Integration of retroviral vectors induces minor changes in the transcriptional activity of T cells from ADA-SCID patients treated with gene therapy.
Gene transfer into hematopoietic stem cells by gamma-retroviral vectors (RVs) is an effective treatment for inherited blood disorders, although potentially limited by the risk of insertional mutagenesis. We evaluated the genomic impact of RV integration in T lymphocytes from adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) patients 10 to 30 months after infusion of autologous, genetically corrected CD34(+) cells. Expression profiling on ex vivo T-cell bulk population revealed no difference with respect to healthy controls. To assess the effect of vector integration on gene expression at the single-cell level, primary T-cell clones were isolated from 2 patients. T-cell clones harbored either 1 (89.8%) or 2 (10.2%) vector copies per cell and displayed partial to full correction of ADA expression, purine metabolism, and T-cell receptor-driven functions. Analysis of RV integration sites indicated a high diversity in T-cell origin, consistently with the polyclonal T-cell receptor-Vbeta repertoire. Quantitative transcript analysis of 120 genes within a 200-kb window around RV integration sites showed modest (2.8- to 5.2-fold) dysregulation of 5.8% genes in 18.6% of the T-cell clones compared with controls. Nonetheless, affected clones maintained a stable phenotype and normal in vitro functions. These results confirm that RV-mediated gene transfer for ADA-SCID is safe, and provide crucial information for the development of future gene therapy protocols. The trials described herein have been registered at http://www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.
Cassani
Barbara
B
San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy.
Montini
Eugenio
E
Maruggi
Giulietta
G
Ambrosi
Alessandro
A
Mirolo
Massimiliano
M
Selleri
Silvia
S
Biral
Erika
E
Frugnoli
Ilaria
I
Hernandez-Trujillo
Vivian
V
Di Serio
Clelia
C
Roncarolo
Maria Grazia
MG
Naldini
Luigi
L
Mavilio
Fulvio
F
Aiuti
Alessandro
A
D000243:Adenosine Deaminase / Q000172:deficiency / Q000235:genetics*; D018952:Antigens, CD34; D014408:Biomarkers, Tumor / Q000235:genetics; D002478:Cells, Cultured; D020869:Gene Expression Profiling; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000627:therapeutic use*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D011687:Purines / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016511:Severe Combined Immunodeficiency / Q000201:enzymology / Q000235:genetics / Q000628:therapy*; D013601:T-Lymphocytes / Q000378:metabolism / Q000473:pathology*; D014161:Transduction, Genetic; D016662:Virus Integration
D018952:Antigens, CD34; D014408:Biomarkers, Tumor; D011687:Purines; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; D000243:Adenosine Deaminase; C030985:purine