Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy
Alessandro Aiuti, Barbara Cassani, Grazia Andolfi, Massimiliano Mirolo, Luca Biasco, Alessandra Recchia, Fabrizia Urbinati, Cristina Valacca, Samantha Scaramuzza, Memet Aker, Shimon Slavin, Matteo Cazzola, Daniela Sartori, Alessandro Ambrosi, Clelia Di Serio, Maria Grazia Roncarolo, Fulvio Mavilio, Claudio Bordignon
Alessandro Aiuti, Barbara Cassani, Grazia Andolfi, Massimiliano Mirolo, Luca Biasco, Alessandra Recchia, Fabrizia Urbinati, Cristina Valacca, Samantha Scaramuzza, Memet Aker, Shimon Slavin, Matteo Cazzola, Daniela Sartori, Alessandro Ambrosi, Clelia Di Serio, Maria Grazia Roncarolo, Fulvio Mavilio, Claudio Bordignon
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Research Article

Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy

Abstract

Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase–deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34+ cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy.

Authors

Alessandro Aiuti, Barbara Cassani, Grazia Andolfi, Massimiliano Mirolo, Luca Biasco, Alessandra Recchia, Fabrizia Urbinati, Cristina Valacca, Samantha Scaramuzza, Memet Aker, Shimon Slavin, Matteo Cazzola, Daniela Sartori, Alessandro Ambrosi, Clelia Di Serio, Maria Grazia Roncarolo, Fulvio Mavilio, Claudio Bordignon

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Figure 5

Vector clonal composition in distinct hematopoietic lineages after GT.

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Vector clonal composition in distinct hematopoietic lineages after GT. (...
(A) Clonal analysis by LM-PCR and I-PCR in purified CD3+ T cells and CD15+ granulocytes from 5 patients with ADA-SCID treated with GT. Nested PCR products were analyzed by electrophoresis on acrilamide gels with a 100-bp ladder as a marker (M). In Pt2, who received a low CD34+ cell dose and displayed poor myeloid engraftment (<0.1%), no RISs could be isolated from multiple samples of granulocytes, thus confirming the lack of potential contamination from T cells. The following representative time points are shown for LM-PCR in T cells: Pt1, 20 months; Pt2, 39 months; Pt3, 26 months; Pt4, 22 months; Pt5, 9 months. The following representative time points are shown for LM-PCR in granulocytes: Pt1, 47 months; Pt3, 33 months; Pt4, 22 months; Pt5, 12 months. The following representative time points are shown for I-PCR in granulocytes: Pt1, 5 months, Pt3, 26 months; Pt4, 4 months; Pt5, 3 months. (B) Evidence for common integrants identified in different cell subpopulations during long-term follow-up. Each RIS from Pt1, Pt2, Pt3, or Pt4 is identified by a unique number. The circles indicate the presence of a specific RIS detected after 6 months of follow-up at 2 or more different time points by random cloning in the indicated population. Numbers in red text refer to the number of independent RISs retrieved by random cloning for the time points in which ≥30 colonies were analyzed. The random cloning analysis in Pt1 (47 months) and Pt3 (33 months) T cells was not carried out because the material was not available. In Pt5, 1 common integration site was detected in T cells at 6 and 8 months after GT. The presence of common integrants was confirmed in selected cases by sequence-specific PCR, which allowed us to unequivocally track integrants in different lineages.