AIMS AND CONTENT

LEARNING OUTCOMES

Module "Molecular Bases of Pathologies"

The course is aimed at exploring specific example pathologies as consequences of   molecular alterations, with particular focus on tumors, and at illustrating novel methodological strategies to investigate disease pathogenetic mechanisms and identify new therapeutic targets.

Module "Gene Therapy"

To allow the student to acquire a detailed knowledge of the current and up-to date gene transfer and therapy approaches, along with their evolution in clinical settings,  and to correlate them with the current knowledge on physiology, molecular biology and genetic engineering technologies; to allow the student to envisage possible future applications of gene therapy based on the current knowledge of monogenic diseases with a relevant clinical prevalence.

TEACHING METHODS

Frontal lessons.

SYLLABUS/CONTENT

Module "Molecular Bases of Pathologies"

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Module "Gene Therapy"

Gene therapy based clinical trials: principles, requirements and optimization: geographical distribution, social and economical aspects and links to the present status of the sanitary system of the industrialized countries; essential principles of the gene therapy approaches and choice of the therapeutic target; application criteria; examples of gene therapy in the clinics: the adenosine deaminase deficiency syndrome (ADA), the severe combined X-linked immunodeficiency syndrome (SCID/X-SCID), Junctional Epidermolysis Bullosa (JEB) and their results and adverse events.

Vireal vectors used in gene therapy approaches: structures and characteristics, use, advantages and/or disadvantages

Herpesviridae:structural characteristics of the viral capsid, of the matrix and envelope proteins; structure and functions of the viral genome: immediate-early genes (IE), early (E) and late (L) genes; viral proteins: structure and characteristics; lytic and lysogenic cycles; viral tropism; helper virus-based and helper virus-free packaging systems; transgene insertion in the HSV vector; viral vector optimization to target cellular sub-types; function of the gB and gD proteins.

Sindbin Virus and Semliki Forest Viruses (SFV): their use as suicidal vectors.

Adenoviridae: capsid structure; functions of the viral genome: E1, E2, and E3 genes, ITR and psi (ψ) regions; effects of the proteins involved in viral protection; risks associated to the clinical use of adenoviral-based vectors; exploitation of the adenoviral vectors through genetic engineering of the capsidic proteins (ex.: RGBs), through promotor-specific activity (ex. tumor-associated promoters), through “pro-drug zymes”, through “vector-spread molecules”and through anti-tumor molecules; pre-clinical studies: positive and negative aspects derived from murine models.

Adeno-associated viruses (AAV): structure of the viral genome and its replication; requirements of the helper virus and of the packaging phase.

Retroviridae: viral particle structure; integrated form; structure of the retroviral genome and of the coded proteins; viral genome replication: LTR duplication of the integrated form; helper viruses for packaging cell lines: strategies and advantages; double-copy “minigene” vectors; packaging lines for retroviral vectors: characteristics, advantages and disadvantages; packaging lines with bi- and tripartite helper viruses; implementation of viral tropism: used strategies.

Lentiviridae: virion structure; function of the lentiviral proteins and comparison with retroviral proteins: accessory proteins; structure and functions of Tet, Rev, Vif, Vpi, Vpx, Vpu and Nef; genome structure and mandatory sequences for gene therapy applications: cPPT, TAR, RRE; role of Nef/CD4, Tat/NfKb and SMARs sequences; viral vectors optimization and packaging cell lines; “self-inactivating (SIN)” vectors and “integrase-mutated” vectors; methods of recovery of LV vectors: ultracentrifugation, poly-lysine-mediate precipitation, ionic-exchange chromatography; comparison between HSV, AV, AAV, Retroviruses and LV for therapeutic transfer of shRNA.

Non –viral approaches of gene therapy: gene transfer without viral vectors; naked DNA injection systems; jet-gas systems; gene-gun systems; electroporation; sonoporation; hydrodynamic transfer; chemical vectors; clinical applications: target tissue characteristics of the non-viral applications in gene therapy.

Examples of clinical applications of gene therapyin the treatment of : haematopoietic stem cells; muscular dystrophies; haemophilia; tumors; neurodegenerative diseases.

RECOMMENDED READING/BIBLIOGRAPHY

Module "Molecular Bases of Pathologies"

Alberts et al. “Biologia molecolare della cellula”,  Zanichelli ; Mendelsohn et al.,  “The molecular basis of cancer”, Elsevier; Robbins e Cotran, “Le basi patologiche delle malattie”, Elsevier.

Module "Gene Therapy"

Giacca M., “Terapia Genica”, Springer-Verlag Italia s.r.l.; Cann A.J., “Elementi di virologia molecolare”, Casa Editrice Ambrosiana.

TEACHERS AND EXAM BOARD

Exam Board

PAOLO GIANNONI (President)

GRAZIA MARIA SPAGGIARI (President)

LESSONS

LESSONS START

March 1, 2018

Class schedule

MOLECULAR BASIS OF DISEASE AND GENE THERAPY

EXAMS

EXAM DESCRIPTION

Oral.

ASSESSMENT METHODS

The student is examined orally; They are asked at least three questions on topics inherent in the form, of increasing difficulty; the first question is always related to a general topic; This allows the student to respond even if the preparation proves only be mnemonic. The second question instead provides a more specific evaluation of some aspects of the topic, and the answer implies, therefore, an organized understanding of the phenomena / mechanisms related to that specific topic (eg. In a pathology or using particular proteins / carriers ); the third question requires the student to make correlations between different course topics, by searching for and presenting independently and critically similarities, differences, similarities, advantages and / or disadvantages, so as to demonstrate a capacity of more extensive vision. The examination can proceed with further questions for further study, in particular to ascertain the proactiveness of the student to provide adequate solutions to possible pathological conditions amenable to different approaches. This aspect is emphasized during the course providing students with numerous examples of clinical applications that are discussed in chorus and critically.
Students are then invited to submit, albeit optional, two scientific papers (one per module) through critical review and presentation that is done collegially with all the teachers and other students of the course, in order to practice an interpretation of current literature, and to use the knowledge which were scanned during the course to motivate / duly criticize the choices made and the results obtained by the authors. This presentation is not mandatory but completes the preparation of the student and allows to increase the overall exam score if viewed positively by teachers.

Exam schedule