U.S. patent application number 11/938906 was filed with the patent office on 2008-03-20 for regulatory elements for delivery to the liver.
This patent application is currently assigned to GENZYME CORPORATION. Invention is credited to Donna ARMENTANO, David W. SOUZA, Samuel C. WADSWORTH.
Application Number | 20080070297 11/938906 |
Document ID | / |
Family ID | 22600808 |
Filed Date | 2008-03-20 |
United States Patent
Application |
20080070297 |
Kind Code |
A1 |
SOUZA; David W. ; et
al. |
March 20, 2008 |
REGULATORY ELEMENTS FOR DELIVERY TO THE LIVER
Abstract
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 from 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.
Inventors: |
SOUZA; David W.; (Waltham,
MA) ; ARMENTANO; Donna; (Bedford, MA) ;
WADSWORTH; Samuel C.; (Shrewsbury, MA) |
Correspondence
Address: |
GENZYME CORPORATION;LEGAL DEPARTMENT
15 PLEASANT ST CONNECTOR
FRAMINGHAM
MA
01701-9322
US
|
Assignee: |
GENZYME CORPORATION
15 Pleasant Street Connector
Framingham
MA
01701-9322
|
Family ID: |
22600808 |
Appl. No.: |
11/938906 |
Filed: |
November 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10139763 |
May 6, 2002 |
7312324 |
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11938906 |
Nov 13, 2007 |
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09712775 |
Nov 14, 2000 |
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10139763 |
May 6, 2002 |
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60165866 |
Nov 16, 1999 |
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Current U.S.
Class: |
435/320.1 ;
536/24.1 |
Current CPC
Class: |
C12N 2830/85 20130101;
C12N 2830/15 20130101; A61K 48/0058 20130101; A61K 48/00 20130101;
C12N 2710/10343 20130101; C12N 2799/022 20130101; C12N 2830/008
20130101; C12N 15/86 20130101; C12N 15/85 20130101 |
Class at
Publication: |
435/320.1 ;
536/024.1 |
International
Class: |
C12N 15/861 20060101
C12N015/861; C12N 15/11 20060101 C12N015/11 |
Claims
1. A recombinant transgene useful for expression of a coding
sequence, comprising a strong constitutive promoter and one or more
liver-specific enhancer elements.
2. A recombinant transgene according to claim 1, wherein the strong
constitutive promoter is selected from the group comprising a
cytomegalovirus (CMV) promoter, a truncated CMV promoter, human
serum albumin promoter and .alpha.-1-antitrypsin promoter.
3. A recombinant transgene according to claim 2, wherein the
promoter is a truncated CMV promoter from binding sites for known
transcriptional repressors have been deleted.
4. A recombinant transgene according to claim 1, wherein the
liver-specific enhancer elements are selected from the group
consisting of human serum albumin enhancers, human prothrombin
enhancers, .alpha.-1microglobulin enhancers and intronic aldolase
enhancers.
5. A recombinant transgene according to claim 1, comprising one or
more human serum albumin (HSA) enhancers and a promoter selected
from the group consisting of a CMV promoter or an HSA promoter.
6. A recombinant transgene according to claim 1, comprising one or
more enhancer elements selected from the group consisting of human
prothrombin (HPrT) enhancers and .alpha.-1microglobulin (A1MB)
enhancers and the promoter is the CMV promoter.
7. A recombinant transgene according to claim 1, wherein the
enhancer elements are selected from the group consisting of HPrT
enhancers and A1MB enhancers, and the promoter is the
.alpha.-1-antitrypsin promoter.
8. A recombinant adenoviral vector useful for transgene expression
comprising a strong constitutive promoter and one or more
liver-specific enhancer elements.
9. A recombinant adenoviral vector according to claim 8, wherein
the strong constitutive promoter is selected from the group
comprising a cytomegalovirus (CMV) promoter, a truncated CMV
promoter, human serum albumin promoter and .alpha.-1-antitrypsin
promoter.
10. A recombinant adenoviral vector according to claim 9, wherein
the promoter is a truncated CMV promoter from binding sites for
known transcriptional repressors have been deleted.
11. A recombinant adenoviral vector according to claim 8, wherein
the liver-specific enhancer elements are selected from the group
consisting of human serum albumin enhancers, human prothrombin
enhancers, .alpha.-1microglobulin enhancers and intronic aldolase
enhancers.
12. A recombinant adenoviral vector according to claim 8,
comprising one or more human serum albumi (HSA) enhancers and a
promoter selected from the group consisting of a CMV promoter or an
HSA promoter.
13. A recombinant adenoviral vector according to claim 8,
comprising one or more enhancer elements selected from the group
consisting of human prothrombin (HPrT) enhancers and
.alpha.-1microglobulin (A1MB) enhancers and the promoter is the CMV
promoter.
14. A recombinant adenoviral vector according to claim 8, wherein
the enhancer elements are selected from the group consisting of
HPrT enhancers and A1MB enhancers, and the promoter is the
.alpha.-1-antitrypsin promoter.
Description
FIELD OF THE INVENTION
[0001] This invention relates to nucleic acid delivery vehicle
constructs that have an enhanced capability of expression in target
cells, namely to hepatocytes and other liver cells.
BACKGROUND OF THE INVENTION
[0002] The ability to deliver nucleic acids carried by delivery
vehicles, e. g., recombinant viruses (adenovirus, adeno-associated
virus, herpesvirus, retrovirus) which are used with nucleic acid
molecules, such as a plasmid, comprising a transgene, to transfect
a target cell; molecular conjugate vectors; and modified viral
vectors are important for the potential treatment of genetic
diseases through gene delivery.
[0003] Adenovirus is a non-enveloped, nuclear DNA virus with a
genome of about 36 kb. See generally, Horwitz, M. S., "Adenoviridae
and Their Replication," in Virology, 2nd edition, Fields et al.,
eds., Raven Press, New York, 1990. Recombinant (adenovirus
dodecahedron and recombinant adenovirus conglomerates) to specific
cell types is useful for various applications in oncology,
developmental biology and gene therapy. Adenoviruses have
advantages for use as expression systems for nucleic acid molecules
coding for, inter alia, proteins, ribozymes, RNAs, antisense RNA
that are foreign to the adenovirus carrier (i.e. a transgene),
including tropism for both dividing and non-dividing cells, minimal
pathogenic potential, ability to replicate to high titer for
preparation of vector stocks, and the potential to carry large
inserts. See Berkner, K. L.,--1992, Curr. Top. Micro Immunol, 158:
39-66, Jolly D., 1994, Cancer Gene Therapy, 1: 5 l-64. Adenoviruses
have a natural tropism for respiratory tract cells, which has made
them attractive vectors for use in delivery of genes to respiratory
tract cells. For example, adenovirus vectors have been and are
being designed for use in the treatment of certain diseases, such
as cystic fibrosis (CF): the most common autosomal recessive
disease in Caucasians. In CF, mutations in the cystic fibrosis
transmembrane conductance regulator (CFTR) gene disturb
CAMP-regulated chloride channel function, resulting in pulmonary
dysfunction. The gene mutations have been found to encode altered
CFTR proteins which cannot be translocated to the cell membrane for
proper functioning. The CFTR gene has been introduced into
adenovirus vectors to treat CF in several animal models and human
patients. Particularly, studies have shown that adenovirus vectors
are fully capable of delivering CFTR to airway epithelia of CF
patients, as well as airway epithelia of cotton rats and primates
See e. g., Zabner et al., 1994, Nature Genetics, 6:75-83; Rich et
al., 1993, Human Gene Therapy, 4: 461-476; Zabner et al., 1993,
Cell, 75:207-216; Zabner et al., 1994, Nature Genetics 6:75-83,
Crystal et al., 1004, Nature Genetics, 8:42-51, Rich et al., 1993,
Human Gene Therapy, 4:461-476.
[0004] However, it would be useful to alter the genome of
adenovirus, to allow it to be used to deliver a nucleic acid
molecule that would be enhanced for expression in the liver,
particularly in hepatocytes.
[0005] It would be useful to mediate expression of the transgene
carried by the adenoviral vector through the use of one or more
specialized regulatory elements. In this way the expression of
transgene within desired cells can be enhanced and the adenovirus
effects can be targeted to certain cells or tissues within an
organism.
[0006] Like adenoviruses, retroviruses have also been used for
delivery of transgenes to target cells. As set forth above, a
transgene is a nucleic acid molecule that codes for, inter alia, a
protein, RNA, ribozyme, antisense RNA not produced by the virus.
