Laboratory for the development of Genetic and Pharmacogenomic Therapy of Thalassemia

NEW FRONTIERS IN MOLECULAR DIAGNOSIS AND EXPERIMENTAL THERAPY OF BETA THALASSEMIA

 

Thal-Lab,
Laboratory for the Development of Pharmacologic and Pharmacogenomic Therapy of Thalassemia,
Biotechnology Center , Ferrara University

Preface - I
What has been done

The LABORATORY FOR THE DEVELOPMENT OF PHARMACOLOGICAL AND PHARMACOGENOMIC THERAPY OF THALASSEMIA (ThalLab) has been founded following an Agreement of collaboration between the Center of Biotechnology of University of Ferrara (CBF), Via Fossato di Mortara n. 64/B and the Veneta Association for the Fight against Thalassaemia (AVTL) of Rovigo . The Laboratory is directed by a Scientific Responsible, with the advice of a Directive Council, currently constituted by the same responsible, the investigators involved in the research and the representatives of AVTL. The Laboratory welcomes adhesion of new partners, whose participation to the Council depends from a contribution for the Laboratory research activities.
The program of the Directive Council is organized following meetings (at least two/year). The Scientific Responsible presents to the Council the milestones of the Program activity, together with a summary of the major results obtained.

The ThalLab is located within the Biotechnology Center of Ferrara University (CBF). This is a partial list of the relevent equipments located at the Biotechnology Center: a DNA ALF DNA Sequencing System (Pharmacia), a DNA ABI PRISM 377 DNA SEQUENCER (Perkin-Elmer), FacSTAR plus cytofluorimeter (FACS = Fluorescente-Activated Cell Sorter) (Becton-Dickinson), 2 class P3 laboratory, a biosensor BIAcore-1000 System (Pharmacia Biosensors), a preparative HPLC (Millipore), a Peptide synthetizer Milligen (Millipore), a Packard microarray scanner.

General objective 

The main general objective of the research activities of the ThalLab is to identify and to characterize biomolecole able to induce the transcription of the globin genes, with particular reference to the genes coding the g -globin and the b -globin. This research is very important for the pharmacological treatment of patients affected by b -thalassemia and sickle-cell anemia aimed at the production of fetal hemoglobin (HbF). This would allow to render thalassemia patients independent from transfusion treatment.

Homepage – talassemiaricerca.it

The activity of ThalLab is reported within a WEB site that, in addition to general informations of thalassaemia, includes the results of th scientific activity of ThalLab and the activity of AVTL (Veneta Association for the fight against Thalassaemia), the major sponsor of the laboratory. An english version of the WEB site has been recently included.

Patents on HbF inducers

• Patent n. PCT/EP01/02804 del 13 marzo 2001: “Synthetic oligonucleotides as inducers of erythroid differentiation”. Inventors: Nicoletta Bianchi, Giordana Feriotto , Roberto Gambari , Carlo Mischiati.
• Patent n. PCT/IB02/02628 del 1° luglio 2002: "Use of heterocyclic and benzoheterocyclic polaymides structurally related to the natural antibiotic distamycin A for the treatment of beta-thalassemia". Inventors : Pier Giovanni Baraldi, Nicoletta Bianchi, Giordana Feriotto , Roberto Gambari , Carlo Mischiati, Romeo Romagnoli.
• Patent n. PCT/IB02/04042 del 26 settembre 2002: “3-deoxy-3-amide derivatives of carbohydrates as inducers of erythroid cell differentiation”. Inventors : Giorgio Catelani , Felicita D'Andrea, Roberto Gambari , Andrea Spitaleri.
• Patent n. PCT/IB03/02632 del 3 luglio 2003: “A novel use of rapamycin and structural analogues thereof”. Inventors : Nicoletta Bianchi, Monica Borgatti, Roberto Gambari , Carlo Mischiati.
• Patent n. PCT/IB03/03462 del 30 luglio 2003: “A novel use of angelicin and structural analogues thereof”. Inventors : Nicoletta Bianchi, Monica Borgatti, Roberto Gambari , Ilaria Lampronti.

