U.S. patent application number 12/251451 was filed with the patent office on 2010-02-18 for compositions and methods for treating lysosomal storage disease.
This patent application is currently assigned to Genzyme Corporation. Invention is credited to Seng H. Cheng, Nelson S. Yew, Robin J. Ziegler.
Application Number | 20100041151 12/251451 |
Document ID | / |
Family ID | 22049811 |
Filed Date | 2010-02-18 |
United States Patent
Application |
20100041151 |
Kind Code |
A1 |
Yew; Nelson S. ; et
al. |
February 18, 2010 |
Compositions and methods for treating lysosomal storage disease
Abstract
The present invention provides recombinant viral and non-viral
vectors comprising a transgene encoding a biologically active human
lysosomal enzyme that are able to infect and/or transfect and
sustain expression of the biologically active human lysosomal
enzyme transgene in mammalian cells deficient therein. In addition,
methods are provided for providing a biologically active human
lysosomal enzyme to cells deficient therein, which comprises
introducing into the cells a vector comprising and expressing a
transgene encoding the biologically active human lysosomal enzyme,
wherein the vector is taken up by the cells, the transgene is
expressed and biologically active enzyme is produced. The cells may
be infected and/or transfected by the vector, dependent upon
whether the vector is a viral vector and/or plasmid or the like.
The invention also provides a method of supplying a biologically
active human lysosomal enzyme to other distant cells deficient
therein wherein the transfected and/or infected cells harboring the
vector secrete the biologically active enzyme which is then taken
up by the other deficient cells. In a preferred embodiment the
present invention provides for sustained production of biologically
human active .alpha.-galactosidase A in cells of Fabry individuals
that are deficient in said enzyme.
Inventors: |
Yew; Nelson S.; (West Upton,
MA) ; Ziegler; Robin J.; (Sterling, MA) ;
Cheng; Seng H.; (Wellesley, MA) |
Correspondence
Address: |
GENZYME CORPORATION;LEGAL DEPARTMENT
15 PLEASANT ST CONNECTOR
FRAMINGHAM
MA
01701-9322
US
|
Assignee: |
Genzyme Corporation
Cambridge
MA
|
Family ID: |
22049811 |
Appl. No.: |
12/251451 |
Filed: |
October 14, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10244700 |
Sep 13, 2002 |
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12251451 |
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09496499 |
Feb 2, 2000 |
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10244700 |
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09182245 |
Oct 29, 1998 |
6066626 |
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09496499 |
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60063527 |
Oct 29, 1997 |
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Current U.S.
Class: |
435/455 |
Current CPC
Class: |
A01K 2217/075 20130101;
C12N 9/2465 20130101; A61K 31/00 20130101; A61P 3/00 20180101; C12N
2799/022 20130101; A61K 48/00 20130101; A61K 38/47 20130101 |
Class at
Publication: |
435/455 |
International
Class: |
C12N 15/64 20060101
C12N015/64 |
Claims
1. A method for providing biologically active acid
.beta.-glucosidase to cells deficient therein, said method
comprising administration of a vector to cells in vivo comprising
and expressing a transgene encoding said biologically active acid
.beta.-glucosidase, wherein the vector is taken up by the cells,
the transgene is expressed therein and said biologically active
acid .beta.-glucosidase is produced.
2. A method according to claim 1, wherein the cells harboring the
vector secrete the biologically active enzyme which is taken up by
other cells deficient in the enzyme.
3. A method according to claim 1, wherein the vector is a viral
vector.
4. A method according to claim 3, wherein the viral vector is
adenovirus.
5. A method according to claim 4, wherein the adenovirus is
complexed with DEAE-dextran.
6. A method according to claim 2, wherein the vector is a viral
vector.
7. A method according to claim 6, wherein the viral vector is
adenovirus.
8. A method according to claim 7, wherein the adenovirus is
complexed with DEAE-dextran.
9. A method according to claim 1, wherein the vector is
plasmid.
10. A method according to claim 9, wherein the plasmid is complexed
with a cationic lipid.
11. A method according to claim 2, wherein the vector is
plasmid.
12. A method according to claim 11, wherein the plasmid is
complexed with a cationic lipid.
13. A composition comprising the plasmid vector of claim 9
complexed with a cationic lipid.
14. A composition according to claim 13, wherein the cationic lipid
is N.sup.4-spermine cholesteryl carbamate.
15. A composition comprising the plasmid vector of claim 11
complexed with a cationic lipid.
16. A composition according to claim 11, wherein the cationic lipid
is N.sup.4-spermine cholesteryl carbamate.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/244,700, filed Sep. 13, 2002, which is a continuation of
U.S. application Ser. No. 09/496,499, filed Feb. 2, 2000, which is
a continuation of U.S. application Ser. No. 09/182,245, filed Oct.
29, 1998, now U.S. Pat. No. 6,066,626, which claims the benefit of
U.S. Provisional Application No. 60/063,527, filed Oct. 29, 1997,
now abandoned. The entire teachings of the above applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Lysosomal storage diseases are a group of over 40 disorders
which are the result of defects in genes encoding enzymes that
break down glycolipid or polysaccharide waste products within the
lysosomes of cells. The enzymatic products, e.g., sugars and
lipids, are then recycled into new products. Each of these
disorders results from an inherited autosomal or X-linked recessive
trait which affects the levels of enzymes in the lysosome.
Generally, there is no biological or functional activity of the
affected enzymes in the cells and tissues of affected individuals.
Table I provides a list of representative storage diseases and the
enzymatic defect associated with the diseases. In such diseases the
deficiency in enzyme function creates a progressive systemic
deposition of lipid or carbohydrate substrate in lysosomes in cells
in the body, eventually causing loss of organ function and death.
