Cytoreductive conditioning intensity predicts clonal diversity in ADA-SCID retroviral gene therapy patients

Aaron R. Cooper, Georgia R. Lill, Kit Shaw, Denise Carbonaro, Alejandra Davila, Robert Sokolic, Fabio Candotti, Matteo Pellegrini and Donald B. Kohn

Key points

  • Retroviral vector clonal diversity and T cell receptor diversity correlated with intensity of busulfan conditioning.

  • Some patients had stable dominant clones with retroviral vectors adjacent to known proto-oncogenes.


Retroviral gene therapy has proven efficacious for multiple genetic diseases of the hematopoietic system, but roughly half of clinical gene therapy trial protocols using gammaretroviral vectors have reported leukemias in some of the patients treated. In dramatic contrast, 39 ADA-SCID patients have been treated with four distinct gammaretroviral vectors without oncogenic consequence. We investigated clonal dynamics and diversity in a cohort of 15 ADA-SCID children treated with gammaretroviral vectors and found clear evidence of genotoxicity, indicated by numerous common integration sites near proto-oncogenes and by increased abundance of clones with integrations near MECOM and LMO2. These clones showed stable behavior over multiple years and never expanded to the point of dominance or dysplasia. One patient developed a benign clonal dominance that could not be attributed to insertional mutagenesis, and instead likely resulted from expansion of a transduced NK clone in response to chronic EBV viremia. Clonal diversity and T-cell repertoire, measured by vector integration site sequencing and T-cell receptor beta chain rearrangement sequencing, correlated significantly with the amount of busulfan preconditioning delivered to patients and to CD34+ cell dose. These data, in combination with results of other ADA-SCID gene therapy trials, suggest that disease background may be a crucial factor in leukemogenic potential of retroviral gene therapy, and underscore the importance of cytoreductive conditioning in this type of gene therapy approach.

  • Submitted December 13, 2016.
  • Accepted March 20, 2017.