 

Publications (partial list)

HbF induction

• Chiarabelli, C., Bianchi, N., Borgatti, M., Prus, E., Fibach, E., Gambari R. Induction of gamma-globin gene expression by tallimustine analogs in human erythroid cells (2003) Haematologica , 88 , 826-7.
• Lampronti I, Martello D, Bianchi N, Borgatti M, Lambertini E, Piva R, Jabbar S, Choudhuri MS, Khan MT, Gambari R. In vitro antiproliferative effects on human tumor cell lines of extracts from the Bangladeshi medicinal plant Aegle marmelos Correa. Phytomedicine. 2003 May;10(4):300-8.
• Fibach E, Bianchi N, Borgatti M, Prus E, Gambari R. Mithramycin induces fetal hemoglobin production in normal and thalassemic human erythroid precursor cells. Blood. 2003 102(4):1276-81.
• Khan MT , Lampronti I, Martello D, Bianchi N, Jabbar S, Choudhuri MS, Datta BK, Gambari R. Identification of pyrogallol as an antiproliferative compound present in extracts from the medicinal plant Emblica officinalis: Effects on in vitro cell growth of human tumor cell lines. Int J Oncol. 2002
• Catelani G, D'Andrea F, Mastrorilli E, Bianchi N, Chiarabelli C, Borgatti M, Martello D, Gambari R. Preparation and evaluation of the in vitro erythroid differentiation induction properties of some esters of methyl 3,4-O-isopropylidene-beta-D-galactopyranoside and 2,3-O-isopropylidene-D-mannofuranose. Bioorg Med Chem. 2002 Feb;10(2):347-53.
• Bianchi N, Chiarabelli C, Borgatti M, Mischiati C, Fibach E, Gambari R. Accumulation of gamma-globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine. Br J Haematol. 2001 Jun;113(4):951-61.
• Gambari R, Feriotto G, Rutigliano C, Bianchi N, Mischiati C. Biospecific interaction analysis (BIA) of low-molecular weight DNA-binding drugs. J Pharmacol Exp Ther. 2000 Jul;294(1):370-7.
• Bianchi N, Ongaro F, Chiarabelli C, Gualandi L, Mischiati C, Bergamini P, Gambari R. Induction of erythroid differentiation of human K562 cells by cisplatin analogs. Biochem Pharmacol. 2000 Jul 1;60(1):31-40.
• Catelani G, Osti F, Bianchi N, Bergonzi MC, D'Andrea F, Gambari R. Induction of erythroid differentiation of human K562 cells by 3-O-acyl-1,2-O-isopropylidene-D-glucofuranose derivatives. Bioorg Med Chem Lett. 1999 Nov 1;9(21):3153-8.
• Bianchi N, Osti F, Rutigliano C, Corradini FG, Borsetti E, Tomassetti M, Mischiati C, Feriotto G, Gambari R. The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells. Br J Haematol. 1999 Feb;104(2):258-65.
• Cortesi R, Gui V, Osti F, Nastruzzi C, Gambari R. Human leukemic K562 cells treated with cytosine arabinoside: enhancement of erythroid differentiation by retinoic acid and retinol. Eur J Haematol. 1998 Nov;61(5):295-301.
  
  
  
  

Development of non-viral gene therapy based on PNA-DNA chimeras

• Borgatti M, Lampronti I, Romanelli A, Pedone C, Saviano M, Bianchi N, Mischiati C, Gambari R. Transcription factor decoy molecules based on a peptide nucleic acid (PNA)-DNA chimera mimicking Sp1 binding sites. J Biol Chem. 2003 Feb 28;278(9):7500-9.
• Piva R, Gambari R. Transcription factor decoy (TFD) in breast cancer research and treatment Technology in Cancer Research and Treatment 2002; 1: 405-416.
• Romanelli A, Pedone C, Saviano M, Bianchi N, Borgatti M, Mischiati C, Gambari R. Molecular interactions with nuclear factor kappaB (NF-kappaB) transcription factors of a PNA-DNA chimera mimicking NF-kappaB binding sites. Eur J Biochem. 2001 Dec;268(23):6066-75.
• Gambari R. Peptide-nucleic acids (PNAs): a tool for the development of gene expression modifiers. Curr Pharm Des. 2001 Nov;7(17):1839-62.
• Saviano M, Romanelli A, Bucci E, Pedone C, Mischiati C, Bianchi N, Feriotto G, Borgatti M, Gambari R. Computational procedures to explain the different biological activity of DNA/DNA, DNA/PNA and PNA/PNA hybrid molecules mimicking NF-kappaB binding sites. J Biomol Struct Dyn. 2000 Dec;18(3):353-62.
• Mischiati C, Borgatti M, Bianchi N, Rutigliano C, Tomassetti M, Feriotto G, Gambari R. Interaction of the human NF-kappaB p52 transcription factor with DNA-PNA hybrids mimicking the NF-kappaB binding sites of the human immunodeficiency virus type 1 promoter. J Biol Chem. 1999 Nov 12;274(46):33114-22.

