Antonia Follenzi

RON is a heterodimeric tyrosine kinase receptor activated by the HGF homologue MSP.

RON, a cDNA homologous to the hepatocyte growth factor (HGF) receptor gene (MET), encodes a putative tyrosine kinase. Here we show that the RON gene is expressed in several epithelial tissues as well as in granulocytes and monocytes. The major RON transcript is translated into a glycosylated single chain precursor, cleaved into a 185 kDa heterodimer (p185RON) of 35 (alpha) and 150 kDa (beta) disulfide‐linked chains, before exposure at the cell surface. The Ron beta‐chain displays intrinsic tyrosine kinase activity in vitro, after immunoprecipitation by specific antibodies. In vivo, tyrosine phosphorylation of p185RON is induced by stimulation with macrophage stimulating protein (MSP), a protease‐like factor containing four ‘kringle’ domains, homologous to HGF. In epithelial cells, MSP‐induced tyrosine phosphorylation of p185RON is followed by DNA synthesis. p185RON is not activated by HGF, nor is the HGF receptor activated by MSP in biochemical and biological assays. p185RON is also activated by a pure recombinant protein containing only the N‐terminal two kringles of MSP. These data show that p185RON is a tyrosine kinase activated by MSP and that it is member of a family of growth factor receptors with distinct specificities for structurally related ligands.

The MET oncogene drives a genetic programme linking cancer to haemostasis

The close relationship between activation of blood coagulation and cancer is an old enigma. In 1865, migrans trombophlebitis (‘a condition of the blood that predisposes it to spontaneous coagulation’) was described as a forewarning of occult malignancy (Trousseaus sign). This pioneering observation emphasized the existence of haemostasis disorders associated with cancer onset; this phenomenon has since been extensively reported in clinical and epidemiological studies, but has so far resisted a mechanistic explanation. Here we report a mouse model of sporadic tumorigenesis based on genetic manipulation of somatic cells. Targeting the activated, human MET oncogene to adult liver caused slowly progressing hepatocarcinogenesis. This was preceded and accompanied by a syndrome manifesting first with blood hypercoagulation (venous thromboses), and then evolving towards fatal internal haemorrhages. The pathogenesis of this syndrome is driven by the transcriptional response to the oncogene, including prominent upregulation of plasminogen activator inhibitor type 1 (PAI-1) and cyclooxygenase-2 (COX-2) genes. In vivo analysis showed that both proteins support the thrombohaemorrhagic phenotype, thus providing direct genetic evidence for the long-sought-after link between oncogene activation and haemostasis.

In Vivo Activation of met Tyrosine Kinase by Heterodimeric Hepatocyte Growth Factor Molecule Promotes Angiogenesis

Hepatocyte growth factor (HGF) is a powerful motogen and mitogen for epithelial cells. The factor is a 90-kD heterodimer composed of an alpha chain containing four kringle motifs and a beta chain showing structural homologies with serine proteases. It is, however, devoid of enzymatic activity. Recently, it has been reported that HGF activates migration and proliferation of endothelial cells and is angiogenic. In this article we discuss (1) the molecular domains of HGF required to activate in vitro and in vivo endothelial cells, studied by use of molecular mutants, and (2) the characteristics of the angiogenic response to HGF in an experimental model system of implanted reconstituted basement membrane (Matrigel). Two groups of mutants were made and used in vitro and in vivo: one with deletions of kringle domains and one with substitution at the cleavage site of the HGF precursor. In vitro, HGF variants containing only the first two (HGF-NK2) or the first three kringles (HGF-NK3) of the alpha chain did not induce proliferation of endothelial cells even if used at concentration 160-fold higher than that optimal for HGF (0.05 nmol/L). High concentrations of these mutants (4 to 8 nmol/L) activated a little endothelial cell motogenic response that was 60% lower than that elicited by HGF. Substitution of Arg 489 with Gln 489 in the HGF precursor generated an uncleavable single-chain factor, unable to induce either endothelial cell migration or proliferation. In vivo, HGF induced a dose-dependent angiogenic response, which was enhanced by heparin.