Retrovirus virions range in diameter from 80 to 130 nm and are made
up of a protein capsid that is lipid encapsulated. The viral genome
is encased within the capsid along with the proteins integrase and
reverse transcriptase. The retrovirus genome consists of two RNA
strands. After the virus enters the cells, the reverse
transcriptase synthesizes viral DNA using the viral RNA as its
template. The cellular machinery then synthesizes the complementary
DNA which is then circularized and inserted into the host genome.
Following insertion, the viral RNA genome is transcribed and viral
replication is completed.
[0007] Examples of retroviruses include Moloney murine leukemia
virus (MO-MuLV), HTLV and HIV retroviruses. MO-MuLV vectors are
most commonly used and are produced simply by replacing viral genes
required for replication with the desired transgenes to be
transferred. The genome in retroviral vectors contains a long
terminal repeat sequence (LTR) at each end with the desired
transgene or transgenes in between. The most commonly used system
for generating retroviral vectors consists of two parts, the
retroviral vector and the packaging cell line. Retroviruses are
typically classified by their host range. For example, ecotropic
viruses are viruses which bind receptors unique to mice and are
only able to replicate within the murine species. Xenotropic
viruses bind receptors found on all cells in most species except
those of Polytropic and amphotropic viruses bind different
receptors found in both murine and nonmurine species. The host
range is determined primarily by the binding interaction between
viral envelope glycoproteins and specific proteins on the host cell
surface that act as viral receptors. For example, in murine cells,
an amino acid transporter serves as the receptor for the envelope
glycoprotein gp70 of ecotropic Moloney murine leukemia virus
(MO-MuLV). The receptor for the amphotropic MoMuLV has recently
been cloned and shows homology to a phosphate transporter. There
are six known receptors for retroviruses: CD4 (for HIV); CAT (for
MLV-E (ecotropic Murine leukemic virus E); RAM1/GLVR2 (for murine
leukemic virus-A (MLV-A)); GLVRI (for Gibbon Ape leukemia virus
(GALV) and Feline leukemia virus B (FeLV-B). RAM1 and GLVR1
receptors are broadly expressed in human tissues.
[0008] Retrovirus packaging cell lines provide all the viral
proteins required for capsid production and the virion maturation
of the vector, i.e., the gag, pol and env genes. For the MMLV
vectors, it is the packaging cell line that determines whether the
vector is ecotropic, xenotropic or amphotropic. The choice of the
packaging cell line determines the cells that will be targeted.
Thus, the usefulness of retroviruses for gene transfer is limited
by the fact that they are receptor specific.
[0009] However, retroviruses are useful for gene delivery systems
because they have a high infection efficiency and the retroviral
nucleic acid (after reverse transcription) integrates into the host
genome resulting in sustained expression of the transgenes carried
by the vector. However, typical retroviral vectors are limited in
that they require dividing cells for infectivity. Furthermore, in
vivo delivery of these vectors is poor and is effective only when
infecting helper cell lines. Thus, it would be useful to have a
system for increasing the efficiency of retroviral infection.
[0010] Certain situations exist where it would be useful to modify
the expression of transgenes carried by viruses. For example,
tissue-specific expression of the transgene in targeted cells
increase the efficiency of infection, and consequently, a lower
volume of virus may be the body. It would be useful to have a
method of up-regulating the expression of the transgene in a
tissue-specific manner.
[0011] One method for targeting specific cell populations to
express a protein of interest is to use heterologous regulatory
elements that are specifically expressed in the desired target
tissue or cell populations. This may be achieved through the use of
combinations of tissue-specific enhancers, promoters and/or other
regulatory elements. The regulatory elements may be constitutive or
inducible, such that they are regulated by the absence or presence
of other DNA sequences, proteins or other elements or environmental
factors. Where the transgene of interest is a cytotoxic gene, leaky
expression would be highly undesirable.
SUMMARY OF THE INVENTION
[0012] 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.
[0013] 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.
[0014] 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 (I-IPrT) enhancers and .alpha.-1 microglobulin
(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.
[0015] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a depiction of the transcription factor binding
sites present in the CMV, HSA and .alpha.-1 antitrypsin promoter
regions.
[0017] FIG. 2 is a depiction of the transcription factor binding
sites present in the 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).