Molecular diagnosis

• Altomare L, Borgatti M, Medoro G, Manaresi N, Tartagni M, Guerrieri R, Gambari R. Levitation and movement of human tumor cells using a printed circuit board device based on software-controlled dielectrophoresis. Biotechnol Bioeng. 2003 May 20;82(4):474-9.
• Feriotto G, Borgatti M, Mischiati C, Bianchi N, Gambari R. Biosensor technology and surface plasmon resonance for real-time detection of genetically modified Roundup Ready soybean gene sequences. J Agric Food Chem. 2002 Feb 27;50(5):955-62.
• Feriotto G, Corradini R, Sforza S, Bianchi N, Mischiati C, Marchelli R, Gambari R. Peptide nucleic acids and biosensor technology for real-time detection of the cystic fibrosis W1282X mutation by surface plasmon resonance. Lab Invest. 2001 Oct;81(10):1415-27.
• Feriotto G, Ferlini A, Ravani A, Calzolari E, Mischiati C, Bianchi N, Gambari R. Biosensor technology for real-time detection of the cystic fibrosis W1282X mutation in CFTR. Hum Mutat. 2001;18(1):70-81.
• Feriotto G, Lucci M, Bianchi N, Mischiati C, Gambari R. Detection of the deltaF508 (F508del) mutation of the cystic fibrosis gene by surface plasmon resonance and biosensor technology. Hum Mutat. 1999;13(5):390-400.

 

Delivery

• Borgatti M, Romanelli A, Saviano M, Pedone C, Lampronti I, Breda L, Nastruzzi C, Bianchi N, Mischiati C, Gambari R. Resistance of decoy PNA-DNA chimeras to enzymatic degradation in cellular extracts and serum. Oncol Res. 2003;13(5):279-87.
• Borgatti M, Breda L, Cortesi R, Nastruzzi C, Romanelli A, Saviano M, Bianchi N, Mischiati C, Pedone C, Gambari R. Cationic liposomes as delivery systems for double-stranded PNA-DNA chimeras exhibiting decoy activity against NF-kappaB transcription factors. Biochem Pharmacol. 2002 Aug 15;64(4):609.

 

 

Induction of apoptosis of primary osteoclasts as a novel approach for treatment of osteoporosis

• Penolazzi L, Elisabetta Lambertini, Monica Borgatti, Roberta Piva, Mauro Cozzani, Ilaria Giovannini, Rosalba Naccari, Giuseppe Siciliani, Roberto Gambari. Decoy oligodeoxynucleotides targeting NF-kappaB transcription factors: induction of apoptosis in human primary osteoclasts. Biochem. Pharmacol., 66: 1189-1198, 2003.

 

Preface - II
What we plan to do (years 2004-2008)

This workplan is constituted by several Projects, each of them to be developed by Research Teams at Thal-Lab.

Project 1: Induction of HbF in adults
Researchers involved: Nicoletta Bianchi, Monica Borgatti, Ilaria Lampronti, Sara Gardenghi , Laura Breda , Cristina Zuccato.

Project 2: Development of non-viral gene therapy based on peptide nucleic acids (PNAs)
Researchers involved: Nicoletta Bianchi, Monica Borgatti, Giordana Feriotto , Giulia Breveglieri, Laura Breda.

Project 3: Pharmacogenomic
Researchers involved: Giulia Breveglieri, Monica Borgatti, Ilaria Lampronti.

Project 4: Molecular diagnosis
Researchers involved: Nicoletta Bianchi, Monica Borgatti, Giordana Feriotto , Giulia Breveglieri, Sara Gardenghi , Martina Baruffa, Leonardo Vizziello, Enrica Fabbri.

Project 5. Experimental animal model systems mimicking thalassemia
Researchers involved: Giordana Feriotto , Giulia Breveglieri, Laura Breda , Sara Gardenghi.

Project 6. Gene expression profile in HPFH subjects
Researchers involved: Nicoletta Bianchi, Alessia Finotti, Cristina Zuccato.

Project 7. Delivery
Researchers involved: Monica Borgatti.

Project 8. Induction of apoptosis of osteoclasts as an experimental therapeutical approach for osteoporosis
Researchers involved: Roberta Piva, Elisabetta Lambertini. Letizia Penolazzi.

