Gene replacement by integrating vectors has been successfully used to treat several inherited diseases, such as Lysosomal Storage Disorders (LSD), Thalassemia and Primary Immunodeficiencies (PIDs). X-linked Combined Immunodeficiency (SCID-X1) is a fatal monogenic disorder, caused by mutation of the Interleukin 2 Receptor common γ-chain (IL2RG) gene. For SCID-X1, the early clinical studies have clearly shown the therapeutic potential of integrating vector based gene replacement therapy, which achieved efficient lymphoid reconstitution thanks to the selective growth advantage of the genetically modified cells. However, these studies also highlighted the potential risk of insertional mutagenesis due to random integration of the vector into the host cell genome and to unregulated transgene expression, thus calling for the development of safer gene therapy approaches. Here, by combining the Zinc Finger Nuclease (ZFNs) technology to induce site-specific DNA double-strand breaks (DSB) and of Integrase-Defective Lentiviral Vector (IDLV) to deliver a corrective donor template, we exploited Homology Driven Repair (HDR) to correct SCID-X1 mutation in situ, restoring both physiological expression and function of the IL2RG gene . By knocking-in a corrective IL2RG cDNA transgene downstream of its endogenous promoter in B-lymphoblastoid cells, which constitutively express IL2RG, and in primary T-lymphocytes, which requires IL2RG for their survival and growth, we provide evidence of physiologic activity of the gene-edited IL2RG gene. By including an excisable GFP- or a Puromycin Resistance (PuroR) expression cassette downstream of the corrective cDNA, we coupled correction with exogenous selection of corrected SCID-X1 primary fibroblasts, which do not physiologically express IL2RG, and obtained an enriched population of gene-corrected cells. We then reverted this population to pluripotency by using a novel reprogramming vector that expresses OCT4, SOX2, KLF4 and microRNA cluster 302-367 to obtain a potentially unlimited source of gene-corrected induced pluripotent stem cells (iPSC). We thus generated several gene-corrected bona-fide iPSCs, as confirmed by molecular analyses for targeted integration, which were characterized for their pluripotent state. IDLV-mediated transient delivery of the Cre-recombinase resulted in the co-excision of the reprogramming vector together with the selector cassette, thus allowing the generation of several gene-corrected, reprogramming-factor free iPSCs with normal karyotypes. Finally, by differentiating corrected iPSC to T-lymphoid progenitor cells, which are lacking in SCID-X1 patients, and showing a selective growth advantage of those derived from corrected iPSCs, we provide evidence of the functional correction of the IL2RG mutant allele. Overall these data demonstrate the feasibility of our targeted gene editing strategy, which couples gene correction with cell reprogramming to generate disease-free IPSC, thus paving the way for the development of novel and safer therapeutic approaches for SCID-X1.

La terapia genica basata sull’utilizzo di vettori integranti è stata già applicata con successo per la cura di varie malattie genetiche come le malattie da accumulo lisosomiale (LSD), la beta-talassemia (β-Thal) e le immunodeficienze primarie (PID). L’immunodeficienza combinata grave legata al cromosoma X (SCID-X1) è una malattia monogenica letale causata da mutazioni del gene codificante la catena comune gamma del recettore per l’interleuchina 2 (IL2RG). I primi studi clinici per la SCID-X1 hanno mostrato il potenziale terapeutico della terapia genica basata su vettori integranti, risultando nella ricostituzione del compartimento linfoide grazie al vantaggio selettivo delle cellule geneticamente modificate. D’altra parte, tali studi hanno evidenziato il rischio di mutagenesi inserzionale dovuto all’integrazione casuale del virus nel genoma della cellula ospite e all’espressione non regolata del transgene, sottolineando la necessità di sviluppare nuove strategie di terapia genica più sicure. In questo lavoro, sfruttando la tecnologia delle Zinc-Finger Nucleasi (ZFN) per indurre una rottura del doppio filamento del DNA in maniera sito specifica e dei vettori lentivirali difettivi per l’integrazione (IDLV) per l’introduzione di un templato donatore, abbiamo impiegato il processo di riparazione del DNA guidata dall’omologia per la correzione delle mutazioni che causano la SCID-X1, ripristinando così la funzione genica e l’espressione fisiologica del gene IL2RG. Mediante l’integrazione di un cDNA correttivo del gene IL2RG a valle del promotore endogeno sia in cellule B linfoblastodi, che esprimono costitutivamente la catena gamma comune, sia in linfociti T da donatori sani, che richiedono IL2RG per la loro sopravvivenza, abbiamo dimostrato la funzionalità e l’attività fisiologica del gene modificato. Abbiamo quindi accoppiato la correzione genica con la selezione delle cellule mediante l’inclusione di una cassetta excidibile di espressione della GFP o della resistenza alla puromicina (PuroR) a valle del cDNA correttivo, al fine di correggere fibroblasti, che normalmente non esprimono IL2R, derivati da pazienti SCID-X1. Abbiamo quindi ottenuto una popolazione di fibroblasti corretti che abbiamo “ riprogrammato” mediante un nuovo vettore di reprogramming che esprime i fattori di trascrizione (SOX2, OCT4, KLf4) e il microRNA cluster 367, generando così una fonte illimitata di cellule staminali pluripotenti indotte (iPSC) geneticamente corrette di interesse terapeutico. L’espressione transiente della Cre-ricombinasi mediante IDLV ha inoltre permesso l’excisione del vettore di reprogramming e della cassetta di selezione, permettendo così l’ottenimento di cellule iPSC corrette, prive di vettore e con un normale cariotipo. Infine, attraverso il differenziamento delle cellule iPSC in progenitori T-linfoidi, un tipo cellulare assente nei pazienti SCID-X1, e l’osservazione di un vantaggio selettivo delle cellule linfoidi derivate dalle iPSC corrette, abbiamo dimostrato la correzione funzionale dell’allele IL2RG mutato. In conclusione questi dati dimostrano la validità della nostra strategia di integrazione sito-specifica che, mediante la correzione e la riprogrammazione cellulare, consente di ottenere cellule iPSC geneticamente corrette, aprendo la strada a nuove opportunità terapeutiche più sicure per il trattamento della SCID-X1.