The genetic etiology, clinical manifestations, molecular biology
and possibility of the lysosomal storage diseases are detailed in
Scriver et al., eds., The Metabolic and Molecular Basis of
Inherited Disease, 7.sup.th Ed., Vol. 11, McGraw Hill, (1995).
TABLE-US-00001 TABLE I Lysosomal storage diseases and associated
enzymatic defects Disease Enzymatic Defect Pompe disease Acid
.alpha.-glucosidase (acid maltese) MPSI* (Hurler disease)
A-L-iduronidase MPSII (Hunter disease) Iduronate sulfatase MPSIII
(Sanfilippo) heparin N-sulfatase MPS IV (Morquio A)
galactose-6-sulfatase MPS IV (Morquio B) Acid .beta.-galactosidase
MPS VII (Sly disease) B-glucoronidase I-cell disease
N-acetylglucosamine-1-phosphotransferase Schindler disease
.alpha.-N-acetylgalactosaminidase (.alpha.-galactosidase B) Wolman
disease acid lipase Cholesterol ester storage acid lipase disease
Farber disease lysosomal acid ceramidase Neimann-Pick disease acid
sphingomyelinase Gaucher disease .beta.-glucosidase
(glucocerebrosidase) Krabbe disease Galactosylceramidase Fabry
disease .alpha.-galactosidase GM1 gangliosidosis acid
.beta.-galactosidase Galactosialidosis .beta.-galactosidase and
neuraminidase Tay-Sach's disease hexosaminidase A Sandhoff disease
hexosaminidase A and B *MPS = mucopolysaccaridosis
[0003] As a representative of the class of lysosomal storage
diseases, Fabry Disease is a recessive, X-linked inherited
recessive disorder caused by a deficiency in the lysosomal enzyme
.alpha.-galactosidase A. Absence of this lysosomal hydrolase
results in progressive deposition of the glycosphingolipid
globotriasylceramide (GL3), or
galactosyl-(.alpha.1->4)-galactosyl-(.beta.1->4)-glucosyl-(.beta.1--
>1')-ceramide, in most tissues of the body. The birefringent
deposits of GL3 are primarily found in the vascular endothelium.
Progressive endothelial accumulation of GL3, leads to ischemia and
infarction in organs such as kidney, heart or brain, causing
excruciating pain, kidney failure, cardiac and cerebrovascular
disease. The average age of death for an affected individual, from
renal, cardiac and/or cerebral complications of the vascular
disease, is 41 years. There are no effective treatments currently
available for this disease. (See, e.g., Desnick et al., in Scriver
et al., eds. The Molecular Basis of Inherited Disease, 7.sup.th
Ed., Chapter 89, pp. 2741-2784, McGraw Hill (1995)).
[0004] Human .alpha.-galactosidase A (.alpha.-D-galactoside
galactohydrolase; .alpha.-gal A; EC 3.2.1.22) is a lysosomal
exoglycosidase encoded by a gene on Xq22. A human liver cDNA that
codes for .alpha.-galactosidase A was isolated from a .lamda.gt11
expression library (Calhoun et al., Proc. Natl. Acad. Sci., USA
82:7364-7368 (1985)). The isolated cDNA encoded the mature amino
acid sequence of .alpha.-galactosidase A but did not contain the
complete signal peptide sequence of the precursor form (Bishop et
al., Proc. Natl. Acad. Sci., USA 83:4859-4863 (1986). This partial
cDNA clone was then used to construct an E. coli expression vector
with the .alpha.-galactosidase A coding sequence under control of
the trp promoter (Hantzopoulos et al., Gene 57:159-169 (1987)). A
genomic clone was later isolated which carried the promoter and
first exon of the protein including the full signal peptide (Quinn
et al., Gene 58:177-188 (1987)). Further, full length cDNA clones
isolated from human fibroblasts were obtained and used to obtain
transient expression of .alpha.-galactosidase A in COS cells (Tsuji
et al., Eur. J. Biochem. 165:275-280 (1987)). Recently, a Fabry
knockout transgenic mouse demonstrating a deficiency in this enzyme
activity has been made (Ohshima et al., Proc. Natl. Acad. Sci., USA
94:2540-2544 (1997) knockout mice display a complete lack of
.alpha.-galactosidase A activity). Lipid analysis of the liver and
kidneys of the knockout mice revealed a marked accumulation of GL3
over time, indicating the similarity of the pathophysiological
process in the mutant mice and in patients-with-Fabry disease. Id.
Thus, the Fabry knockout mice provide an excellent model for the
human disease.
[0005] De Duve first suggested that replacement of the missing
lysosomal enzyme with exogenous biologically active enzyme might be
a viable approach to treatment of lysosomal storage diseases. De
Duve, Fed Proc. 23:1045 (1964). Since that time, various studies
have suggested that enzyme replacement therapy may be beneficial
for treating various lysosomal storage diseases. The best success
has been shown with individuals with type 1 Gaucher disease, who
have been treated with exogenous enzyme
(.beta.-glucocerebrosidase), prepared from placenta (Ceredase.RTM.)
or, more recently, recombinantly (Cerezyme.RTM.). It has been
suggested that enzyme replacement may also be beneficial for
treating Fabry's disease, as well as other lysosomal storage
diseases. See, for example, Dawson et al., Ped. Res. 7(8):684-690
(1973) (in vitro) and Mapes et al., Science 169:987 (1970) (in
vivo). Clinical trials of enzyme replacement therapy have been
reported for Fabry patients using infusions of normal plasma (Mapes
et al., Science 169:987-989 (1970)); .alpha.-galactosidase A
purified from placenta (Brady et al., N. Eng. J. Med. 279:1163
(1973)); or .alpha.-galactosidase A purified from spleen or plasma
(Desnick et al., Proc. Natl. Acad. Sci., USA 76:5326-5330 (1979))
demonstrated the biochemical effectiveness of direct enzyme
replacement for Fabry disease. These studies indicated the
potential for eliminating, or significantly reducing, the
pathological glycolipid storage by repeated enzyme replacement. For
example, in one study (Desnick et al., supra), intravenous
injection of purified enzyme resulted in a transient reduction in
the plasma levels of the stored lipid substrate,
globotriasylceramide.
