The invention is directed to novel combinations of liver specific enhancers and promoter elements for achieving persistent transgene expression in the liver. The liver specific enhancer elements may be derived from either the human serum albumin, prothrombin, .alpha.-1microglobulin or aldolase genes in single copies or in multimerized form linked to elements derived from the cytomegalovirus intermediate early (CMV), .alpha.-1-antitrypsin or albumin promoters. In a preferred embodiment of the invention, an adenoviral vector comprising a liver specific enhancer/promoter combination operably linked to a transgene is administered to recipient cells. In other embodiments of the invention, adeno-associated viral vectors, retroviral vectors, lentiviral vectors or a plasmid comprising the liver specific enhancer/promoter combination linked to a transgene is administered to recipient cells. Also within the scope of the invention are promoter elements derived from the human prothrombin gene and the .beta.-fibrinogen gene.

Description of the Invention

SUMMARY OF THE INVENTION

Accordingly, the present invention provides improved regulatory elements that are useful for targeting transgene expression to the liver. In preferred embodiments, the regulatory elements comprise combinations of promoter and enhancer elements that are able to direct transgene expression preferentially in liver. In particular embodiments, the regulatory elements are used in recombinant vectors, such as such as nonviral plasmid based vectors or such as viral vectors, including adenovirus, adeno-associated virus, retrovirus and lentivirus, including the human immunodeficiency [HIV] virus. In other embodiments, the invention comprises recombinant vectors useful for transgene expression, particularly for high and sustained expression in the liver, such as viral vectors. The vectors comprise combinations of a constitutive or high-expressing promoter and one or more liver-specific enhancer elements.

Thus, the present invention comprises recombinant transgenes comprising strong constitutive promoters and one or more liver-specific enhancer elements. The transgenes may be used in recombinant vectors, such as recombinant viral vectors, for targeting expression of the associated coding DNA sequences preferentially in liver. In preferred embodiments, the strong constitutive promoter is selected from the group comprising a CMV promoter, a truncated CMV promoter, human serum albumin promoter and .alpha.-1-antitrypsin promoter. In other preferred embodiments, the promoter is a truncated CMV promoter from which binding sites for known transcriptional repressors have been deleted.

In other embodiments, the liver-specific enhancer elements are selected from the group consisting of human serum albumin [HSA] enhancers, human prothrombin [HPrT] enhancers, .alpha.-1microglobulin enhancers and intronic aldolase enhancers. One or more of these liver-specific enhancer elements may be used in combination with the promoter. In one preferred embodiment of the invention, one or more HSA enhancers are used in combination with a promoter selected from the group consisting of a CMV promoter or an HSA promoter. In another preferred embodiment, one or more enhancer elements selected from the group consisting of human prothrombin (HPrT) enhancers and .alpha.-1microglobulin (A1MB) enhancers are used in combination with the CMV promoter. In yet another preferred embodiment, the enhancer elements are selected from the group consisting of HPrT enhancers and A1MB enhancers, and are used in combination with the .alpha.-1-antitrypsin promoter.

The preferred embodiments of the present invention are recombinant viral vectors, particularly adenoviral vectors. In the preferred embodiments, the coding DNA sequence may encode a therapeutic protein that is most effective when delivered to the liver. The adenoviral vectors may comprise, in addition to the promoters and enhancers of the present invention, one or more adenoviral genes in order to support the efficient expression of the coding DNA sequence.

DETAILED DESCRIPTION OF THE INVENTION

The delivery of genes to the liver for therapeutic purposes has been explored extensively. This includes investigation aimed at correction of genetic diseases of the liver as well as systemic diseases that might be corrected by using the liver as a depot for therapeutic protein production. For this gene therapy approach to be feasible, expression of the therapeutic gene must be long-lived and approach appropriate levels. In several reports, the use of a variety of viral, non-viral, and liver specific promoters as well as various enhancer/promoter combinations has been explored in the context of adenoviral, AAV, retroviral and plasmid-based vectors for gene expression in cultured cells and in vivo. In many of these examples transgene expression was transient and/or not sufficient to achieve therapeutic benefit. In the context of adenoviral vectors, the CMV promoter and RSV promoter direct high levels of transgene expression however the longevity of expression is dependent upon retention of the adenoviral E4 region in the vector. The development of an enhancer/promoter combination that can direct sustained and appropriate levels of transgene expression in the context of a variety of vector systems would therefore be of benefit.

Promoters which are suitable for the present invention may be any strong constitutive promoter which is capable of promoting expression of an associated coding DNA sequence in the liver. Such strong constitutive promoters include the human and murine cytomegalovirus [CMV] promoter, truncated CMV promoters, human serum albumin promoter [HAS] and .alpha.-1-antitrypsin promoter. In a specific embodiment, the promoter used is a truncated CMV promoter from which binding sites for known transcriptional repressors have been deleted.

