Diagram of the BB305 LVV with human β^A-T87Q substitution and corresponding HIV-derived sequences. (A) The β^A-T87Q substitution (ACA [Thr] to CAG [Gln]) in the human β-globin gene is indicated by an asterisk and arrow. Safety modifications included mutation of the gag gene and a 400-bp deletion in the U3 of the right HIV LTR.⁴ HIV-derived sequences are shown in gray boxes and are aligned with corresponding genes in the HIV genome (top; adapted from Frankel and Young).⁹ 3'enh/pA, 3' enhancer/polyadenylation signal from the β-globin gene; ΔU3, promoter/enhancer-deleted unique 3'; ψ, packaging signal; cPPT, central polypurine tract; E, exon; gag, HIV-1 partial gag sequence; Globin LCR, human globin locus control regions; LTR, long terminal repeat; LVV, lentiviral vector; P, β-globin promoter; Q, gluatmine; R, repeat; RRE, rev-response element; T, threonine; U5, unique 5'. (B) WB assay of BB305 LVV for HIV-1 proteins. Lane 1 of each blot contains 50 to 100 ng of each analyzed protein (from left to right: [A] recombinant (r)-gp120, [B] r-gp41, [C] r-RT, [D] r-p24); lane 2 of each blot contains a molecular weight ladder; lane 3 of each blot contains 4 ng BB305 LVV. Primary mAbs include anti-p24 mAb 63917 (Abcam); anti-gp41 mAb MA1-7590 (Invitrogen); anti-RT mAb 63911 (Abcam); anti-gp120 mAb AHP2204 (Bio-Rad). Secondary mAb is IRDye 800CW (925-32211; Li-Cor Biosciences). The HIV p24 and reverse transcriptase proteins are not encoded in the BB305 LVV but are present as part of the purified vector particle and may be identifiable via WB analysis of drug product. The HIV-1 env protein gp160 cleavage products gp41 and gp120 are not present in the BB305 LVV production system and thus are not detectable.^4,9 mAbs, monoclonal antibodies; MW, molecular weight; RT, reverse transcriptase; WB, western blot.