Sistema ETD

Archivio digitale delle tesi discusse presso l'Università di Pisa


Tesi etd-02112016-100137

Tipo di tesi
Tesi di laurea magistrale
Redirecting gamma-retroviral vectors for safer gene therapy A study on p12 fusion peptides to redirect retroviral vector integration preference
Corso di studi
relatore Prof. Pistello, Mauro
Parole chiave
  • vettori retrovirali
  • terapia genica
  • ottimizzazione trasduzione
  • suntag
Data inizio appello
Data di rilascio
Riassunto analitico
In the last twenty-five years gene therapy has been used successfully to cure life-threatening monogenic inherited disorders, like ADA-SCID, X-SCID, WAS syndrome, X-CGD and β-thalassaemia. Retroviral vectors are frequently used to deliver the therapeutic genes into patients, because of their stable integration. In this thesis I will focus on gammaretroviral-based vectors, based on the Murine Leukemia Virus (MLV) platform. Gammaretroviral vectors were the first vectors used in gene therapy applications and are among the best studied and characterized. Despite successful outcome in a wide variety of clinical trials, adverse events in a subset of patients (20%) resulted in the malignant proliferation of blood cells and leukemia, due to insertional mutagenesis. Indeed, MLV-based viral vectors preferentially integrate in active promoters, enhancers, DNase hypersensitive sites (DHS) and transcription start sites (TSS). Viral vector integration in these specific regions can occasionally lead to the deregulation of the transcription of genes nearby the integration site; for example when the integration takes place near proto-oncogenes (as LMO2) it can lead to oncogenesis.
Retroviral integration is not a random process, but is coordinated by cellular host factors that are co-opted by the invading viral PIC. The integration pattern of MLV and its derived viral vectors is determined by BET (bromo and ET-SEED domain containing family) proteins, which are cellular cofactors that dock to active enhancers and promoters in the chromatin and that can also bind the MLV integrase (IN). By binding to active enhancer and promoter regions, BET proteins tether the MLV viral vector integration to these regions. In 2013 it was proven that a single mutation in MLV IN (W390A) hampers the interaction between IN and BET proteins, generating a BET-independent integrase (Bin). Bin-MLV vectors have a more random and safer integration profile (from 20% to 10% integrations near TSS) compared to WT MLV vectors.
In addition to integrase, the viral particle depends on p12 for efficient integration in the invaded host cell.
Since MLV and derived vectors can’t pass the nuclear membrane, they require the p12 protein to tether the PIC to chromatin during mitosis, when the nuclear membrane is degraded.
In this thesis I evaluated the role of the viral p12 protein on the integration sites preference of the viral vector. Previous studies showed that mutation of p12 (PM14 mutant) hampers the ability of the p12 protein to tether the PIC to the mitotic chromatin, resulting in a dead MLV virus. Introduction of different alternative chromatin binding peptides in the C-terminal domain of the p12 protein rescued this lethal PM14 mutant. In this thesis I studied the role of the p12 chromatin interaction in integration sites choice. I generated several p12-fusions and combined these with either a wild-type IN or a Bin-IN protein. Assuming that the MLV IN and BET protein interaction plays a dominant role in determining the MLV integration profile, combination of the Bin-IN together with p12-peptide fusions, might lead to an additional shift of the integration of the MLV vector to more safe regions.
Briefly, I generated Bin-MLV vectors and WT vectors with the p12-PM14 protein complemented with various chromatin binding peptides. We chose to target chromatin in general with peptides that bind H2A-H2B nucleosome core histones (peptides derived from Kaposi Sarcoma-associated Herpes Virus and Prototype Foamy Virus) or the genomic DNA backbone (peptides derived from Human Papilloma Virus 8), as well as specific chromatin modifications, such as methylated histone side-chains (like Me3 of K36 at histone H3 that is recognized by a domain of LEDGF/p75).
MLV vectors were produced by using a GFP transfer plasmid, a VSV-G envelope plasmid and a packaging plasmid encoding the p12-peptide fusions. WT-IN MLV and W390A-IN MLV vectors were taken as control.
Both WT-IN and W390A-IN MLV vectors showed comparable transduction efficiencies, whereas the lethal PM14 p12 mutant showed no transduction, in line with previous studies. Genomic DNA of transduced SupT1 cells was also used to perform qPCR experiments to evaluate the number of integrated copies compared with the WT vector, and was used to prepare a library for Illumina Next Generation Sequencing (NGS). The protocol for library preparation was optimized and samples were sent for sequencing.
The data obtained in this thesis will help to make one more step towards the engineering of safer retroviral vectors for gene therapy.