Feline leukemia virus integrase and capsid packaging functions do not change the insertion profile of standard Moloney retroviral vectors.
Adverse events linked to perturbations of cellular genes by vector insertion reported in gene therapy trials and animal models have prompted attempts to better understand the mechanisms directing viral vector integration. The integration profiles of vectors based on MLV, ASLV, SIV and HIV have all been shown to be non-random, and novel vectors with a safer integration pattern have been sought. Recently, we developed a producer cell line called CatPac that packages standard MoMLV vectors with feline leukemia virus (FeLV) gag, pol and env gene products. We now report the integration profile of this vector, asking if the FeLV integrase and capsid proteins could modify the MoMLV integration profile, potentially resulting in a less genotoxic pattern. We transduced rhesus macaque CD34+ hematopoietic progenitor cells with CatPac or standard MoMLV vectors, and determined their integration profile by LAM-PCR. We obtained 184 and 175 unique integration sites (ISs) respectively for CatPac and standard MoMLV vectors, and these were compared with 10 000 in silico-generated random IS. The integration profile for CatPac vector was similar to MoMLV and equally non-random, with a propensity for integration near transcription start sites and in highly dense gene regions. We found an IS for CatPac vector localized 715 nucleotides upstream of LMO-2, the gene involved in the acute lymphoblastic leukemia developed by X-SCID patients treated by gene therapy using MoMLV vectors. In conclusion, we found that replacement of MoMLV env, gag and pol gene products with FeLV did not alter the basic integration profile. Thus, there appears to be no safety advantage for this packaging system. However, considering the stability and efficacy of CatPac vectors, further development is warranted, using potentially safer vector backbones, for instance those with a SIN configuration.
Métais
J-Y
JY
Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Topp
S
S
Doty
R T
RT
Borate
B
B
Nguyen
A-D
AD
Wolfsberg
T G
TG
Abkowitz
J L
JL
Dunbar
C E
CE
D000818:Animals; D002213:Capsid; D036022:Capsid Proteins / Q000235:genetics; D018014:Gene Transfer Techniques / Q000009:adverse effects*; D005822:Genetic Vectors / Q000009:adverse effects*; D006412:Hematopoietic Stem Cells / Q000821:virology*; D019426:Integrases / Q000235:genetics*; D005256:Leukemia Virus, Feline / Q000235:genetics* / Q000378:metabolism; D008253:Macaca mulatta; D008979:Moloney murine leukemia virus / Q000235:genetics*; D014161:Transduction, Genetic; D016662:Virus Integration
D036022:Capsid Proteins; D019426:Integrases