Project 9. Technology transfer

Research Team	% (months/year)
Coordinator: Prof. Roberto Gambari	4
University Researchers:
Giordana Feriotto (Projects 2, 4, 5)	5
Roberta Piva (Project 8)	7
Technicians:
Nicoletta Bianchi (Projects 1, 2, 4)	8
Research Assistants:
Ilaria Lampronti (Project 1)	8
Research Fellows:
Monica Borgatti (Projects 1, 2, 4, 7)	4
Laura Breda (Projects 1, 5)	11
Elisabetta Lambertini (Project 8)	8
Letizia Penolazzi (Project 8)	7
PhD Students:
Sara Gardenghi (Biochimica e Biologia Molecolare, Projects 1 and 5)	11
Giulia Breveglieri (Biochimica e Biologia Molecolare, Projects 4 and 5)	7
Alessia Finotti (Biochimica, Project 6)
Cristina Zuccato (Biochimica, Biologia Molecolare e Biotecnologie, Project 1)	11
Irene Mancini (Biochimica, Biologia Molecolare e Biotecnologie, Project 4)	4
Enrica Fabbri (Biochimica, Biologia Molecolare e Biotecnologie, Project 4)	4
Total months/year	115

Short Curriculum Vitae: Prof. Roberto Gambari

Roberto Gambari is full Professor of Biochemistry (Degree in Chemical and Pharmaceutical Technologies, CTF, Ferrara University , Faculty of Pharmacy) since 2001.

He is chairman of PhD course in Biotechnology and is Director of the Biotechnology Center of Ferrara University. In September 1991 he was elected as member of the Council of European Tissue Culture Society (ETCS). Prof. Gambari is member of the Scientific Committee of the Interuniversity Consortium for Biotechnologies (C.I.B.).

Chief-Editor of the Journal Minerva Biotecnologica. Roberto Gambari is member of American Society for Biochemistry and Molecular Biology. Member of the Editorial Board of the American Journal of Pharmacogenomics, Technology in Cancer Research and Treatment, International Journal of Oncology, Drugs Design Reviews, Medicinal Chemistry.

Referee of the following Journals: FASEB J., Biochemical Pharmacoloy, Human Gene Therapy, BioTechniques, Biological Chemistry, J. of Clinical Investigations, Nucleic Acids Research, J. Agricultural and Food Chemistry.

Chairman of Workshops on Synthetic Oligonucleotides in Biotechnology and Molecular Biology, Ferrara 1992-1995.

Coordinator of several Research Projects, including Target Project Genetic Engineering (1992-1995), ISS-AIDS (1992-1993), Telethon, Target Project ACRO (1992-1995), Fondazione Italiana per la Guarigione dalla Thalassemia, AIRC (1992-1994; 2003). Coordinator of the Network "Use of PNA, ribozymes and peptides for the study of gene functions and possible diagnostic and therapeutic applications " (COFIN-1998). Coordinator of the Network " Applications of a dielectrophoresis-based Lab-on-a-chip to diagnosis and drug research and development " (COFIN-2002).

More than 160 papers, published by Roberto Gambari , are present in MedLine.

July 2003

 

 

PROGETTI DI RICERCA (2006-2009)

1) EU-FP6 (COCHISE) 2006-2009

Project title: Cell−on−chip Biosensor for detection of cell−to−cell interactions 

Brief description: The COCHISE project will develop a novel class of biosensors able to detect single cell−to−cell interactions and will provide a tool for improved monitoring and treatment of cancer. The strategic objectives achieved by this project are: •Demonstration of a cell−based biosensor and its macro−to−micro interfaces allowing delivery, detection and recovery of effector and target cells without interfering with their gene expression. •Demonstration of a new microtechnology which integrates on the same platform microfluidics, electronics, sensing and living cell management. •Sensing of cell−to−cell interactions at single cell level, overcoming the low signal levels. •Evaluation of this biosensor using in vitro and animal models. In addition to the main objective of the project, other important fields will benefit from this work. As an example, several drugs are supposed to force or prevent the disruption of cells. Hence, such a technology would open up new opportunities in the field of targeted drug delivery, where each patient would receive drugs that are known to be useful for his/her specific situation. Finally, the detection of lytic phenomena provides an important marker of the presence of toxins in the environment. This is a key issue for any security agency.

Personnel involved: Roberto Gambari, Monica Borgatti, Nicoletta Bianchi, Federica Destro.

 

2) EU-FP6 (ITHANET) 2006-2008

Project title: eInfrastructure for Thalassemia Research Network (ITHANET)

Brief description: The aim of ITHANET is to strengthen the Euromediterranean community of researchers in molecular and clinical research into thalassaemia and related haemoglobinopathies and to enhance its scientific potential using the existing and emerging infrastructures and tools of European research networks (GEANT, Grids). Participants of the project include all major European research institutions active in haemoglobinopathies research as well as a number of collaborating partner institutions from non−EU Mediterranean and Black Sea countries.

