7.3 to 13.4) or by adding heparin. The stability against degradation was tested in vitro, by incubating the complexes at 37 degrees C in the presence of DNase I and sera from the three species. Both high pH and heparin were able to release DNA from its complexes. Naked DNA formed aggregates with serum proteins that delayed electrophoresis migration, and this effect was reversed in the presence of heparin. However, these aggregates did not protect DNA from digestion by serum DNase, and the DNA digesting ability of serum was: mouse>rat>human. The DNA from the complexes was resistant to degradation by DNase I, although a low proportion of DNA from the complexes was partially digested, as determined by electrophoresis. In contrast, PEI-DNA and DOTAP-DNA complexes were stable in the presence of all sera. Heparin and high pH release DNA from its complexes. The order of DNA degradation is: mouse>rat>human, but DOTAP and PEI avoid degradation of DNA by serum compounds.

Characterization of a Novel Type of Serine/Threonine Kinase That Specifically Phosphorylates the Human Goodpasture Antigen

Goodpasture disease is an autoimmune disorder that occurs naturally only in humans. Also exclusive to humans is the phosphorylation process that targets the unique N-terminal region of the Goodpasture antigen. Here we report the molecular cloning of GPBP (Goodpasture antigen-bindingprotein), a previously unknown 624-residue polypeptide. Although the predicted sequence does not meet the conventional structural requirements for a protein kinase, its recombinant counterpart specifically binds to and phosphorylates the exclusive N-terminal region of the human Goodpasture antigen in vitro. This novel kinase is widely expressed in human tissues but shows preferential expression in the histological structures that are targets of common autoimmune responses. The work presented in this report highlights a novel gene to be explored in human autoimmunity.

Polyethyleneimine-based immunopolyplex for targeted gene transfer in human lymphoma cell lines

Specific and efficient delivery of genes into targeted cells is a priority objective in non‐viral gene therapy. Polyethyleneimine‐based polyplexes have been reported to be good non‐viral transfection reagents. However, polyplex‐mediated DNA delivery occurs through a non‐specific mechanism. This article reports the construction of an immunopolyplex, a targeted non‐viral vector based on a polyplex backbone, and its application in gene transfer over human lymphoma cell lines.

Goodpasture Antigen-binding Protein Is a Soluble Exportable Protein That Interacts with Type IV Collagen IDENTIFICATION OF NOVEL MEMBRANE-BOUND ISOFORMS

Goodpasture-antigen binding protein (GPBP) is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Various studies have questioned these findings and proposed that GPBP serves as transporter of ceramide between the endoplasmic reticulum and the Golgi apparatus. Here we show that cells expressed at least two GPBP isoforms resulting from canonical (77-kDa) and noncanonical (91-kDa) mRNA translation initiation. The 77-kDa polypeptide interacted with type IV collagen and localized as a soluble form in the extracellular compartment. The 91-kDa polypeptide and its derived 120-kDa polypeptide associated with cellular membranes and regulated the extracellular levels of the 77-kDa polypeptide. A short motif containing two phenylalanines in an acidic tract and the 26-residue Ser-rich region were required for efficient 77-kDa polypeptide secretion. Removal of the 26-residue Ser-rich region by alternative exon splicing rendered the protein cytosolic and sensitive to the reduction of sphingomyelin cellular levels. These and previous data implicate GPBPs in a multicompartmental program for protein secretion (i.e. type IV collagen) that includes: 1) phosphorylation and regulation of protein molecular/supramolecular organization and 2) interorganelle ceramide trafficking and regulation of protein cargo transport to the plasma membrane.

Antitumor effect of B16 melanoma cells genetically modified with the angiogenesis inhibitor rnasin.

The growth of new blood vessels is an essential condition for the development of tumors with a diameter greater than 1–2 mm and also for their metastatic dissemination. RNasin, the placental ribonuclease inhibitor, is known to have antiangiogenic activity through the inhibition of angiogenin and basic fibroblast growth factor. Nevertheless, the administration of the recombinant form of a protein poses several limitations; as a result, we have studied the antitumor effect of RNasin in a murine gene therapy model. RNasin cDNA was subcloned into the pcDNA3 expression vector, and the resulting recombinant plasmid was used to transfect the B16 murine melanoma cell line. An RNasin inverted construction was used as control. Mice intravenously injected with clones expressing RNasin showed a significant inhibition of tumor metastatic progression with respect to control groups (P<.001) and survived longer (P<.001). Tissue sections from RNasin-expressing cell tumors showed a lower number of blood vessels when compared to tissue sections from mice lungs that had been inoculated with control cell lines. The results of these experiments show that the genetic modification of tumor cells with RNasin cDNA yields a significant antitumor effect, and suggest that this effect is at least partially the result of angiogenesis inhibition. Cancer Gene Therapy (2001) 8, 278–284

