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Blood, 1 June 2006, Vol. 107, No. 11, pp. 4257-4265.
Prepublished online as a Blood First Edition Paper on February 9, 2006; DOI 10.1182/blood-2005-10-4047.
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GENE THERAPY
Proteasome activity restricts lentiviral gene transfer into hematopoietic stem cells and is down-regulated by cytokines that enhance transduction
Francesca Romana Santoni de Sio,
Paolo Cascio,
Anna Zingale,
Mauro Gasparini, and
Luigi Naldini
From the San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) and San Raffaele Vita Salute University, San Raffaele Scientific Institute, Milan; Department of Morphophysiology, School of Veterinary Medicine, University of Turin; Politecnico di Torino, Turin; and University Center for Statistics in the Biomedical Sciences, San Raffaele Vita-Salute University, Milan, Italy.
The therapeutic potential of hematopoietic stem cell (HSC) gene therapy can be fully exploited only by reaching efficient gene transfer into HSCs without compromising their biologic properties. Although HSCs can be transduced by HIV-derived lentiviral vectors (LVs) in short ex vivo culture, they display low permissivity to the vector, requiring cytokine stimulation to reach high-frequency transduction. Using stringent assays of competitive xenograft repopulation, we show that early-acting cytokines synergistically enhanced human HSC gene transfer by LVs without impairing engraftment and repopulation capacity. Using S-phase suicide assays, we show that transduction enhancement by cytokines was not dependent on cell cycle progression and that LVs can transduce quiescent HSCs. Pharmacologic inhibition of the proteasome during transduction dramatically enhanced HSC gene transfer, allowing the reach of very high levels of vector integration in their progeny in vivo. Thus, LVs are effectively restricted at a postentry step by the activity of this proteolytic complex. Unexpectedly, cytokine stimulation rapidly and substantially down-regulated proteasome activity in hematopoietic progenitors, highlighting one mechanism by which cytokines may enhance permissiveness to LV gene transfer. These findings demonstrate that antiviral responses ultimately mediated by proteasomes strongly limit the efficiency of HSC transduction by LVs and establish improved conditions for HSC-based gene therapy.
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