Retroviral transduction and expansion of peripheral blood lymphocytes for the treatment of mucopolysaccharidosis type II, Hunter's syndrome

BACKGROUND: Gene therapy using autologous peripheral blood lymphocytes (PBL s) has been used to produce adenosine deaminase with which to treat patient s with severe combined immunodeficiency. Patients with mucopolysaccharidosi s type II (MPS II) lack iduronate-2-sulfatase (IDS), and serial PBL gene th erapy may benefit these patients. STUDY DESIGN AND METHODS: The purpose of these studies was to develop a met hod to transduce PBLs from a patient with MPS If by using a retroviral vect or, LS2N, containing the IDS gene. PBLs were collected by apheresis and cry opreserved in aliquots for the performance of multiple transductions and ex pansions. The PBLs were expanded in number and then transduced in a hollow- fiber bioreactor (HFBR). Additional culture allowed for further expansion. RESULTS: Fresh PBLs (6.2 x 10(7)) from a patient with MPS II were transduce d with L2SN and expanded in an HFBR with an extracapillary space of 11 mL. After 10 days of culture, 4.1 x 10(9) cells were harvested. Cryopreserved M PS It PBLs could not be reliably expanded in they were placed in the HFBR i mmediately after being thawed; however, cells were successfully transduced and expanded in the HFBR if they were first cultured in a bag. To increase the cell yield, PBLs were expanded in a 60-mL HFBR after transduction and e xpansion in an 11-mL HFBR. In four separate experiments, 2 x 10(8) cryopres erved PBL were cultured for 3 days in a bag and transferred to an 11-mL HFB R, where they were transduced daily with L2SN for 3 days and then expanded for 4 additional days. Cells were then transferred into a 60-mL HFBR and ex panded for an additional 7 days. In the four experiments, 5.5 x 10(9), 7.4 x 10(9), 1.12 x 10(9), and 19.4 x 10(9) cells were produced. The vector was detected in the harvested cells, but the proportion of cells transduced wa s less than 2.5 percent, the lowest standard used in the assay. In two of t he experiments, cells harvested from the HFBR were used in a gene therapy c linical trial. CONCLUSION: Autologous cryopreserved PBLs can be transduced and expanded to produce >1 x 10(10) cells. This procedure is being used for a Phase I/II c linical trial of lymphocyte gene therapy.