(2015). Targeted Gene Correction and Reprogramming of SCID-X1 Fibroblasts to Rescue IL2RG Expression in iPSC-derived Hematopoietic Cells. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).

Targeted Gene Correction and Reprogramming of SCID-X1 Fibroblasts to Rescue IL2RG Expression in iPSC-derived Hematopoietic Cells

FIRRITO, CLAUDIA
2015

Abstract

Gene replacement by integrating vectors has been successfully used to treat several inherited diseases, such as Lysosomal Storage Disorders (LSD), Thalassemia and Primary Immunodeficiencies (PIDs). X-linked Combined Immunodeficiency (SCID-X1) is a fatal monogenic disorder, caused by mutation of the Interleukin 2 Receptor common γ-chain (IL2RG) gene. For SCID-X1, the early clinical studies have clearly shown the therapeutic potential of integrating vector based gene replacement therapy, which achieved efficient lymphoid reconstitution thanks to the selective growth advantage of the genetically modified cells. However, these studies also highlighted the potential risk of insertional mutagenesis due to random integration of the vector into the host cell genome and to unregulated transgene expression, thus calling for the development of safer gene therapy approaches. Here, by combining the Zinc Finger Nuclease (ZFNs) technology to induce site-specific DNA double-strand breaks (DSB) and of Integrase-Defective Lentiviral Vector (IDLV) to deliver a corrective donor template, we exploited Homology Driven Repair (HDR) to correct SCID-X1 mutation in situ, restoring both physiological expression and function of the IL2RG gene . By knocking-in a corrective IL2RG cDNA transgene downstream of its endogenous promoter in B-lymphoblastoid cells, which constitutively express IL2RG, and in primary T-lymphocytes, which requires IL2RG for their survival and growth, we provide evidence of physiologic activity of the gene-edited IL2RG gene. By including an excisable GFP- or a Puromycin Resistance (PuroR) expression cassette downstream of the corrective cDNA, we coupled correction with exogenous selection of corrected SCID-X1 primary fibroblasts, which do not physiologically express IL2RG, and obtained an enriched population of gene-corrected cells. We then reverted this population to pluripotency by using a novel reprogramming vector that expresses OCT4, SOX2, KLF4 and microRNA cluster 302-367 to obtain a potentially unlimited source of gene-corrected induced pluripotent stem cells (iPSC). We thus generated several gene-corrected bona-fide iPSCs, as confirmed by molecular analyses for targeted integration, which were characterized for their pluripotent state. IDLV-mediated transient delivery of the Cre-recombinase resulted in the co-excision of the reprogramming vector together with the selector cassette, thus allowing the generation of several gene-corrected, reprogramming-factor free iPSCs with normal karyotypes. Finally, by differentiating corrected iPSC to T-lymphoid progenitor cells, which are lacking in SCID-X1 patients, and showing a selective growth advantage of those derived from corrected iPSCs, we provide evidence of the functional correction of the IL2RG mutant allele. Overall these data demonstrate the feasibility of our targeted gene editing strategy, which couples gene correction with cell reprogramming to generate disease-free IPSC, thus paving the way for the development of novel and safer therapeutic approaches for SCID-X1.
FERRARI, GIULIANA
endonucleases/; iPSC/; lentiviral vector; lentivirali
endonucleasi; iPSC; vettori lentivirali
BIO/11 - BIOLOGIA MOLECOLARE
English
30-nov-2015
Scuola di Dottorato in Medicina Traslazionale e Molecolare
SCUOLA DI DOTTORATO IN MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 72R
28
2014/2015
open
(2015). Targeted Gene Correction and Reprogramming of SCID-X1 Fibroblasts to Rescue IL2RG Expression in iPSC-derived Hematopoietic Cells. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/94656
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