Efficiency of Onco-Retroviral and Lentiviral Gene Transfer into Primary Mouse and Human B-Lymphocytes Is Pseudotype Dependent

B lymphocytes are attractive targets for gene therapy of genetic diseases associated with B-cell dysfunction and for immunotherapy. Transduction of B lymphocytes was evaluated using green fluorescent protein (GFP)-encoding onco-retroviral and HIV-derived lentiviral vectors which were pseudotyped with ecotropic, amphotropic or vesicular stomatitis virus (VSV-G) envelopes. Transduction of mouse B lymphocytes activated with lipopolysaccharides (LPS) or by cross-linking CD40 in conjunction with interleukin-4 (IL-4) was significantly more efficient (p < 0.003) with ecotropic (11%) than with VSV-G pseudotyped onco-retroviral vectors (1%). Using high-titer cell-free ecotropic viral supernatant or by coculture with ecotropic onco-retroviral vector-producing cells, transduction efficiency increased significantly (p < 0.001) to approximately 50%, whereas transduction efficiency by coculture with VSV-G pseudotyped vector-producing cells remained low (< 2%). Similarly, transduction of mouse B lymphocytes was significantly more efficient (twofold, p < 0.01) with the ecotropic (7%) than with the VSV-G pseudotyped lentiviral vectors although gene transfer efficiency remained low because of dose-limiting toxicity of the concentrated vector preparations on the LPS-activated murine B cells. Consistent with murine B-cell transduction, human B cells activated with CD40L and IL-4 were also found to be relatively refractory to VSV-G pseudotyped onco-retroviral vectors (< 1%). However, higher transduction efficiencies could be achieved in activated primary human B lymphocytes using VSV-G pseudotyped lentiviral vectors instead (5%-6%). Contrary to the significant increase in mouse B-cell transduction efficiency with ecotropic vectors, the use of amphotropic onco-retroviral or lentiviral vectors did not increase transduction efficiency in primary human B cells. The present study shows that the transduction efficiency of onco-retroviral and lentiviral vectors in human and mouse B lymphocytes is pseudotype-dependent and challenges the widely held assumption that VSV-G pseudotyping facilitates gene transfer into all cell types.

Correction of mucopolysaccharidosis type IIIb fibroblasts by lentiviral vector-mediated gene transfer

Mucopolysaccharidosis type IIIB (MPS IIIB; or Sanfilippo syndrome type B) is a lysosomal disease, due to glycosaminoglycan storage caused by mutations on the alpha-N-acetylglucosaminidase (NAGLU) gene. The disease is characterized by neurological dysfunction but relatively mild somatic manifestations. No effective treatment is available for affected patients. In the present study, we evaluated the role of a lentiviral vector as the transducing agent of NAGLU cDNA in MPS IIIB fibroblasts. The vector expressed high transduction efficiency and high levels of enzymic activity, 20-fold above normal levels, persisting for at least 2 months. PCR experiments confirmed the integration of the viral vector into the target genome. The NAGLU activity restored by virus infection was sufficient to normalize glycosaminoglycan accumulation, which is directly responsible for the disease phenotype. Metabolic labelling experiments on transduced fibroblasts exhibited, in the medium and in cellular lysates, polypeptide forms of 84 and 80 kDa respectively related to the precursor and mature forms of the enzyme. The enzyme secreted by transduced MPS IIIB fibroblasts was endocytosed in deficient cells by the mannose 6-phosphate system. Thus we show that lentiviral vectors may provide a therapeutic approach for the treatment of MPS IIIB disease.

Intraspinal stem cell transplantation for amyotrophic lateral sclerosis: Ready for efficacy clinical trials?

Intraspinal stem cell (SC) transplantation represents a new therapeutic approach for amyotrophic lateral sclerosis (ALS) clinical trials. There are considerable difficulties in designing future efficacy trials, some related to the field of ALS and some that are specific to SCs or the mode of delivery. In October 2015, the most controversial points on SC transplantation were addressed during an international workshop intended to bring together international SC and ALS researchers in a public discussion on a topic for which expertise is limited. During the meeting, a discussion was started on the basic structure of the ideal clinical trial testing the efficacy and safety of SC transplantation. The current document includes a number of consensus points reflecting the design of phase II/III clinical trials.