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 2

RIS hot spots in patients with ADA-SCID without clonal expansion or overexpression.

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RIS hot spots in patients with ADA-SCID without clonal expansion or over...
(A) Schematic representation of RISs in the most frequently hit genes. Additional RISs outside these intervals in the proximity of CCND2 and BCL2 are also indicated. In vitro RISs are shown as green arrows and in vivo RISs as blue arrows; the direction of the arrow denotes orientation of the retroviral vector. The upper or lower position of the arrow refers to an LTR-genomic junction sequence mapping to the positive (above line) or negative (below line) chromosomal strand, respectively. Genes are depicted in red, with filled boxes and red arrows indicating exons and TSSs, respectively. RISs in the LMO2 locus were detected in T cells (Pt1, S1_049, 27 months after GT; Pt3, S3_042, 18 months after GT; Pt4, S4_048, 26 months after GT), granulocytes (Pt5, S5_144 and S5_163, 6 and 12 months after GT, respectively), and in Pt5 preinfusion CD34+ cells (S5_P048). (B) Quantification of the LMO2-containing clones within the total T cell population. The frequency of insertions was measured by sequence-specific real-time PCR in purified CD3+ T cells isolated at different time points after GT. Dotted lines show the frequencies of nonrecurrent (neutral) RISs retrieved from Pt3 as a control of physiologic clonal fluctuations. (C) Expression of LMO2 in peripheral blood T cells and granulocytes. Total RNA was isolated from the indicated cell subsets of GT-treated patients, patients with ADA-SCID undergoing polyethylene glycol-modified bovine ADA treatment (UT, not treated with GT), or age-matched healthy controls (ND). LMO2 gene expression was measured by quantitative RT-PCR using the standard curve of a reference sample and normalized for the expression of the housekeeping gene HPRT.