[0006] However, to date, the biochemical and clinical effectiveness
of enzyme replacement in Fabry disease, as well as other lysosomal
storage diseases, has not been demonstrated due to the lack of
sufficient human enzyme for adequate doses and long-term
evaluation.
[0007] Accordingly, there exists a need in the art for methods for
providing sufficient quantities of biologically active lysosomal
enzymes, such as human .alpha.-galactosidase A, to deficient cells.
Additionally, there exists a need for new vector compositions that
allow for efficient transfer of genes encoding lysosomal enzymes,
such as .alpha.-galactosidase A, to deficient cells and at the same
time direct expression of the transferred gene. Recently,
recombinant approaches have attempted to address these needs, see,
e.g., U.S. Pat. No. 5,658,567 issued Aug. 19, 1997 for Recombinant
alpha-galactosidase A therapy for Fabry disease; U.S. Pat. No.
5,580,757 issued Dec. 3, 1996 for Cloning and Expression of
Biologically Active alpha-galactosidase A as a Fusion Protein;
Bishop, D. F. et al., Proc. Natl. Acad Sci., USA. 83:4859-4863,
(1986); Medin, J. A. et al., Proc. Natl. Acad. Sci., USA.
93:7917-7922, (1996); Novo, F. J., Gene Therapy 4:488-492, (1997);
Ohshima, T. et al., Proc. Natl. Acad. Sci., USA. 94:2540-2544,
(1997); and Sugimoto Y. et al., Human Gene Therapy 6:905-915,
(1995). In addition, in allowed U.S. patent application Ser. No.
08/466,597, filed Jun. 6, 1995, incorporated herein by reference,
retroviral expression vectors containing a gene encoding human
.beta.-glucocerebrosidase were shown to infect autologous
hematopoietic stem cells, which when retransplanted into a Gaucher
patient provided sustained production of biologically active enzyme
to the patient.
[0008] To date, however, there has not been a vector composition
that has proven capable of transducing and sustaining expression of
the human. .beta.-galactosidase A gene, or most other genes
encoding lysosomal enzymes to cells that are deficient therein. The
present invention satisfies these needs and provides related
advantages as well.
SUMMARY OF THE INVENTION
[0009] Accordingly, the present invention provides recombinant
viral and non-viral vectors comprising a transgene encoding a
biologically active human lysosomal enzyme that are able to infect
and/or transfect and sustain expression of the biologically active
human lysosomal enzyme transgene in mammalian cells deficient
therein. In a preferred embodiment, the expressed transgene encodes
.alpha.-galactosidase and the deficient cells are those of an
individual with Fabry's disease.
[0010] The present invention further provides a method for
providing a biologically active human lysosomal enzyme to cells
deficient therein, which comprises introducing into the cells a
vector comprising and expressing a transgene encoding the
biologically active human lysosomal enzyme, wherein the vector is
taken up by the cells, the transgene is expressed and biologically
active enzyme is produced. The cells may be infected and/or
transfected by the vector, dependent upon whether the vector is a
viral vector and/or plasmid or the like.
[0011] In a preferred embodiment the present invention provides for
sustained production of biologically human active
.alpha.-galactosidase A in cells of Fabry individuals that are
deficient in said enzyme.
[0012] In a still further aspect, the invention also provides a
method of supplying a biologically active human lysosomal enzyme to
other distant cells deficient therein wherein the transfected
and/or infected cells harboring the vector secrete the biologically
active enzyme which is then taken up by the other deficient cells.
In a preferred embodiment, the enzyme is human
.alpha.-galactosidase A and the cells are those of a Fabry
individual.
[0013] In a still further aspect, the biologically active enzyme,
preferably .alpha.-galactosidase A, is secreted into the
circulation of an individual (e.g., a Fabry individual).
[0014] The present invention also provides a recombinant E1 deleted
adenoviral vector, Ad2/CEH .alpha.-gal, and a recombinant plasmid
expression vector, pCFA-hAGA, both of which comprised and express a
transgene encoding .alpha.-galactosidase A.
[0015] The present invention further provides a method for
providing biologically active human .alpha.-galactosidase A to the
cells of an individual with Fabry disease comprising introducing
into the cells of a Fabry individual an amount of
Ad2/CEH.alpha.-gal effective to infect and sustain expression of
the biologically active human .alpha.-galactosidase A transgene in
cells deficient therein.
[0016] The present invention further provides a method for
providing biologically active human .alpha.-galactosidase A to the
cells of an individual with Fabry disease comprising introducing
into the cell of a Fabry individual an amount of pCFA-hAGA
effective to transfect and sustain expression of biologically
active human .alpha.-gal A gene in cells deficient therein.
[0017] Other features and advantages of the present invention will
be apparent from the following detailed description as well as from
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows the plasmid expression vector pCFA-hAGA.
[0019] FIG. 2 shows the adenovirus expression vector
Ad2/CEH.alpha.-gal.
[0020] FIGS. 3A-3B show uptake of .alpha.-galactosidase A produced
from Ad2/CEH.alpha.-gal by Fabry cells. FIG. 3A shows uptake of
.alpha.-galactosidase A expressed in Ad2/CEH.alpha.-gal infected
fibroblasts (GM02775). FIG. 3B shows uptake of
.alpha.-galactosidase A expressed in Ad2/CEH.alpha.-gal infected
skeletal muscle cells (SkMC).
