Development of a novel trans-lentiviral vector that affords predictable safety.
Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) hold great promise for gene therapy. However, the possibility of generating replication-competent retrovirus (RCR) through genetic recombination raises concerns for safety. Here we describe a novel HIV-based packaging system (trans-lentiviral) that splits gag/gag-pol into two parts: One that expresses gag/gag-pro and another that expresses reverse transcriptase and integrase as fusion partners of viral protein R (Vpr). Using a sensitive assay developed to specifically detect recombinant lentiviral DNA mobilization, we demonstrated that the trans-lentiviral vector prevents the generation of recombinants that contain a functional gag-pol structure, while the lentiviral vector generates env-minus recombinant lentivirus that mobilizes recombinant genomes to other cells when pseudotyped with an exogenous envelope. Since an intact gag-pol structure is absolutely required for retroviral DNA mobilization and RCR, the trans-lentiviral vector design significantly reduces this risk. Moreover, it makes it possible to assess the risk of RCR and DNA mobilization using an in vitro assay that monitors trans-lentiviral vector stocks for the regeneration of the gag-pol structure. Therefore, the trans-lentiviral vector design will ensure the greatest predictable level of safety for the clinical application of retroviral vectors, including HIV-based vectors.
Wu
X
X
Department of Medicine, University of Alabama, Birmingham, AL 35294, USA.
Wakefield
J K
JK
Liu
H
H
Xiao
H
H
Kralovics
R
R
Prchal
J T
JT
Kappes
J C
JC
D018952:Antigens, CD34 / Q000378:metabolism; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000821:virology; D002460:Cell Line; D002469:Cell Separation; D005434:Flow Cytometry; D015685:Fusion Proteins, gag-pol / Q000235:genetics; D016336:Gene Products, vpr / Q000235:genetics; D005822:Genetic Vectors / Q000009:adverse effects; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000378:metabolism; D008264:Macrophages / Q000821:virology; D008954:Models, Biological; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics; D054325:vpr Gene Products, Human Immunodeficiency Virus
D018952:Antigens, CD34; D015685:Fusion Proteins, gag-pol; D016336:Gene Products, vpr; D008164:Luminescent Proteins; D014764:Viral Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins