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Gene Therapy Expression Vectors Based on the Clotting Factor IX Promoter

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Gene Therapy Expression Vectors Based on the Clotting Factor IX Promoter Gene Therapy (1999) 6, 1584–1589 1999 Stockton Press All rights reserved 0969-7128/99 $15.00 http://www.stockton-press.co.uk/gt Gene therapy expression vectors based on the clotting Factor IX promoter H Hoag, J Gore, D Barry and CR Mueller Department of Biochemistry and Cancer Research Laboratories, Queen’s University, Kingston, Ontario, Canada The liver is one of the prime targets for gene therapy, and moter. Introduction of this element increases promoter the correction of defects in a variety of clotting factor genes activity at least 20-fold over the proximal promoter alone is one of the main goals of liver-directed therapies. The use when assayed in the human liver cell line Hep G2. This of transcriptional regulatory elements derived from these optimized promoter is significantly more active than the genes may provide for the optimal expression of trans- SV40 enhancer/early promoter. The expression of the opti- duced genes. We have applied our knowledge of the pro- mized Factor IX promoter is also more persistent in the moter structure of the clotting Factor IX gene to design short term. The inclusion of a liver-specific locus control optimized expression vectors for use in gene therapy. The region, derived from the apolipoprotein E/C locus, did not activity of the proximal promoter has been augmented by further augment expression levels. These Factor IX vectors the introduction of a multimerized upstream site which we also exhibit a high degree of tissue specificity, as meas- have previously shown to be a prime regulator of the pro- ured by transfection into breast and muscle cell lines. Keywords: gene therapy; liver; Factor IX; gene expression; transcription; tissue specific Introduction transduction in vivo.9 Liver-specific promoters have also been assessed in the context of adenoviral transduction, The targeting of therapeutic genes to the liver is of parti- where various combinations of tissue-specific regulatory cular importance for the treatment of a wide variety of elements were shown to produce sustained expression.4 diseases (reviewed in Ref. 1). Considerable advances In order to develop expression vectors with optimal have been made in the ability to direct transducing characteristics, we turned to promoter elements associa- 2 viruses to the liver using a variety of strategies. Retro- ted with one of the main targets for gene therapy, the 3 viruses, adenoviruses and adenovirus-associated virus clotting Factor IX gene. Factor IX synthesis is confined to have all been used to deliver genes to the liver. Most of the liver and the gene is expressed at high levels in the these systems have relied on various viral promoters for adult liver. Its structure is somewhat different from other expression of the transduced gene. This has led to limited strong liver-specific promoters such as albumin and ␣1- levels of gene expression, a lack of tissue specificity, and antitrypsin as it is a TATA-less promoter.10 As a gene that problems with the long-term persistence of expression. is naturally expressed in the liver, it should not be subject Hepatic expression of the CMV promoter has been to the type of negative regulation which suppresses viral shown to be limited when incorporated into an adenovi- promoters, and once introduced may be more stably 4 ral vector, as was expression in an AAV vector in adult expressed. mice, although some expression in newborn mice was Inherited defects in the clotting Factor VIII and IX 5 observed. CMV repression appears to be at the level of genes result in hemophilia A and B, respectively, and cor- gene regulation as its expression can be reactivated in rection of these defects using gene therapy is a high pri- 6 response to NF kappaB induction. In transfected liver ority. Both of these genes are normally expressed in the ␤ cells, non-liver-specific promoters such as CMV and - liver, and their protein products must be post-trans- actin produce higher levels of expression than the albu- lationally processed and produced in significant quan- ␣ 7 min and 1-antitrypsin promoters. When assayed in the tities in order to be effective. The use of the natural pro- context of a retroviral transduction, the inclusion of natu- moters from these genes to drive their expression in gene ␣ ral promoters such as the 1-antitrypsin, albumin, therapy would ensure the appropriate tissue-specific PEPCK and fatty acid binding protein promoters resulted expression and, possibly, appropriate levels of these fac- ␣ in high expression levels, with the -antitrypsin promoter tors. We have carried out extensive characterization of 8 resulting in the greatest expression. In combination with the Factor IX promoter in the context of understanding ␣ the Apo E enhancer, the 1-antitrypsin promoter pro- the basis of the Leiden