[0021] FIG. 4 shows tissue distribution of .alpha.-galactosidase A
in normal vs. Fabry's knockout mice.
[0022] FIG. 5 shows tissue distribution of .alpha.-galactosidase A
after intranasal, intravenous and intramuscular administration of
plasmid.
[0023] FIGS. 6A-6B show tissue distribution of
.alpha.-galactosidase A after administration of
Ad2/CEH.alpha.-gal/CEH.alpha..-gal vector to Fabry's knockout mice.
FIG. 6A shows distribution after viral injection into the tail vein
of female Fabry's knockout mice. FIG. 6B shows distribution after
viral injection into the right quadriceps muscle group of female
Fabry's mice.
[0024] FIGS. 7A-7B show a time course of .alpha.-galactosidase A
expression after intravenous injection of Ad2/CEH.alpha.-gal into
C57BL/6n mice. FIG. 7A shows expression of .alpha.-galactosidase A
over time. FIG. 7B shows persistence of .alpha.-galactosidase A
relative to day 3.
[0025] FIGS. 8A-8B show levels of .alpha.-galactosidase A in whole
blood after intravenous injection of Ad2/CEH.alpha.-gal into
C57BL/6n and BALB/c(nu/nu) mice. FIG. 8A shows expression of
.alpha.-galactosidase A over time. FIG. 8B shows persistence of
.alpha.-galactosidase A relative to day 3.
[0026] FIGS. 9A-9B show levels of .alpha.-galactosidase A in
tissues of Fabry mice after intravenous injection of a low level
dose (1.65.times.10.sup.10 particles) of Ad2/CEH.alpha.-gal. FIG.
9A shows a .alpha.-galactosidase A expression over time. FIG. 9B
shows persistence of .alpha.-galactosidase A relative to day 3.
[0027] FIGS. 10A-10B show levels of .alpha.-galactosidase A in
tissues of Fabry mice a intravenous injection of a high level dose
(1.65.times.10.sup.11 particles) of Ad2/CEH.alpha.-gal. FIG. 10A
shows a .alpha.-galactosidase A expression over time. FIG. 10B
shows persistence of .alpha.-galactosidase A relative to day 3.
[0028] FIGS. 11A-11F show levels of GL3 in Fabry mouse tissues
after intravenous injection of high and low doses of
Ad2/CEH-.alpha.-gal over time.
[0029] FIG. 12 shows effect of DSG on .alpha.-galactosidase A
levels in mice after repeat administration of adenovirus
vector.
[0030] FIG. 13 shows effect of DSG on anti-adenovirus antibody
levels in mice following repeat administration of adenovirus
vector.
[0031] FIG. 14 shows the effect of MRI antibody directed to CD154
on .alpha.-galactosidase A levels in mouse tissues following repeat
administration of adenovirus vector.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention provides recombinant viral and
non-viral vectors comprising a transgene encoding a biologically
active human lysosomal enzyme that are able to infect and/or
transfect and sustain expression of the biologically active human
lysosomal enzyme transgene in mammalian cells deficient therein. In
a preferred embodiment, the expressed transgene encodes
.alpha.-galactosidase A.
[0033] The present invention further provides a method for
providing a biologically active human lysosomal enzyme to cells
deficient therein which comprises introducing into the cells a
vector comprising and expressing a transgene encoding the
biologically active human lysosomal enzyme, wherein the vector is
taken up by the cells, the transgene is expressed and biologically
active enzyme is produced. The cells may be infected and/or
transfected by the vector, dependent upon whether the vector is a
viral vector and/or plasmid or the like.
[0034] In a still further aspect, the invention provides a method
of supplying a biologically active human lysosomal enzyme to other
distant cells deficient therein wherein the transfected and/or
infected cells harboring the vector secrete the biologically active
enzyme which is then taken up by the other deficient cells.
[0035] Vectors that may be used in the present invention include
viruses, such as adenoviruses, adeno associated virus (AAV),
vaccinia, herpesviruses, baculoviruses and retroviruses,
bacteriophages, cosmids, plasmids, fungal vectors and other
recombination vehicles typically used in the art which have been
described for expression in a variety of eukaryotic and prokaryotic
hosts, and may be used for gene therapy as well as for simple
protein expression.
[0036] Polynucleotides/transgenes are inserted into vector genomes
using methods well known in the art. Transgenes are defined herein
as nucleic acids molecules or structural genes that encode a
particular protein--in the present invention, a human lysosomal
enzyme and nucleic acids encoding said enzymes. Representative
lysosomal enzymes in accordance with the present invention are
provided in Table I above. References relating to isolation and
characterization of the lysosomal enzymes in Table I may be found
in Scriver et al., The Metabolic Basis of Inherited Disease,
7.sup.th Ed., vol. 11, pp. 2427-2879, McGraw Hill (1995),
incorporated herein by reference.
[0037] By way of example, in order to insert the transgene into the
vector, transgene and vector nucleic can be contacted, under
suitable conditions, with a restriction enzyme to create
complementary ends on each molecule that can pair with each other
and be joined together with a ligase. Alternatively, synthetic
nucleic acid linkers can be ligated to the termini of the
restricted polynucleotide. These synthetic linkers contain nucleic
acid sequences that correspond to a particular restriction site in
the vector nucleic acid. Additionally, an oligonucleotide
containing a termination codon and an appropriate restriction site
can be ligated for insertion into a vector containing, for example,
some or all of the following: a selectable marker gene, such as the
neomycin gene for selection of stable or transient transfectants in
mammalian cells; enhancer/promoter sequences from the immediate
early gene of human CMV for high levels of transcription;
transcription termination and RNA processing signals from SV40 for
mRNA stability; SV40 polyoma origins of replication and ColE1 for
proper episomal replication; versatile multiple cloning sites; and
T7 and SP6 RNA promoters for in vitro transcription of sense and
antisense RNA. Other means are well known and available in the
art.
