Liver-directed gene therapy using adeno-associated viral (AAV) vectors holds significant promise for providing long-term transgene expression following a single systhemic administration. However, both the limited cargo capacity (~4.7 kb) and the non-integrative nature of AAV vectors prevent their application to conditions caused by either mutations in large genes (i.e. F8 whose coding sequence exceeds ~ 4.7 kb) or to those that require early stage of intervention, such as early-onset inborn errors of metabolism. My PhD project aims to overcome these shortcomings and broaden the therapeutic applications of AAVs. To overcome the issue of limited cargo capacity, I explored an AAV-mediated protein trans-splicing (PTS) strategy utilizing split inteins. PTS is a natural mechanism employed by organisms across all domains of life to reconstitute large proteins from shorter precursors. Split inteins facilitate this process by selfexcising from the host protein in an energy-independent manner. I used this approach for haemophilia A (HemA), the most common X-linked bleeding disorder (affecting 1 in 5,000 males), caused by a deficiency of clotting factor 8 (F8, ~7 kb). I first demonstrated the successful full-length reconstitution of the large (~5 kb) and highly active B-domain deleted (B-DD) N6-F8 variant in vitro. Subsequently, I designed two separate AAV vectors, with regular size genomes, each encoding for half of the N6-F8, flanked by split inteins. The AAV-N6 intein system led to successful reconstitution of functional full-length N6-F8 in the mouse liver, achieving therapeutic F8 activity in haemophilic mice without eliciting anti-F8 immune responses. In parallel, I developed an AAV-mediated CRISPR/Cas9 homology-independent targeted integration (AAV-HITI) platform to achieve stable transgene integration at the 3’ end of the mouse Albumin locus, which is highly expressed in hepatocytes. This approach mitigates the loss of episomal AAV genomes in proliferative tissues, thereby preserving therapeutic efficacy over time. 14 I demonstrated AAV-HITI efficacy, in mouse models of HemA and Mucopolysaccharidosis type VI (MPS VI), a lysosomal storage disorder, achieving stable therapeutic levels of systemic proteins even at low AAV doses. Importantly, I performed comprehensive molecular analyses to characterize the AAV-HITI outcomes within the target site confirming successful targeted integration. Moreover, in AAV-HITI treated livers nor gross chromosomal rearrangements or significant off-target insertions/deletions were observed 1-year post neonatal delivery. Furthermore, I demonstrated the effectiveness of this platorm in adult MPS VI and HemA mice, using vector doses that are within the safe range for human translation. Overall, my findings support two distinct AAV-mediated liver-directed platforms to address major limitations of current AAV-based therapies. The AAV-intein N6-F8 system holds promise as a potential therapeutic option for HemA, while the AAV-HITI platform could serve as a durable therapeutic option for livertargeted diseases, particularly those requiring early intervention. These innovations represent significant advancements in overcoming challenges related to cargo capacity, stable transgene expression, and long-term efficacy in gene therapy.
ADVANCED AAV-MEDIATED LIVER-DIRECTED GENE THERAPIES FOR HAEMOPHILIA A AND MUCOPOLYSACCHARIDOSIS TYPE VI / F. Esposito ; tutor: A. Auricchio ; internal supervisor: N. Brunetti-Pierri ; external supervisor: A. Follenzi ; coordinator: S. Minucci. Dipartimento di Oncologia ed Emato-Oncologia, 2024 Dec. 36. ciclo, Anno Accademico 2023/2024.
ADVANCED AAV-MEDIATED LIVER-DIRECTED GENE THERAPIES FOR HAEMOPHILIA A AND MUCOPOLYSACCHARIDOSIS TYPE VI
F. Esposito
2024
Abstract
Liver-directed gene therapy using adeno-associated viral (AAV) vectors holds significant promise for providing long-term transgene expression following a single systhemic administration. However, both the limited cargo capacity (~4.7 kb) and the non-integrative nature of AAV vectors prevent their application to conditions caused by either mutations in large genes (i.e. F8 whose coding sequence exceeds ~ 4.7 kb) or to those that require early stage of intervention, such as early-onset inborn errors of metabolism. My PhD project aims to overcome these shortcomings and broaden the therapeutic applications of AAVs. To overcome the issue of limited cargo capacity, I explored an AAV-mediated protein trans-splicing (PTS) strategy utilizing split inteins. PTS is a natural mechanism employed by organisms across all domains of life to reconstitute large proteins from shorter precursors. Split inteins facilitate this process by selfexcising from the host protein in an energy-independent manner. I used this approach for haemophilia A (HemA), the most common X-linked bleeding disorder (affecting 1 in 5,000 males), caused by a deficiency of clotting factor 8 (F8, ~7 kb). I first demonstrated the successful full-length reconstitution of the large (~5 kb) and highly active B-domain deleted (B-DD) N6-F8 variant in vitro. Subsequently, I designed two separate AAV vectors, with regular size genomes, each encoding for half of the N6-F8, flanked by split inteins. The AAV-N6 intein system led to successful reconstitution of functional full-length N6-F8 in the mouse liver, achieving therapeutic F8 activity in haemophilic mice without eliciting anti-F8 immune responses. In parallel, I developed an AAV-mediated CRISPR/Cas9 homology-independent targeted integration (AAV-HITI) platform to achieve stable transgene integration at the 3’ end of the mouse Albumin locus, which is highly expressed in hepatocytes. This approach mitigates the loss of episomal AAV genomes in proliferative tissues, thereby preserving therapeutic efficacy over time. 14 I demonstrated AAV-HITI efficacy, in mouse models of HemA and Mucopolysaccharidosis type VI (MPS VI), a lysosomal storage disorder, achieving stable therapeutic levels of systemic proteins even at low AAV doses. Importantly, I performed comprehensive molecular analyses to characterize the AAV-HITI outcomes within the target site confirming successful targeted integration. Moreover, in AAV-HITI treated livers nor gross chromosomal rearrangements or significant off-target insertions/deletions were observed 1-year post neonatal delivery. Furthermore, I demonstrated the effectiveness of this platorm in adult MPS VI and HemA mice, using vector doses that are within the safe range for human translation. Overall, my findings support two distinct AAV-mediated liver-directed platforms to address major limitations of current AAV-based therapies. The AAV-intein N6-F8 system holds promise as a potential therapeutic option for HemA, while the AAV-HITI platform could serve as a durable therapeutic option for livertargeted diseases, particularly those requiring early intervention. These innovations represent significant advancements in overcoming challenges related to cargo capacity, stable transgene expression, and long-term efficacy in gene therapy.File | Dimensione | Formato | |
---|---|---|---|
phd_unimi_R13154_1.pdf
accesso aperto
Tipologia:
Altro
Dimensione
10.05 MB
Formato
Adobe PDF
|
10.05 MB | Adobe PDF | Visualizza/Apri |
phd_unimi_R13154_2.pdf
accesso aperto
Tipologia:
Altro
Dimensione
10.16 MB
Formato
Adobe PDF
|
10.16 MB | Adobe PDF | Visualizza/Apri |
phd_unimi_R13154_3.pdf
accesso aperto
Tipologia:
Altro
Dimensione
10.09 MB
Formato
Adobe PDF
|
10.09 MB | Adobe PDF | Visualizza/Apri |
Pubblicazioni consigliate
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.