Personnel involved: Roberto Gambari, Giulia Breveglieri, Francesca Salvatori.

 

3) Fondazione CARIPARO 2005-2010

Project title: Pharmacology and pharmacogenics therapy of the thalassemia.

Brief description: The Project has the following objectives: (a) Induction of fetal hemoglobin (HbF) in adults. The aim is the identification of novel molecules able to induce erythroid differentiation and expression of HbF in erythroid precursor cells. In addition, we will continue our study on molecules that we were able to demonstrate as HbF inducers; (b) Development of non−viral gene therapy based on peptide nucleic acids (PNAs) and PNA−DNA chimeras. The aim is to modify gene transcription (with decoy molecules and transcriptional activators) and splicing of the primary transcript using peptide nucleic acids (PNAs) and PNA−DNA chimeras; (c) Molecular diagnosis. The aim is the analysis of the beta−thalassemia mutations using the BIAcore−1000 biosensor. We will develop multiplex diagnosis of the most frequent beta−thalassemia mutations. This study will permit the identification and recruitment of patients with specific mutations to be cured with specific molecular biology approaches; (d) Delivery. The aim is the development of delivery systems for molecules of some interest for therapy of beta−thalassemia. We will consider cationic liposomes, microspheres, electroporation technologies, Lab−on−a−chip technology; (e) Induction of apoptosis in osteoclasts to interfere with osteoporosis in beta−thalassemia patients. The issue is to induce apoptosis in human osteoclasts to the aim to develop a therapeutic approach to osteoporosis associated to beta−thalassemia. We will use the decoy strategy to the aim of interfering with transcription factors which, if bound to selective molecules, are associated to activation of the apoptotic process.

Personnel involved: Roberto Gambari , Nicoletta Bianchi, Cristina Zuccato, Giulia Breveglieri, Francesca Salvatori.

 

4) COFIN 2005 (2006-2008)

Project title: Applications  of  novel  Peptide  Nucleic  Acids  (PNA)−based  molecules  for  alteration  of  gene expression and molecular diagnosis. Brief description:  The PNA−based molecules will be used to characterize the molecular interactions with target molecules. The general aim of the project is to study the effects of PNA−based molecules on (a) apoptosis of human osteoclasts; (b) erythroid differentiation and HbF production in erythroid precursor cells; (c) expression of IL−6, IL−8, ICAM−1 in cystic fibrosis cells.

Personnel involved: Roberto Gambari, Michele Saviano, Monica Borgatti, Laura Piccagli, Alessandra Romanelli.

 

5) Telethon (2007-2009)

Project title: Modifiers of globin gene expression for therapy of beta−thalassemia. Brief description: The general aim of this proposal is to analyse the gene expression of erythroid progenitor cells from beta−thalassemia patients, for the identification of inducers of fetal hemoglobin (HbF), of possible interest for the therapy of beta−thalassemia. Task 1 is dedicated to the characterization of genotype and HbF production of beta−thalassemic patients for classification with respect to the molecular defects causing thalassemia. Tasks 2 and 3 are dedicated to studies on the effects of different HbF inducers on globin gene expression in erythroid precursor cells from beta−thalassemia patients. In this section of the proposal we will verify the possibility that hydroxyurea (HU) resistant erythroid precursor cells from a sub−set of beta−thalassemia patients might be induced to HbF production with treatment with alternative HbF inducers. For each beta−thalassemia patients, the best HbF inducer of erythroid progenitors will be identified. The aim of Task 4 is the employment of globin−gene expression modifiers in gene therapy, for the identification of modifiers of beta−globin gene expression able to maximize HbA production during retroviral mediated gene therapy of erythroid precursor cells from beta−thalassemia patients. Task 5 is dedicated to studies of the effects of siRNAs or antisense molecules on mutated unfunctional beta−globin or alpha−globin mRNAs. Aim of Task 6 is the analysis of gene expression in HPFH patients, for the identification of molecules able to mimic up− or down−regulation associated with HbF induction or HPFH (High Persistence of Fetal Hemoglobin) phenotype. Task 7 is dedicated to exploitation, for the identification of Biotech companies interested in the developed protocols and patents.

Personnel involved: Roberto Gambari, Nicoletta Bianchi, Cristina Zuccato, Giulia Breveglieri, Francesca Salvatori.