[0038] As used herein, "expression" refers to the process by which
polynucleotides/transgenes are transcribed into mRNA and then
translated into peptides, polypeptides, or proteins. If the
polynucleotide is derived from genomic DNA, expression may include
splicing of the mRNA, if an appropriate eukaryotic host is
selected. Regulatory elements required for expression include
promoter sequences to bind RNA polymerase and transcription
initiation sequences for ribosome binding. For example, a bacterial
expression vector includes a promoter such as the lac promoter and
for transcription initiation the Shine-Dalgarno sequence and the
start codon AUG (Sambrook et al., Molecular Cloning, A Laboratory
Manual 2d Ed. (Cold Spring harbor, N.Y., 1989), or Ausubel et al.,
Current Protocols in Molecular Biology (Greene Assoc., Wiley
Interscience, New York, N.Y., 1995). Similarly, a eukaryotic
expression vector, be it a virus or a plasmid, includes a
heterologous or homologous promoter for RNA polymerase II, a
downstream polyadenylation signal, the start codon AUG, and a
termination codon for detachment of the ribosome. Such vectors can
be obtained commercially or assembled by the sequences described in
methods well known in the art, for example, the methods described
above for constructing vectors in general. Expression vectors are
useful to produce cells that express the protein encoded by the
polynucleotide/transgene.
[0039] Preparations of the transgene encoding a human lysosomal
enzyme, e.g., .alpha.-galactosidase A, can be incorporated in a
suitable vector for delivery into an individual's cells, e.g., a
Fabry individual, using methods that are known in the art. See, for
example, Finkel and Epstein, FASEB J. 9:843-851 (1995); Feldman and
Steg, Cardiovascular Res. 32:194-207 (1996).
[0040] Naked nucleic--Naked plasmid DNA can be introduced into
muscle cells, for example, by direct injection into the tissue.
(Wolff et al., Science 247:1465 (1989)).
[0041] Nucleic acid-Lipid Complexes--Lipid carriers can be
associated with naked nucleic acids (e.g., plasmid DNA) to
facilitate passage through cellular membranes. Cationic, anionic,
or neutral lipids can be used for this purpose. However, cationic
lipids are preferred because they have been shown to associate
better with DNA which, generally, has a negative charge. Cationic
lipids have also been shown to mediate intracellular delivery of
plasmid DNA (Felgner and Ringold, Nature 337:387 (1989)).
Intravenous injection of cationic lipid-plasmid complexes into mice
has been shown to result in expression of the DNA in lung (Brigham
et al., Am. J. Med. Sci. 298:278 (1989)). See also, Osaka et al.,
J. Pharm. Sci. 85(6):612-618 (1996); San et al., Human Gene Therapy
4:781-788 (1993); Senior et al., Biochemica et Biophysica Acta
1070:173-179 (1991); Kabanov and Kabanov, Bioconjugate Chem. 6:7-20
(1995); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Behr,
J-P., Bioconjugate Chem 5:382-389 (1994); Behr et al., Proc. Natl.
Acad. Sci., USA 86:6982-6986 (1989); and Wyman et al., Biochem.
36:3008-3017 (1997).
[0042] Cationic are known to those of ordinary skill in the art.
Representative cationic lipids include those disclosed, for
example, in U.S. Pat. No. 5,283,185; and e.g., U.S. Pat. No.
5,767,099, the disclosures of which are incorporated herein by
reference. In a preferred embodiment, the cationic lipid is
N.sup.4-spermine cholesteryl carbamate (GL-67) disclosed in U.S.
Pat. No. 5,767,099. Additional preferred lipids include
N.sup.4-spermidine cholestryl carbamate (GL-53) and
1-(N.sup.4-spermine)-2,3-dilaurylglycerol carbamate (GL-89).
[0043] Adenovirus--Adenovirus-based vectors for the delivery of
transgenes are well known in the art and may be obtained
commercially or constructed by standard molecular biological
methods. Recombinant adenoviral vectors containing exogenous genes
for transfer are, generally, derived from adenovirus type 2 (Ad2)
and adenovirus type 5 (Ad5). They may also be derived from other
non-oncogenic serotypes. See, for example, Horowitz, "Adenoviridae
and their Replication" in VIROLOGY, 2d ed., Fields et al. Eds.,
Raven Press Ltd., New York, 1990, incorporated herein by
reference.
[0044] The adenoviral vectors of the present invention are
incapable of replicating, have minimal viral gene expression and
are capable of expressing a transgene in target cells. Adenoviral
vectors are generally rendered replication-defective by deletion of
the E1 region genes. The replication-defective vectors maybe
produced in the 293 cell line (ATCC CRL 1573), a human embryonic
kidney cell line expressing E1 functions. The deleted E 1 region
may be replaced by the transgene of interest under the control of
an adenoviral or non-adenoviral promoter. The transgene may also be
placed in other regions of the adenovirus genome. See, Graham et
al., "Adenovirus-based Expression Vectors and Recombinant Vaccines"
in VACCINES: NEW APPROACHES to IMMUNOLOGICAL PROBLEMS pp 363-390,
Ellis, Ed., Butterworth-Heinemann, Boston, (1992) for a review of
the production of replication-defective adenoviral vectors, also
incorporated herein by reference.
