Gene therapy expression vectors based on the clotting Factor IX promoter

Chimeric transcriptional control units for improved liver-specific transgene expression

“…Promoter sequences were chosen for analyses on the basis that their function as basal promoters had been defined in previous studies. These included the promoters of the alcohol dehydrogenase 6 (ADH6) (Yasunami et al, 1991;Zhi et al, 2000), the human alpha-antitrypsin (hAAT) (Shen et al, 1989;Hafenrichter et al, 1994), the cholesterol 7alpha-hydroxylase (CYP) (Molowa et al, 1992) and the clotting factor IX (FIX) (Boccia et al, 1996;Hoag et al, 1999) genes. Transient transfection experiments using the human hepatoma cell line HepG2 (Fig.…”

“…For this purpose, we constructed enhancer modules containing elements from three different liverspecific genes: the HCR-1 region of the apoE gene (Allan et al, 1997), a far upstream enhancer region of the ABP gene (Rouet et al, 1992) and the site 5 enhancer domain of the FIX gene (Hoag et al, 1999). In total 19 transcriptional control units containing these enhancer elements in different configurations were designed, i.e.…”

“…The promoters contained the following sequences: ADH6, À 342 to + 57 (Yasunami et al, 1991;Zhi et al, 2000); hAAT, À 347 to + 56 (Shen et al, 1989;Hafenrichter et al, 1994;Bowling et al, 1996;Miao et al, 2000); CYP, À 370 to + 63 (Molowa et al, 1992); and FIX, À 219 to + 21 (Boccia et al, 1996). The core promoters were linked to the following enhancer sequences in the pGL3-basic luciferase vector by standard ligation techniques: a 319-bp apoE enhancer containing the hepatic control region (HCR-1) (Simonet et al, 1993;Dang et al, 1995;Allan et al, 1997); a 190-bp ABP enhancer region spanning nucleotides À 2824 to À 2634 (Rouet et al, 1992); a quadruplet of the site 5 enhancer element of the FIX gene spanning nucleotides À 219 to À 190 (Hoag et al, 1999). The simian virus 40 (SV40) promoter/enhancer luciferase vector pGL3 was obtained from Promega.…”

“…In the present study, we have addressed this problem by constructing a total of 25 synthetic transcriptional control units consisting of core promoters from liverspecific genes linked in various combinations and configurations to hepatocyte-specifc enhancer elements, and analyzing these constructs for transcriptional activity in different cell types in cell culture and in mouse liver in vivo. The transcription regulatory elements used in this study were derived from the human genes coding for alcohol dehydrogenase 6 (ADH6) (Yasunami et al, 1991;Zhi et al, 2000), human alpha-antitrypsin (hAAT) (Shen et al, 1989;Hafenrichter et al, 1994), cholesterol 7alpha-hydroxylase (CYP) (Molowa et al, 1992), clotting factor IX (FIX) (Boccia et al, 1996;Hoag et al, 1999), apolipoprotein E/C-I (apoE) (Simonet et al, 1993;Allan et al, 1997) and alpha1-microglobulin/bikunin (ABP) (Rouet et al, 1992). These studies led to the clear conclusion that the ADH6 basal promoter linked to two tandem copies of an apoE enhancer element allows for the highest level of liver-specific transcription.…”

Hybrid vector designs to control the delivery, fate and expression of transgenes

“…Also, the augmentation of transgene expression may depend on the distance between the LCR and its target genes, as well as other types of DNA elements required to form a stable transcription complex. Hence, inclusion of a liver‐specific LCR did not improve the transgene expression level in hepatocytes under control of a liver specific promoter109.…”