form of hemophilia.11 This is a rare duced even higher levels of expression during retroviral form of hemophilia B in which mutations in the promoter result in a severe deficit in Factor IX production.12 This disease is unique in that spontaneous recovery of Correspondence: CR Mueller, Cancer Research Laboratories, 3rd Floor expression occurs following puberty, leading to resol- 13 Botterell Hall, Queen’s University, Kingston, Ontario, Canada, K7L 3N6 ution of the disease. It has been suggested that recovery Received 1 February 1999; accepted 4 May 1999 is correlated with the induction of sex steroid hormones Factor IX-based vector H Hoag et al 1585 at puberty and their activation of an androgen receptor Results site in the proximal promoter.14 Alternatively, we have demonstrated that a site approximately 200 nucleotides Our previous analysis of the clotting Factor IX promoter upstream of the transcription start site (site 5), plays a has characterized an upstream element which has a key role in the recovery of transcriptional activity during dominant effect on the expression of this promoter. This puberty.11 This element was seen to be a prime regulator element is referred to as site 5, and is comprised of both of the Factor IX promoter, with point mutations in this C/EBP␣ and GABP␣/␤ binding elements.15 It appears to element resulting in a dramatic decrease in promoter play a role in the recovery of Factor IX expression in the activity.15 The factors binding to this site were identified Leiden form of hemophilia B.11 This type of hemophilia as C/EBP␣, a factor expressed in a limited number of is the result of a variety of mutations in the proximal tissues including liver, intestine and fat cells, and promoter. The importance of interactions between site 5 GABP␣/␤, which is a ubiquitous ets protein. Either alone, and elements in the proximal promoter suggested that or as a multimerized site, this element is able to direct increasing site 5 function could lead to greater promoter efficient transcription initiation from a heterologous pro- activity. This was done by cloning multimerized site 5 moter. Additional transcription factors which have been elements at the 5′ end of the proximal promoter shown to regulate the Factor IX promoter include HNF- (Figure 1). By increasing the local concentration of a site 4,16 the expression of which is tissue restricted, and and allowing both physical and functional co-operativity, NF-1, another ubiquitous protein. multimerization often produces optimal transcriptional Given the apparent dominant effect of the site 5 activity of a given site. The transcriptional activity of the element on the proximal promoter, and its ability to act wild-type Factor IX promoter (FIX) and the site 5 multi- as a strong activator when multimerized, we tested the mer (GTV) were tested by transient transfection into the effect of inclusion of a site 5 multimer on the activity of human hepatocellular carcinoma cell line, Hep G2. This the proximal promoter. Insertion of five site 5 repeats at line was chosen as it has served as an in vitro model of the 5′ end of the proximal Factor IX promoter increased liver specificity, and while it does not exhibit all of the expression over 20-fold when assayed in the human liver characteristics of differentiated liver cells it expresses line, Hep G2. The level of expression of this plasmid was many essential liver-specific transcription factors. An higher than that mediated by the SV40 early promoter in SV40 enhancer/promoter construct was used for com- these cells. Inclusion of site 5 repeats at both the 5′ and parison as SV40 expression shows little tissue specificity 3′ ends of the transcription unit had no additional effect and expression in the mouse liver has been shown to be on expression and liver specificity. A liver-specific LCR robust when incorporated into an adenovirus vector.17 derived from the apolipoprotein E/C locus was also Inclusion of the site 5 elements increased expression of incorporated into the 3′ end of the transcription unit, but the promoter by a factor of 20- to 35-fold, depending did not result in a significant increase in expression. upon the individual transfection (Figure 2). The absolute Expression of the site 5 containing constructs is highly activity of the GTV construct was higher than that of a liver specific and, over the short term, does not decay as comparable amount of an SV40 enhancer/early promoter rapidly as the SV40 early promoter directed expression. reporter (pGL2-CTRL), which is considered to be a highly Figure 1 Structure of the Factor IX-based plasmid constructs and of pGL2-CTRL. Schematics of the various plasmid constructs are shown. The region of the human Factor IX promoter from nt −219 to +21 was cloned in the luciferase reporter construct pGL2-BASIC to generate the FIX plasmid. The five binding elements characterized, to date, are indicated by the boxed numbers with the arrow indicating the transcriptional start site.
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