[0045] Skilled artisans are also aware that other non-essential
regions of the adenovirus can be deleted or repositioned within the
viral genome to provide an adenoviral vector suitable for delivery
of a transgene in accordance with the present invention. For
example, U.S. Pat. No. 5,670,488, incorporated herein by reference,
discloses that some or all of the E1 and E3 regions may be deleted,
and non-essential open reading frames (ORFs) of E4 not required for
in vitro virus propagation can also be deleted. Other
representative adenoviral vectors are disclosed, for example, by
Rich et al., Human Gene Therapy 4:461 (1993); Brody et al., Ann. NY
Acad. Sci. 716:90 (1994); Wilson, N. Eng. J. Med. 34:1185 (1996);
Crystal, Science 270:404 (1995); O'Neal et al., Hum. Mol. Genet.
3:1497 (1994); and Graham et al., supra., incorporated herein by
reference. In a preferred embodiment of the present invention, the
adenoviral vector is an E1 deleted Ad2-based vector, e.g. as
disclosed in U.S. Pat. No. 5,670,488, incorporated herein by
reference. Other adenoviral vectors that may be used include those
that have been designed to prevent the generation of replication
competent adenovirus in vivo (U.S. Pat. No. 5,707,618, incorporated
herein by reference). In addition, pseudoadenovirus vectors (PAV),
which are deleted for early and late genes, as disclosed in U.S.
Pat. No. 5,670,488, are also contemplated for use herein.
[0046] As defined above, a transgene, as used herein, is a nucleic
acid or structural gene coding for a human lysosomal enzyme.
Moreover the transgene is foreign or non-native to adenovirus. Any
nucleic acid coding for a human lysosomal enzyme that can be
transcribed in the adenoviral vector is contemplated. In a
preferred embodiment, the transgene encodes a biologically active
or functional .alpha.-galactosidase A protein. A biologically
active or functional protein or peptide is a protein or peptide
that affects the cellular mechanism of a cell in which it is
expressed, or the function of a tissue or organism. In the case of
.alpha.-galactosidase A, the enzyme cleaves the lipid substrate
globotriasylceramide (galactosyl-galactosyl-glucosyl-ceramide) or
GL3.
[0047] In the adenoviral vectors of the present invention, the
transgene is operably linked to expression control sequences, e.g.,
a promoter that directs expression of the transgene. As used
herein, the phrase "operatively linked" refers to the functional
relationship of a polynucleotide/transgene with regulatory and
effector sequences of nucleotides, such as promoters, enhancers,
transcriptional and translational stop sites, and other signal
sequences. For example, operative linkage of a nucleic acid to a
promoter refers to the physical and functional relationship between
the polynucleotide and the promoter such that transcription of DNA
is initiated from the promoter by an RNA polymerase that
specifically recognizes and binds to the promoter, and wherein the
promoter directs the transcription of RNA from the
polynucleotide.
[0048] Promoter regions include specific sequences that are
sufficient for RNA polymerase recognition, binding and
transcription initiation. Additionally, promoter regions include
sequences that modulate the recognition, binding and transcription
initiation activity of RNA polymerase. Such sequences may be cis
acting or may be responsive to transacting factors. Depending upon
the nature of the regulation, promoters may be constitutive or
regulated. Examples of promoters are SP6, T4, T7, SV40 early
promoter, cytomegalovirus (CMV) promoter, mouse mammary tumor virus
(MMTV) steroid-inducible promoter, Moloney murine leukemia virus
(MMLV) promoter, phosphoglycerate kinase (PGK) promoter, and the
like. Alternatively, the promoter may be an endogenous adenovirus
promoter, for example the E1 a promoter or the Ad2 major late
promoter (MLP). Similarly, those of ordinary skill in the art can
construct adenoviral vectors utilizing endogenous or heterologous
poly A addition signals. In particular, the use of a CMV
promoter/transgene, together with adenovirus E4 region, preferably
ORF3, which as disclosed in PCT/US98/07841, filed Apr. 14, 1998 and
incorporated herein by reference, has been shown to provide
increased persistence of transgene expression is preferred. Also,
E1 deleted, partially E3 deleted vectors capable of providing
persistent expression of a transgene, as disclosed in
PCT/US98/07840, filed Apr. 14, 1998 and incorporated herein by
reference, are also contemplated.
[0049] Other viral vectors for use in the present invention include
vectors derived from vaccinia, herpesvirus, AAV and retroviruses.
In particular, herpesviruses, especially herpes simplex virus
(HSV), such as those disclosed in U.S. Pat. No. 5,672,344, the
disclosure of which is incorporated herein by reference, are
particularly useful for delivery of a transgene to a neuronal cell,
which has importance for those lysosomal storage diseases in which
the enzymatic defect manifests in neuronal cells, e.g, Hurler's,
Hunter's, and Tay-Sach's diseases. AAV vectors, such as those
disclosed in U.S. Pat. Nos. 5,139,941, 5,252,479 and 5,753,500 and
PCT publication WO 97/09441, the disclosures of which are
incorporated herein, are also useful since these vectors integrate
into host chromosomes, with a minimal need for repeat
administration of vector.
[0050] Retroviruses may also find use in the present invention,
especially for transgene delivery to cells that can be removed from
an individual, infected ex vivo and readministered back to the
individual for production of biologically active enzyme.
[0051] The viral and non-viral vectors of the present invention are
useful for transferring a transgene encoding a lysosomal enzyme to
a target cell. The target cell may be in vitro or in vivo. Use of
invention vectors in vitro allows the transfer of a transgene to a
cultured cell and is useful for the recombinant production of the
transgene product. Use of invention vectors to deliver a transgene
to a cell in vivo is useful for providing biologically active
enzyme to cells deficient therein, for example, in the case of
Fabry disease, a cell in which .alpha.-galactosidase A is absent,
insufficient or nonfunctional.
