curriculum vitae - sns activities and responsibilities my research is mainly focused on the...

/5 - Curriculum vitae of David Porciani

Curriculum Vitae

Personal information First name and Surname David Porciani

Nationality Italian

Date of birth 03.08.1984

Gender Male

Occupational field PhD student in Molecular Biophysics at NEST, Scuola Normale Superiore

Work experience Dates June 2014 - present

Occupation or position held Six-month internship at Burke Lab, Bond Life Sciences Center, University of Missouri http://burkelab.missouri.edu/people.html

Main activities and responsibilities My research is focused on the selection of aptamers against cell-surface receptor over-expressed in different type of cells using a cell-SELEX approach.

Dates November 2010 - present Occupation or position held PhD student in “Molecular biophysics” at Laboratorio NEST, Scuola Normale Superiore

Main activities and responsibilities My research is mainly focused on the development of aptamers for in vitro and in vivo use. Reported sequences are examined and engineered in order to: i) create novel enhanced aptamer molecules and ii) develop efficient biomolecular architectures able to effectively recognize and target specific biomarkers. This will ultimately lead to systems that allow targeted drug delivery.

Name and address of employer National Enterprise for nanoScience and nanoTechnology (NEST) Piazza San Silvestro 12 - 56127 Pisa – Italy Scuola Normale Superiore

http://www.sns.it/ricerca/scienze/fisica/nest/people/porciani/

Dates August 2010 - October 2010 Occupation or position held Research Fellowship

Main activities and responsibilities The research was focused on improving the several phases of a procedure known as SELEX (Systematic Evolution of Ligands by EXponential enrichment), which is an iterative process to obtain DNA or RNA molecules with high affinity and selectivity against the desired target starting from a very random library. As biomolecular target was chosen the enzyme, myeloperoxidase (MPO) whose activity is increased in coronary artery disease. The final goal was to generate new inhibitory ligands of MPO.

Name and address of employer Institute of Clinical Physiology, National Research Council, CNR, Via Moruzzi, 1, 56124 Pisa, Italy

Education and training

Dates July 2010 Title of qualification awarded Abilitation to the pharmacist profession


Name and type of organisation providing education and training

Faculty of Pharmacy, University of Pisa

Dates June 2010

Title of qualification awarded Master Degree in Pharmaceutical Chemistry and Technology (CTF), with emphasis on biochemistry and pharmacology at the University of Pisa (Italy) approved with 110/110 cum laude.

Principal subjects/occupational skills covered

Thesis title: “The strategy of the gene knockdown for key factors indentification involved in physiopathological processes: design, synthesis and characterization of a hammerhead ribozyme”

Original Italian Title: “La strategia del knock-down genico per l’identificazione di fattori chiave di processi fisiopatologici: progettazione, sintesi e caratterizzazione di un ribozima hammerhead”.

Relators: Dr.Lorenzo Citti and Prof Paola Nieri

Activities carried out at Institute of Clinical Physiology, National Research Council, CNR, Italy Pisa from October 2009 to June 2010.

Principal Subjects: Chemistry, Molecular Biology and Genetics, Human Anatomy, Microbiology and General Pathology, Applied Biochemistry, Biochemistry, Advanced Biochemical Techniques Laboratory, Organic Chemistry I, Organic Chemistry II, Physics, Physics in Organic Chemistry, Chemical Physics, Pharmacology and Pharmacotherapy I, Pharmacology and Pharmacotherapy II, Pharmaceutical Chemistry and Toxicologically I, Pharmaceutical Chemistry and Toxicologically II.


Faculty of Pharmacy, University of Pisa

Level in national or international classification

ISCED 5B (Master degree)

Dates October 2008 - September 2009

Principal subjects/occupational skills covered

Professional training in Pharmacy


Farmacia Attias, via Marradi 2 - 57126 Livorno Tel: +39 0586810048

Dates July 2003 Title of qualification awarded Industrial chemistry expert graduation


Industrial Technical Institute, I.T.I.S. “G. Galilei” – Livorno

Level in national or international classification

ISCED 3B

Dates May 2002 Title of qualification awarded Level 5th - Course of Spoken English for Foreigners passed with merit


Trinity College of London (UK) - The International Examination Board

Pubblications Two interconvertible folds modulate the activity of a DNA aptamer against transferrin receptor D. Porciani, G. Signore, L. Marchetti, P. Mereghetti, R. Nifosì and F. Beltram Molecular Therapy – Nucleic Acids (2014) 3, e144; doi:10.1038/mtna.2013.71 http://www.nature.com/mtna/journal/v3/n1/abs/mtna201371a.html


Conferences and schools Aptamers 2014: 1st Oxford Symposium on Aptamers, March 24-25, 2014 Oxford, UK, StHedmund’s Hall & St Hilda’s College Poster: “An enhanced aptamer sequence as tool for targeted drug delivery: rational engineering of a DNA aptamer against transferrin receptor” D. Porciani, G. Signore, R. Nifosì, L. Marchetti, P. Mereghetti, and F. Beltram

