Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection.
Retroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 10(7) transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common gamma chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 x 10(7) TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).
Throm
Robert E
RE
Department of Hematology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
Ouma
Annastasia A
AA
Zhou
Sheng
S
Chandrasekaran
Anantharaman
A
Lockey
Timothy
T
Greene
Michael
M
De Ravin
Suk See
SS
Moayeri
Morvarid
M
Malech
Harry L
HL
Sorrentino
Brian P
BP
Gray
John T
JT
D002460:Cell Line; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006678:HIV / Q000235:genetics; D006801:Humans; D053631:Interleukin Receptor Common gamma Subunit / Q000008:administration & dosage* / Q000235:genetics; D016511:Severe Combined Immunodeficiency / Q000628:therapy*; D020079:Terminal Repeat Sequences; D014162:Transfection / Q000379:methods*
C507941:IL2RG protein, human; D053631:Interleukin Receptor Common gamma Subunit; D049452:Green Fluorescent Proteins