[0052] The vectors of the invention may be targeted to specific
cells by linking a targeting molecule to the vector. A targeting
molecule is any agent that is specific for a cell or tissue type of
interest, including for example, a ligand, antibody, sugar,
receptor, or other binding molecule. The ability of targeted
vectors renders invention vectors particularly useful in the
treatment of lysosomal storage disorders. For example, including a
targeting molecule, such as VEGF or an antibody to a VEGF receptor
can provide targeting to vascular endothelial cells in individuals
with Fabry's disease.
[0053] In addition, viral vectors, especially adenoviral vectors
that have been complexed with a cationic amphiphile, such as a
cationic lipid as described above, polyL-lysine (PLL), and
diethylaminoethyldextran (DELAE-dextran) provide increased
inefficiency of viral infection of target cells (See, e.g.,
PCT/US97/21496 filed Nov. 20, 1997, incorporated herein by
reference).
[0054] Adenoviral vectors complexed with DEAE dextran are
particularly preferred. In addition, since repeat administration of
a viral vector can result in an immune response to the vector,
thereby limiting its effectiveness in delivering the gene to
affected cells, adenovirus and other viral vectors may be
polymer-modified, e.g. complexed with polyethylene glycol (PEG), to
reduce viral immunogenicity and allow for repeat administration of
the vector (See, e.g., PCT/US98/06609 filed Apr. 3, 1998,
incorporated herein by reference). Alternatively, the vector may be
administered with an immunosuppressive agent to reduce the immune
response to repeated vector administration. In addition,
combinations of the above approaches may be used.
[0055] Transfer of the transgene to the target cells by invention
vectors can be evaluated by measuring the level of the transgene
product (biologically active enzyme) in the target cell. The level
of transgene product in the target cell directly correlates with
the efficiency of transfer of the transgene by invention vectors.
Any method known in the art can be used to measure enzyme levels,
such as ELISA, radioimmunoassay, assays using an fluorescent and
chemiluminescent enzyme substrates.
[0056] Expression of the transgene can be monitored by a variety of
methods known in the art including, inter alia, immunological,
histochemical and activity assays. Immunological procedures useful
for in vitro detection of the transgene product in a sample include
immunoassays that employ a detectable antibody. Such immunoassays
include, for example, ELISA, Pandex microfluorimetric assay,
agglutination assays, flow cytometry, serum diagnostic assays and
immunohistochemical staining procedures which are well known in the
art. An antibody can be made detectable by various means well known
in the art. For example, a detectable marker can be directly or
indirectly attached to the antibody. Useful markers include, for
example, radionuclides. enzymes, fluorogens, chromogens and
chemiluminescent labels.
[0057] For in vivo imaging methods, a detectable antibody can be
administered to a subject and the binding of the antibody to the
transgene product can be detected by imaging techniques well known
in the art. Suitable imaging agents are known and include, for
example, gamma-emitting radionuclides such as .sup.111In,
.sup.99mTc, .sup.51Cr and the like, as well as paramagnetic metal
ions, which are described in U.S. Pat. No. 4,647,447. The
radionuclides permit the imaging of tissues by gamma scintillation
photometry, positron emission tomography, single photon emission
computed tomography and gamma camera whole body imaging, while
paramagnetic metal ions permit visualization by magnetic resonance
imaging.
[0058] The present invention is exemplified using vectors
comprising an .alpha.-galactosidase A transgene to deliver
biologically active .alpha.-galactosidase A to cells and tissues of
individuals with Fabry's disease. The efficacy of this approach has
been demonstrated using a mouse model system, e.g., a Fabry
knockout mouse. Thus, active human .alpha.-galactosidase A is
provided to the cells of an individual with Fabry disease by
introducing into a Fabry individual an amount of invention vectors
effective to infect and/or transfect and sustain expression of
biologically active human .alpha.-gal A gene in cells deficient
therein. Invention vectors may be delivered to the target cells in
a suitable composition, either alone, or complexed, as provided
above, comprising the vector and a suitably acceptable carrier.
Plasmid vectors are preferably complexed with a cationic lipid such
as GL67. Adenoviral vectors are preferably complexed with DEAE
dextran. The vector may be delivered to target cells by methods
known in the art, for example, intravenous, intramuscular,
intranasal, subcutaneous, intubation, lavage, and the like.
[0059] The terms transgene encoding .alpha.-galactosidase A
includes a nucleic acid (DNA) or a structural gene that encodes
.alpha.-galactosidase A that, when expressed in deficient cells of
a Fabry individual, alleviate the .alpha.-galactosidase A
deficiency therein.
[0060] As used herein the terms effective amount refers to an
amount that alleviates the deficiency by the production of
biologically active .alpha.-galactosidase A in the cells of a Fabry
individual. Production of biologically active .alpha.-galactosidase
A in Fabry individuals can be evaluated by the alleviation of the
symptoms associated with Fabry disease. The precise effective
amount of vector to be used in the method of the present invention
can be determined by one of ordinary skill in the art with
consideration of, for example, individual differences in age,
weight, extent of disease and condition of the individual.
[0061] In particular, the present invention provides both viral and
non-viral approaches for delivering biologically active
.alpha.-galactosidase A to cells of individuals with Fabry disease.
A recombinant adenoviral vector (pAd2/CEH.alpha.-gal) and a plasmid
expression vector (pCFA-hAGA) that express human
.alpha.-galactosidase A (.alpha.-gal) have been constructed. A
human airway epithelial cell line that was either infected or
transfected with these vectors expressed active enzyme at levels
more than a log higher than endogenous levels, with a significant
proportion of the activity being secreted into the medium. The
.alpha.-galactosidase A secreted from either infected fibroblasts
(GM02775) or infected primary human skeletal muscle cells (SkMC)
was shown to be taken up by Fabry fibroblasts. This indicates that
enzyme can be secreted by cells that have taken up the vector in
vivo, and that the secreted enzyme can be taken up by untransfected
cells, thus correcting the genetic defect in a large percentage of
cells in the body.
