logo SBA

ETD

Archivio digitale delle tesi discusse presso l’Università di Pisa

Tesi etd-11242014-013833


Tipo di tesi
Tesi di laurea magistrale
Autore
TAHERINASAB, AKRAM
URN
etd-11242014-013833
Titolo
Excision of HIV-1 provirus as novel approach to eradicate the infection
Dipartimento
BIOLOGIA
Corso di studi
BIOTECNOLOGIE MOLECOLARI E INDUSTRIALI
Relatori
relatore Prof. Pistello, Mauro
Parole chiave
  • eradicate
  • gene therapy
  • HIV
  • TALEN
Data inizio appello
11/12/2014
Consultabilità
Non consultabile
Data di rilascio
11/12/2084
Riassunto
Background: Despite potent antiretroviral drugs, HIV-1 infection cannot be eliminated and patients must be treated lifetime to avoid AIDS-related illnesses. Decades of exposure to antiretroviral drugs may cause severe metabolic disorders and lead to a chronic HIV disease with various immunodeficiency complications and other clinically relevant comorbidities. Novel strategies to eradicate the infection are clearly needed. Gene editing is an emerging technology that inserts, replaces, or deletes pieces of DNA from a genome. Engineering is achieved by means of artificially constructed endonucleases that bind specific sequences and work as molecular scissors.
Aim: To develop Transcription Activator Tale Effector Nucleases (TALEN)to cut off (excise) the HIV-1 provirus from infected cells.
Methods: We designed and constructed TALEN targeting an internal conserved region of HIV-1 LTRs. TALEN were tested for specificity and efficiency of cleavage in cells transduced with replication competent HIV-1 molecular clones produced ad hoc and carrying green fluorescent protein(GFP) and luciferase (Luc). Proviral excision was measured by flow cytometry, western blot, and Luc assay. Fate of excised HIV-1 genome and repair of host cell genome were monitored by rolling circle amplification and standard molecular assays.Mock and irrelevant TALEN (irTALEN) transduced cells were tested in parallel as controls.
Results: Compared to pre-treatment levels, TALEN reduced GFP and Luc activities to 20% and 15% at day 3 and 7 post-treatment (pt), respectively. GFP became undetectable by western blot from day 3, and Luc activity was almost negligible at day 7. No reduction was observed in irTALEN treated cells. Circular, excised HIV-1 DNA was monitored for molecular conformation , persistence, integration activity, and GFP/Luc expression up to one month pt. Excised provirus , mostly organized in concatamers, devoid of LRTs –and therefore unable to integrate -, and with no expression activity persisted for several weeks. Genome DNA double strand breaks were repaired by cellular DNA repair enzymes.
Conclusion. TALEN testing against clinical and laboratory isolates will commence shortly. Activity Should be confirmed, this proof-of-concept study will pave the way to exploit gene editing to cure HIV-1 infected cells.
File