ETD

Archivio digitale delle tesi discusse presso l'Università di Pisa

Tesi etd-06272016-112236


Tipo di tesi
Tesi di laurea magistrale
Autore
MATTEOLI, GIULIA
URN
etd-06272016-112236
Titolo
Excision of human immunodeficiency virus as a novel approach to eradicate the infection: analysis of intracellular persistence and residual activity of the cleaved provirus within the cell.
Dipartimento
BIOLOGIA
Corso di studi
BIOTECNOLOGIE MOLECOLARI E INDUSTRIALI
Relatori
relatore Prof. Pistello, Mauro
Parole chiave
  • provirus
  • gene therapy
  • HIV
  • excision
Data inizio appello
18/07/2016
Consultabilità
Completa
Riassunto
The Human Immunodeficiency Virus (HIV) is a retrovirus evolved from the zoonotic infections with Simian Immunodeficiency Virus (SIV) that has been discovered in the 80s as the etiologic agent of the AIDS. HIV is the cause of the acquired immune deficiency syndrome (AIDS), which impairs the normal function of the immune system. Nowadays the only therapy available is the antiretroviral therapy (cART) that consists in a cocktail of drugs that interferes with different steps during the HIV replication cycle. The cART therapy has improved the life quality of patients, leading to low viral loads along with a normal CD4+ T-cell count, but cART cannot eradicate the infection. This is actually the main problem: latent infection develops when the virus became transcriptionally inactive, but a low viral production is still maintained, and some cells become viral reservoirs. Latent infection can shift to productive one and, if the cART is interrupted, the infection can spread again in a short time.

One strategy that can be used to remove the HIV from the host consists in the proviral genome eradication via gene editing techniques. Different gene editing (GE) techniques have been developed in the last years including the clustered regularly interspaced palindromic repeats (CRISPRs). The elimination of the proviral DNA from infected cells can be achieved targeting the proviral genome. The full-length proviral sequence can be excised targeting 5’ and 3’ long terminal repeats (LTRs). The CRISPR/Cas9 system cleaves efficiently these regions.

This work is focused on the elimination of the full proviral sequence and on which is the fate of the excised proviral sequence after the targeting of LTRs via CRISPR/Cas9 technique. We have observed that the excised provirus can be re-ligated together in tandem by the host reparation machinery creating circular structures named concatamers that we show persist for weeks after treatment.
The next step, and the main aim of this thesis, is to understand if these structures, that have a rearranged sequence of the LTRs, can have some level of transcription and protein expression.

To study this process we have isolated HIV concatamers in a stable way, creating concatamers with plasmid capabilities.

To understand concatamer activity we evaluated Pol and Gag RNA presence by RT PCR and p24 by ELISA immunoassay, transfecting 293T with concatamers. Our data show a residual transcription of concatamer. Then we evaluated the interference of concatamers with HIV-1 infected cells, transfecting concatamers in HIV-1 infected cells.

These data shed new lights on the fate of HIV concatamers and cast a warning sign on the whole approach. Should these data been confirmed, excision of provirus to cure HIV infection warrants further studies.
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