Potential mode of action of the 5′HS4/IFN-SAR combination. (A) Standard lentiviral vector showing negative (heterochromatin spreading) and positive (vector enhancer activation of endogenous gene expression) integration site effects. P indicates promoter. (B) SIN lentiviral vector containing the 5′HS4/IFN-SAR combination evaluated in this study. The enhancer-blocking function of the 5′HS4 element requires CTCF binding sites.²⁷  It has been suggested that barrier activity depends on other signals within the 5′HS4 element that recruit histone acetyltransferases, which would acetylate (AC) histone H3 on lysine 9 and prevent encroachment of methylation (Me) by histone methyltransferases (Suv39h).^28,29  The IFN-SAR could antagonize histone H1–mediated repression of gene expression by preferential interaction with high mobility group proteins (HMG-I/Y).^53,54  X indicates block of function. (C) The IFN-SAR associates with the nuclear scaffold/matrix and, in conjunction with the pair of 5′HS4 elements, could facilitate formation of chromatin loops conducive to transgene expression.^24,31,40  Arrows indicate direction in which the various elements exert their influence.