The liver-specific enhancer elements useful for the present invention may be any liver-specific enhancer that is capable of enhancing tissue-specific expression of an associated coding DNA sequence in the liver. Such liver-specific enhancers include one or more human serum albumin enhancers, human prothrombin enhancers, .alpha.-1 microglobulin enhancers and an intronic aldolase enhancers. In preferred embodiments, multiple enhancer elements may be combined in order to achieve higher expression.

Among the preferred embodiments of the present invention are vectors comprising one or more HSA enhancers in combination with either a CMV promoter or an HSA promoter; one or more enhancer elements selected from the group consisting of the human prothrombin (HPrT) enhancer and the .alpha.-1microglobulin (A1MB) enhancer in combination with a CMV promoter; and one or more enhancer elements selected from the group consisting of HPrT enhancers and A1MB enhancers, in combination with an .alpha.-1-antitrypsin promoter.

The strategy for achieving high and sustained levels of transgene expression involves combining promoter elements that have the potential to direct effective and sustained levels of expression with liver specific enhancer elements that can further increase expression. The promoter fragments preferred for use in the present invention include a truncated version of the CMV promoter (mCMV, nucleotides -245 to -14), human serum albumin promoter (-486 to +20) and .alpha.-1-antitrypsin promoter (-844 to -44). The truncated CMV promoter is missing binding sites for known transcriptional repressors and is thus a preferred version of this promoter. The human serum albumin and the .alpha.-1-antitrypsin promoter contain elements that direct basal yet liver specific expression. The transcription factor binding sites in these promoter regions are depicted in FIG. 1 (see Original Patent). The enhancer elements used here include two HSA enhancers (HSA-1.7, nucleotides -1806 to -1737; HSA-6, nucleotides -6081 to -6000), a human prothrombin enhancer (-940 to -860), an .alpha.-1microglobulin enhancer (-2806 to -2659) and an intronic aldolase enhancer (+1916 to +2329). Each of these enhancers has been shown to greatly increase transgene expression when linked to a minimal promoter and transcription factor binding sites in these enhancer elements is depicted in FIG. 2 (see Original Patent). FIG. 3 (see Original Patent) is a schematic representation of an initial series of enhancer/promoter combinations. Group A indicates the combinations of the HSA enhancers that were linked to either the mCMV or HSA promoter. Groups B and C represent the combinations of either the human prothrombin (HPrT) or .alpha.-1microglobulin (A1MB) enhancer linked to the mCMV promoter. Groups D and E represent the combinations of either HPrT or A1MB linked to the .alpha.-1-antitrypsin promoter.

Each of these enhancer/promoter combinations was linked to .alpha.-galactosidase and was tested for activity in Hep3B cells by measuring the levels of .alpha.-galactosidase in the supernatant medium following transient transfection. As shown in FIG. 4 (see Original Patent), expression from the mCMV promoter is reduced compared to the CMV promoter. However, the combination of five copies of the HSA-1.7 enhancer with one copy of the HSA-6 enhancer linked to the mCMV promoter yielded expression that was higher than that obtained with the CMV promoter. The expression results from constructs containing the HPrT enhancer are shown in FIG. 5 (see Original Patent). Linkage of this enhancer to the mCMV promoter (Panel B) elevated expression to near levels achieved with the CMV promoter but did not exceed it. Expression from the .alpha.-l-antitrypsin promoter was rather poor, however when two copies of the HPrT enhancer are added expression from this combination exceeds that from the CMV promoter. The expression results from constructs containing the A1MB enhancer are shown in FIG. 6 (see Original Patent). Progressively increased expression is seen with increasing copy number of this enhancer (up to eight copies) linked to the mCMV promoter (Panel C). All copy combinations of this enhancer linked to the .alpha.-1-antitrypsin promoter yielded expression levels comparable to that obtained with the CMV promoter (Panel E). Representative candidates from each vector series that yielded equivalent or higher levels of .alpha.-galactosidase expression compared to the CMV promoter were retested in a single experiment. As shown in FIG. 7 (see Original Patent), all enhancer/promoter combinations yielded comparable expression with expression from the HSA-1.7(5) HSA-6(1)mCMV and HPrT(2)A1AT promoters being the highest. These results demonstrate that high levels of expression are achievable by combining multiple copies of liver specific enhancers with various promoter elements.
 

Claim 1 of 11 Claims

1. A recombinant DNA vector, comprising a human serum albumin promoter and two human prothrombin enhancers.

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