9th Annual Meeting of the Oligonucleotide Therapeutics Society, October 6-8, 2013 Naples Italy, Royal Continental Hotel Poster: “Two interconvertible folds modulate the activity of a DNA aptamer against transferrin receptor” D. Porciani, G. Signore, L. Marchetti, P. Mereghetti, R. Nifosì and F. Beltram

1st Aptamers Workshop: Aptamers in Medicine and Perspectives, October 4-5, 2013 Naples Italy, Centro Congressi Federico II Poster: “Two interconvertible folds modulate the activity of a DNA aptamer against transferrin receptor” D. Porciani, G. Signore, L. Marchetti, P. Mereghetti, R. Nifosì and F. Beltram

Biophysical Society 57th Annual Meeting: February 2-6, 2013 Philadelphia, Pennsylvania Late Poster: “From sequence to function: a study on DNA aptamer interaction with transferrin receptor” D. Porciani, G. Signore, L. Marchetti and F. Beltram

ESONN 2011, European School On Nanosciences and Nanotechnologies: August 21 – September 10 2011, Grenoble France. Organized by Université Joseph Fourier (UJF) and Grenoble Institut National Polytechnique (Grenoble INP). Poster: “Aptamers for intracellular delivery in nanodiagnostics and therapeutics” D. Porciani, A. Bifone and F. Beltram

Personal skills and competences

Mother tongue Italian

Other language English Self-assessment Understanding Speaking Writing

European level (*) Listening Reading Spoken interaction

Spoken production

English B1 INDEPENDENTUSER B1 INDEPENDENT

USERB1 INDEPENDENT

USERB1 INDEPENDENT

USERB1 INDEPENDENT

USER

(*) Common European Framework of Reference for Languages

Social skills and competences Team spirit gained through my work and sport experiences

Organisational skills and competences

Sense of organization Good experience in work on collaborative projects

Technical skills and competences Good ability in synthesis with DNA synthesizer ABI 3000 (Applied Biosystem) and purification of DNA/RNA oligonucleotides with PAGE or HPLC techniques. Good ability and knowledge in oligonucleotide labelling techniques with fluorescent tags or other biomolecules by NHS chemistry, EDC chemistry, Maleimide-Thiol coupling chemistry. Good knowledge of most instruments for biochemical research and analyses (reverse phase-HPLC, anion exchange HPLC, exclusion chromatography, electrophoresis, dialysis, UV-VIS spectrophotometry, fluorescence spectroscopy, mass-spectrometry) Good ability and knowledge of microscopy: in particular in the use of fluorescence microscopy and confocal microscopy (LEICA SP5) Good ability and knowledge of fluorescence anisotropy assay as in vitro binding assay Good ability and knowledge of the main techniques of tissue culture (good experience in manipulation of many cancer cell lines such as HeLa, MIA-PaCa 2, U2OS and mouse fibroblasts NIH-3T3) Good ability and knowledge of the main techniques of molecular biology (PCR, transcription, retro-transcription, molecular cloning, Western blotting, transfection) Good knowledge in SELEX technology for in vitro selection of specific aptamers


Computer skills and competences European Computer Driving Licence (ECDL) “Full” Good knowledge of the Microsoft Office tools Good knowledge of the MAC OS X and Windows 7 Good knowledge of ImageJ and Fiji

Basic knowledge of Geneious for the study of DNA and RNA sequences Basic knowledge of Origin 8.0 and Graphpad Prism

