A lentiviral vector system for globin gene transfer.

Several modifications were introduced to improve vector production and/or enhance safety as the vector genome was assembled to derive pCL10.1.²² The 5’ LTR is chimeric in that the U3 region of the HIV-1 LTR is replaced with the CMV enhancer. A deletion in the U3 region of the 3’ LTR renders it self-inactivating; in addition, a portion of the R region and the U5 region have been removed and replaced with the rabbit β-globin gene polyadenylation site to enhance safety and improve the efficiency of vector production. The cPPT and CTS were added to improve vector integration. All coding sequences for accessory proteins have been eliminated from the vector. The γ-globin gene with a 720 bp deletion in the second intron was linked to the β-globin gene promoter (-130 to +54) and placed downstream from HS 2 (374 bp), 3 (898 bp), and 4 (445 bp) from the β-globin locus control region. The cassette is placed in the reverse transcriptional orientation of the vector transcript to prevent loss of the globin gene introns during passage of the vector genome (D. A. Persons, P. W. Hargrove, E. R. Allay, H. Hanawa, A. W. Nienhuis, unpublished data, 2002). The expression plasmid pCAGGS was used to construct the cassettes expressing HIV structural and regulatory proteins and the VSV-G envelope protein.²² This expression plasmid contains a powerful chimeric CMV enhancer/β-actin promoter, the β-globin large intron, which ensures splicing of most transcripts, and the rabbit β-globin polyadenylation sequence, which includes an RNA stability element in the 3’ untranslated region and the SV40 origin of replication to increase plasmid copy number in 293T cells. The accessory proteins, tat and rev, were expressed together on a plasmid separate from that expressing the GAG-POL-precursor polyprotein. The RRE element was included to enhance rev-dependent, nuclear-to-cytoplasmic transfer of RNA molecules encoding gag/pol and to modulate expression of rev and tat by fostering transport of unspliced transcripts.

Abbreviations: LTR, long terminal repeat; CMV, cytomegalovirus; cPPT, central polypurine tract; CTS, central termination sequence; HS, hypersensitive site; HIV, human immunodeficiency virus.