[0018] FIG. 3 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.
[0019] FIG. 4 depicts expression from mCMV promoter compared to
that of hCMV promoter, and the effects of adding multiple HSA
enhancers (HSA-1.7 and HSA-6).
[0020] FIG. 5 depicts expression from constructs containing the
HPrT enhancer. 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.-1-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.
[0021] FIG. 6 depicts expression results from constructs containing
the A1MB enhancer. Progressively increased expression is seen with
increasing copy number of this enhancer (up to 25 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).
[0022] FIG. 7 depicts expression results obtained with
representative candidates from each vector series that yielded
equivalent or higher levels of .alpha.-galactosidase expression
compared to the CMV promoter.
[0023] FIG. 8 demonstrates the expression of FVIII in SCID Beige
Mice. The Factor VIII expression cassettes used contained either
the CMV promoter or a hybrid promoter composed of two copies of the
hprt enhancer linked to a human .alpha.-1-antitrypsin promoter
fragment (Hprt(2)AAT). Ten .mu.g of plasmid DNA containing either
cassette was injected via the tail vein of SCID beige mice using
Mirus plasmid delivery technology. Mice were bled at one, seven and
fourteen days post-injection and FVIII levels in the plasma were
determined by an ELISA that is specific for human FVIII. Each bar
represents the average FVIII expression in the plasma of four
mice.
[0024] Expression levels detected on day one were comparable in
mice that received either plasmid indicating that the hybrid
promoter could yield expression levels that approximate that from
the CMV promoter. However, expression from the CMV promoter was
transient and plummeted to undetectable by day seven whereas
expression from the hybrid promoter persisted to day 14 (the last
time point this experiment). This suggests that this hybrid
promoter is a better choice for achieving long-term transgene
expression in the liver.
[0025] FIG. 9 demonstrates the AAV mediated expression of
recombinant human erythropoietin [EPO] in mice. From promoter
studies in cultured Hep3B cells, several enhancer/promoter
combinations were identified as promising candidates for achieving
long term expression in the liver. From this panel of combinations,
several were cloned into AAV vectors to test their ability to drive
expression of recombinant human EPO. 1.times.10e11 particles of
each AAV vector was administered to NCR nude mice by portal vein
injection. On days 14, 28 and 42 post-injection, the mice were bled
retro-orbitally and EPO levels were determined by an ELISA specific
for human EPO. All promoters shown in the above figure gave rise to
persistent expression out to day 42. However from this analysis,
three enhancer/promoter combinations emerge as being the most
promising for yielding high persistent levels of expression. Two
copies of the hprt enhancer linked to either the .alpha.-1AT or HSA
promoters and two copies of the .alpha.-1-microglobulin enhancer
linked to the .alpha.-1-AT promoter yielded expression ranging from
1500 to 3000 mU EPO/ml or from 300 .mu.g to 600 .mu.g
protein/ml.
DETAILED DESCRIPTION OF THE INVENTION
[0026] 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 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.
[0027] 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 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 binding sites for known transcriptional repressors have been
deleted.
[0028] 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 albumin enhancers, human prothrombin enhancers,
.alpha.-1microglobulin enhancers and an intronic aldolase
enhancers. In preferred embodiments, multiple enhancer elements may
be combined in order to achieve higher expression.
[0029] 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 HPrT enhancers
of and A1MB enhancers, in combination with an .alpha.-1-antitrypsin
promoter.
[0030] 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 C 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 known transcriptional
repressors and is thus a preferred version of this promoter. The
human serum albumin and the .alpha.-1-antitrypsin in promoter
contain elements that direct basal yet liver specific expression.
The transcription factor binding sites in these promoter regions
are depicted in FIG. 1. 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.11microglobulin 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. FIG. 3 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.-1 microglobulin (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.
[0031] 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,
expression from CMV 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. Linkage of this enhancer to
the mCMV promoter (Panel B) elevated expression to near levels
achieved with the CMV promoter but did exceed it. Expression from
the .alpha.-1-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. 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, 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.
EXAMPLES
1. Plasmid Constructions
[0032] The alpha one antitrypsin promoter (-1200 to +44) was
PCR-amplified with Vent DNA polymerase (New England Bio Labs,
Beverly, Mass. USA) from an in-house pBr 322 vector that contains a
19-kb genomic Sal I fragment which includes human PI derived from
phage clone .alpha.NN (Dycaico et al. Science 242:1409-1412, 1988).