Annexes Detailed description of research

DETAILED DESCRIPTION OF RESEARCH

During the MD thesis, my work was mainly devoted to the design, synthesis, and evaluation of a specific hammerhead ribozyme against the Platelet Derived Growth Factor (PDGF). The research was conducted under the framework of a project aiming to experiment the gene knockdown technique in the vascular smooth muscle cells (VSMC), which represent a well-accepted model system of cardiovascular diseases. Gene knockdown refers to techniques by which the expression of one or more organism's gene is reduced, thus allowing for a fine and extremely selective modulation of the physiological status of an organism. Hammerhead ribozymes can be used in gene knockdown because they are small catalytic RNAs capable to cleave a specific mRNA target with activity of trans-cleavage. This reduces the gene expression of the target gene. My research was focused on development of an experimental methodology for the design, synthesis and characterization of a specific hammerhead ribozyme against the mRNA of the PDGF, a key factor in many cardiovascular diseases. The process required a computational study, which was performed with the use of computational softwares. These allowed selecting many accessible sequences of cleavage on the mRNA target and, in turn, the most appropriate sequence for the hammerhead ribozyme, i.e. the sequence that corresponds to a stable structure of the ribozyme from point of view thermodynamic, still retaining its 3D structure. Subsequently, an automated DNA/RNA synthesizer based on phosphoramidite chemistry was used to synthesize the selected sequences of the ribozyme. The products obtained were purified by High Performance Liquid Chromatography (HPLC) method, using an anion exchange column and quantified by spectrophotometric measure at 260 nm. The ribozyme cleavage properties were evaluated through in vitro experiments. In particular, multiple turnover reactions were carried out using a high mRNA target/ribozyme molar ratio. These reactions follow the Michaelis-Menten equation, typical of protein enzyme kinetics. This allowed to determine kinetic parameters such as the catalytic constant (Kcat) and the observed constant (Kobs) for the designed hammerhead ribozyme. The reactions were performed in the presence of different concentrations of Mg2+, in order to assess its role as cofactor in the cleavage reaction. The results evidenced a strong dependence between ribozyme catalytic activity and [Mg2+] concentration. The catalytic activity was found to increase with [Mg2+]. The results obtained show that our hammerhead ribozyme is able to cleave its molecular target with good kinetic parameters and that the experimental methodology developed is effective for the design, synthesis and characterization of new hammerhead ribozymes. During the internship at the Istituto di Fisiologia Clinica (IFC-CNR), I had the opportunity to setup, from an experimental point of view, the SELEX technology applied to purified proteins (in vitro SELEX). SELEX technology is a combinatorial chemistry technique used to identify, from a large pool of nucleic acids, one or more oligonucleotide sequences (aptamers) able to bind a specific molecular target. SELEX consists of repeated rounds of in vitro selection. The fundamental steps of this technique are: 1) Synthesis of a DNA library with high complexity containing a random sequence of 20-70 mer.2) Amplification by PCR and trascription by T7 RNA polymerase of the DNA library.3) Incubation with specific molecule target4) Separation of the bound molecules from unbound and recovery of the bound oligonucleotide molecules.5) Amplification of selected RNAs by RT-PCR.During the fellowship, I successfully performed the setup of the experimental conditions for each step of the SELEX round. Finally, I joined NEST laboratory after winning a PhD position in Molecular Biophysics. My PhD project is related to the development of aptamers as nanomedical tools of potential interest for diagnostics and therapeutics. Aptamers are single stranded DNA or RNA oligonucleotides endowed with antibody-like properties. Aptamers fold into well-defined 3D structure, which is able to recognize a target molecule with high affinity and specificity. This feature allows many roles for aptamers in nanomedicine. In the simplest case, aptamers can vehiculate molecular payload into specific domains with high specificity and efficiency. Additionally, depending on the specific biomolecular target, aptamers can play a critical role as therapeutic agents, selectively interfering with the physiologic role of target sequences. My PhD research is mainly focused on the development of aptamers for in vitro and in vivo use. Reported aptamer sequences are first examined in order to increase the strictly oligonucleotide properties. Then, these enhanced sequences are engineered in order to develop efficient biomolecular architectures able to effectively recognize and target specific biomolecules. To date, the main aptamers investigated have been DNA and RNA aptamers that bind cell-surface receptors; particularly transmembrane proteins (e.g. transferrin receptor) that are highly express in some diseases, such as some solid tumors or in critical districts of the organism (e.g. blood brain barrier). The final aim of this work is the development of molecular systems aptamer-based that allow targeted drug delivery. Notably, in the first two years I focused my work in the development of an enhanced sequence of a DNA aptamer against mouse transferrin receptor (TfR). Particularly, a rational engineering approach was employed to study the folding properties of this DNA aptamer and to shed light on the critical domains involved in the active fold conformation. Mutated labeled sequences were tested both in vitro (by fluorescence anisotropy measurements) and in living cells (endocytosis assay monitored by confocal microscopy) in order to evaluate aptamer binding properties and internalization ability, respectively. Notably, a mutated DNA aptamer sequence showed higher affinity than the parent aptamer molecule and most importantly was able to bind both mouse and human TfR. Thus, this novel enhanced aptamer sequence represents a valid and promising tool for targeted drug delivery and in vivo applications (http://www.nature.com/mtna/journal/v3/n1/abs/mtna201371a.html). In the second part of my PhD research activity, I developed an aptamer nanocarrier to selectively deliver a traditional anticancer drug (Doxorubicin) in pancreatic tumor cells (MIA PaCa-2) reducing its off-target effect. Two structural domains constitute this nanocarrier:


(i) an anti-TfR aptamer as targeting motif and (ii) a DNA duplex region as drug loading region. Doxorubicin release was triggered by physiological stimuli found in endo-lysosomal compartment i.e. acid pH and nucleases activity. The cytotoxicity and the apoptotic effect of Doxorubicin were evaluated using two in vitro assays: cellular viability assay (WST-8) and Annexin-PI assay, respectively. Preliminary results showed that aptamer-based nanocarrier preferentially release Doxorubicin in cancer pancreatic cells (high TfR expression) thus reducing the uptake in healthy cells (low TfR expression).

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