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.