Figure 9.

General scheme of 3rd generation globin lentiviral vectors. Vectors have deletions of all dispensable and potentially harmful HIV sequences (tat, vif, vpr, vpu, nef). The partial ORF of GAG that can not be removed because overlaps the packaging signal (Y) contains 2 in frame stop codons. The 400 bp deletion in the U3 region blocks provirus transcription after chromosomal integration (self inactivating vector). POL-GAG, REV and VSV-G envelopes are provided in trans to avoid recombination that could reconstitute wild type replication competent HIV particles. The b-globin cassette in the current vectors contains from 2 to 4 HSs, β-globin promoters of 260–600 bp, a stabilizing RsaI deletion of IVS2, variable length of the 3′ UTR and an enhancer region contained in a PstI fragment 3′ to the β-globin gene. As an additional safety feature some vector are insulated with the chicken HS4 or other equivalent insulator. Insulators that are inserted into the 3′ U3 region are duplicated and moved to the 5′ U3 region during viral reverse transcription achieving insulation of both sides of the vector. Insulation is expected to restrict the activity of the internal LCR enhancer to the globin cassette and to create a barrier to the invasion of adjacent heterochromatin that ensures persistent expression. ORF, Open reading frame; GAG, group-specific Antigen; 3′ UTR, 3′ untranslated region; LCR, locus control region.