The promoter was then cloned between the Hind III-EcoR I sites of
pBluescript II SK+(Stratagene, La Jolla, Calif. USA) to generate
pBs A1AT. The sequence was analyzed using a PE Biosystems 377
automated sequencer. The hybrid alpha-galactosidase cassette from
an in-house vector was cloned into the Spe I site of pBs of A1AT to
generate pBs A1AT HI AGAL. The alpha one antitrypsin hybrid intron
alpha-galactosidase cassette was then subcloned into the pAdQuick
(formerly pAdvantage) shuttle vector Sv2 ICEU I to generate Sv2
A1AT HI AGAL.
[0033] Human liver specific enhancer elements from albumin 60 bp
and 81 bp; (1.7 kb and 6 kb from the transcription initiation site,
respectively); prothrombin 81 bp (-940 to -860); and Alpha-1
microglobulin/Bikunin 154 bp (-2806 to -2653) were obtained via PCR
from genomic DNA or through oligo synthesis. Multiple copies were
cloned into Bluescript II SK+(Stratagene, La Jolla, Calif., USA).
These enhancer elements were then subcloned into Sv2 A1AT HI AGAL
via Cla 1-Stu 1, reducing the alpha one antitrypsin promoter to
(-844 to +44) or subcloned into the in-house vector Sv2 CMV HI AGAL
II via Cla 1-SnaB 1, truncating the wild-type cytomegalovirus
promoter to (-245 to -14).
2. Hep3 B Transfections
[0034] Six well plates were seeded with Hep3 B cells at
2.times.10.sup.5 cells per well. Diluted 2.5 .mu.g enhancer
construct +2.5 .mu.g CMV B (Stratagene, La Jolla, Calif. USA) in
1.5 mls opti-mem reduced serum media (Gibco BRL, Gaithersburg, Md.
USA). Diluted 20 .mu.l lipofectamine 2000 (Gibco BRL, Gaithersburg,
Md. USA) in 1.5 mls opti-mem reduced serum media. The two solutions
were mixed and then incubated at room temperature for 30 min. While
complexes formed, cells were rinsed twice with opti-mem reduced
serum media. Incubated cells with the lipid solution (1.5 mls
solution per well) for 3-4 hrs at 37.degree. C. in 5% co.sub.2
incubator. Cells were rinsed once with 1.times.PBS and the lipid
solution was replaced with 2 mls Mem media containing 1 mM Sodium
pyruvate and 10% Fetal Bovine Serum. (Gibco BRL, Gaithersburg, Md.
USA).
3. Alpha-Galactosidase Fluorescent Assay
[0035] One hundred microliters of supernatant from hepatoma
transfections were transferred into 96-well plate (Coming flat
bottom). Five-fold dilutions were prepared to 1:125.
Alpha-galactoside A enzyme (Genzyme, Framingham, Mass. USA) was
diluted two fold 1250 uU/ml to 19.5 uU/ml to generate a standard
curve. Substrate solution (1.69 mg/ml
4-methylumbelliferyl-a-D-galactoside and 26 mg/ml
N-acetyl-D-galactosamine) in a buffer containing 27 mM citric acid,
46 mM sodium phosphate dibasic pH4.4 was added to the samples.
Samples were incubated at 37.degree. C. for 3 hours. The reactions
were terminated with the addition of fifty microliters of a one
molar sodium hydroxide solution. Spectra Max Gemini (Molecular
Devices Co. Sunnyvale, Calif. USA) were read with excitation filter
365 nm and emission filter 450 nm. Alpha-galactosidase activity was
normalized to .alpha.-galactosidase activity in transfection cell
lysates using Galacto-light Plus kit. (Tropix, Bedford, Mass.,
USA). All .alpha.-galactosidase assay reagents were obtained from
Sigma, St. Louis, Mo., USA.
[0036] The disclosures of all references disclosed herein are
hereby incorporated by reference. The invention has been described
in detail with particular reference to preferred embodiments
thereof. However, it is contemplated that modifications and
improvements within the spirit and teachings of this invention may
be made by those in the art upon considering the present
disclosure. Such modifications and improvements constitute part of
the present claimed invention.
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