A human-specific TNF-responsive promoter for Goodpasture antigen-binding protein

The Goodpasture antigen‐binding protein, GPBP, is a serine/threonine kinase whose relative expression increases in autoimmune processes. Tumor necrosis factor (TNF) is a pro‐inflammatory cytokine implicated in autoimmune pathogenesis. Here we show that COL4A3BP, the gene encoding GPBP, maps head‐to‐head with POLK, the gene encoding for DNA polymerase kappa (pol κ), and shares with it a 140‐bp promoter containing a Sp1 site, a TATA‐like element, and a nuclear factor kappa B (NFκB)‐like site. These three elements cooperate in the assembly of a bidirectional transcription complex containing abundant Sp1 and little NFκB that is more efficient in the POLK direction. Tumour necrosis factor cell induction is associated with Sp1 release, NFκB recruitment and assembly of a complex comparatively more efficient in the COL4A3BP direction. This is accomplished by competitive binding of Sp1 and NFκB to a DNA element encompassing a NFκB‐like site that is pivotal for the 140‐bp promoter to function. Consistently, a murine homologous DNA region, which contains the Sp1 site and the TATA‐like element but is devoid of the NFκB‐like site, does not show transcriptional activity in transient gene expression assays. Our findings identify a human‐specific TNF‐responsive transcriptional unit that locates GPBP in the signalling cascade of TNF and substantiates previous observations, which independently related TNF and GPBP with human autoimmunity.

Human Biliverdin Reductase Suppresses Goodpasture Antigen-binding Protein (GPBP) Kinase Activity THE REDUCTASE REGULATES TUMOR NECROSIS FACTOR-α-NF-κB-DEPENDENT GPBP EXPRESSION

The Ser/Thr/Tyr kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-α). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-κB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that in HEK293A cells hBVR binds to GPBP and down-regulates its TNF-α-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with small interfering RNA to hBVR and to the p65 regulatory subunit of NF-κB show the hBVR role in the initial stimulation of GPBP expression by TNF-α-activated NF-κB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the 281CX10C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC281, corresponding to the core of the consensus D(δ)-Box motif in the interacting domain, was as effective as the intact 296-residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-α dependent NF-κB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-α-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis.

Human biliverdin reductase suppresses goodpasture antigen binding protein (GPBP) kinase activity: the reductase regulates TNF-α- NF-κB-dependent GPBP expression

The Ser/Thr/Tyr kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-α). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-κB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that in HEK293A cells hBVR binds to GPBP and down-regulates its TNF-α-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with small interfering RNA to hBVR and to the p65 regulatory subunit of NF-κB show the hBVR role in the initial stimulation of GPBP expression by TNF-α-activated NF-κB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the 281CX10C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC281, corresponding to the core of the consensus D(δ)-Box motif in the interacting domain, was as effective as the intact 296-residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-α dependent NF-κB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-α-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis.

Pharmacodynamic approach to study the gene transfer process employing non-viral vectors.

In the present work we set out to apply pharmacodynamic concepts derived from dose-response curves (Potency and Efficacy) to characterize the gene transfer efficiency of a vector:DNA complex. We employed two widely used vectors, the cationic lipid DOTAP (N,N, N-trimethyl 1-2-3-bis (1-oxo-9-octa-decenyl)oxy-(Z, Z)-1-propanaminium methyl sulfate) and the cationic polymer PEI (polyethylenimine, 800 kDa) to transfect several constructions of the green fluorescent protein cDNA. The analysis of dose-response curves indicated that in all cases the goodness-of-fit was > 0.99. Potency is a measure that provides information on gene activity per amount of DNA. Efficacy is a measure of maximum gene expression achievable using a specific vector:DNA complex, and depends on both the intrinsic efficacy of the gene (evaluated using different vectors to transfer the same gene construct) and on vector efficacy in DNA delivery (evaluated using a single vector to deliver different gene constructs). The results suggest that Potency and Efficacy are objective parameters for describing and comparing the goodness of vectors, as well as the intrinsic efficacy of a given gene construct. Furthermore, they are useful tools that may contribute to a better understanding of the mechanistic gene transfer process of each vector.

Goodpasture Antigen-binding Protein (GPBP) Directs Myofibril Formation: IDENTIFICATION OF INTRACELLULAR DOWNSTREAM EFFECTOR 130-kDa GPBP-INTERACTING PROTEIN (GIP130)*

Background: GPBP-1 is a non-conventional kinase that regulates glomerular basement membrane collagen organization. Results: GPBP-1 targets GIP130, a new myosin-binding protein, and regulates myofibrillogenesis in cultured myoblasts. Conclusion: GPBP-1 regulates myofibril formation. Significance: GPBP-1 is a kinase for structural protein organization at both intracellular and extracellular compartments. Goodpasture antigen-binding protein-1 (GPBP-1) is an exportable non-conventional Ser/Thr kinase that regulates glomerular basement membrane collagen organization. Here we provide evidence that GPBP-1 accumulates in the cytoplasm of differentiating mouse myoblasts prior to myosin synthesis. Myoblasts deficient in GPBP-1 display defective myofibril formation, whereas myofibrils assemble with enhanced efficiency in those overexpressing GPBP-1. We also show that GPBP-1 targets the previously unidentified GIP130 (GPBP-interacting protein of 130 kDa), which binds to myosin and promotes its myofibrillar assembly. This report reveals that GPBP-1 directs myofibril formation, an observation that expands its reported role in supramolecular organization of structural proteins to the intracellular compartment.