[0062] Studies have been undertaken in mice using pCFA-hAGA to
compare the efficacy of three potential routes of
delivery--intranasal, intravenous, and intramuscular
administration. Intranasal instillation into the lung of plasmid
DNA complexed with the cationic lipid GL-67 resulted in low level
expression (up to 1800 pg .alpha.-gal per 100 mg tissue) in the
lung. Intravenous administration of plasmid DNA complexed with
lipid GL-67 also showed low levels of expression in the lung (up to
700 pg per 100 mg tissue). Intramuscular injection of plasmid DNA
alone in the absence of cationic lipid produced low levels of
expression (up to 1200 pg per 100 mg tissue) in the injected
muscle. Experiments performed using the adenovirus vector show very
high levels of activity in all of the tissues assayed (up to 100
.mu.g per 100 mg tissue in the liver, 10 .mu.g per 100 mg tissue in
most other organs). The level of enzyme assayed in liver from
normal mice was 400 ng per 100 mg tissue. The tissue samples from
the virus treated mice were assayed by two different methods, an
activity assay and an ELISA assay, with similar results.
[0063] In addition, intravenous administration of viral vectors to
Fabry mice has been shown to result in a decrease in accumulated
GL3 substrate in a wide variety of tissues in treated animals. It
has been shown that small quantities of lysosomal enzymes are
normally secreted and that these can be recaptured by distant cells
through the mannose-6-phosphate receptors. Indeed, the results
presented show that .alpha.-galactosidase A collected from
supernatants of cells transfected with viral and non-viral vectors
encoding the enzyme are capable of being internalized by Fabry
cells. These results further suggest that gene transfer of
.alpha.-galactosidase A to an appropriate depot organ can
facilitate reversion of the biochemical defect and storage of GL3
in the affected tissues of Fabry patients.
[0064] The present invention is further illustrated by the
following examples which in no way should be construed as being
further limiting. The contents of all references cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLES
Example 1
Vector Construction
[0065] pCFA-hAGA
[0066] This plasmid expression vector utilizes the cytomegalovirus
immediate early promoter to drive expression of the human
.alpha.-galactosidase A cDNA. A hybrid intron was included after
the promoter to provide splice sites to enhance expression. The
polyadenylation signal was taken from the bovine growth hormone
gene. The ColE1 replicon from pUC was used as a backbone for
replication in E. coli. The kanamycin resistance gene was used to
select for plasmid maintenance. The construction of the pCFA-hAGA
is analogous to the construction of the pCFI vector containing a
CFTR transgene disclosed, e.g., in U.S. Pat. No. 5,783,565, the
disclosure of which is incorporated herein by reference. In the
pCFA-hAGA vector, an .alpha.-galactosidase A transgene is
substituted for the CFTR transgene in pCFI.
Ad2/CEH.alpha.-gal
[0067] The E1-deleted adenovirus expression vector using an Ad2
serotype viral backbone was constructed as provided in U.S. Pat.
No. 5,670,488, the disclosure of which is incorporated herein by
reference. The E1 region of the virus genome was deleted to allow
space for an expression cassette. Deleting the E1 region also makes
the virus incapable of replication. The adenovirus E1 promoter was
used to drive expression of the human .alpha.-galactosidase A cDNA.
The hybrid intron was included after the promoter. The
polyadenylation signal was taken from the SV40 virus. (FIG. 2).
Example 2
Uptake of Human .alpha.-galactosidase A Produced from
Ad2/CEH.alpha.-gal by Fabry Fibroblasts
[0068] Human primary cells were infected with Ad2/CEH.alpha.-gal at
the following MOIs (Fabry fibroblast cell line GM02775: 0, 2, 4, 6
and 8 .mu.U .alpha.-gal/.mu.g protein; skeletal muscle cell line
SkMC: 0, 0.5, 1, 1.5, 2, 2.5 and 3 .mu.U .alpha.-gal/.mu.g
protein). Three days after infection conditioned culture medium was
collected and filtered to remove virus particles. Filtered
conditioned medium was applied to uninfected Fabry fibroblasts
(GM02775). After a five hour incubation, medium was removed, cells
were washed with PBS, and harvested in 0.5 ml lysis buffer.
Fibroblasts from normal (GM02770B) and Fabry donors which had not
been exposed to conditioned medium were harvested and assayed as
controls. Cell lysates were assayed using the fluorescent substrate
4-methylumbelliferyl-.alpha.-D-galactopyranoside
(4-mu-.alpha.-gal). (FIGS. 3A and 3B). The assays showed that human
primary cells infected with Ad2/CEH.alpha.-gal secreted
biologically active .alpha.-galactosidase A that was taken up by
Fabry fibroblasts.
Example 3
Tissue Distribution of .alpha.-galactosidase A in Normal Vs.
Fabry's Knockout Mice
[0069] Normal (C57BL/6n) and Fabry knockout mice (provided by Dr.
Robert Desnick, Mount Sinai School of Medicine, New York, N.Y.)
were assayed for levels of .alpha.-galactosidase A using the
4-mu-.alpha.-gal activity assay. A full body perfusion was
performed at the time of sacrifice and the organs were harvested
and stored at -80.degree. C. Tissues were homogenized in assay
buffer and put through several freeze-thaw cycles. Fabry mice
showed significantly reduced levels of .alpha.-galactosidase A
activity when compared to normal mice in all organs tested. (FIG.
4).
Example 4
Tissue Distribution of .alpha.-galactosidase A after Intranasal,
Intravenous and Intramuscular Administration of pCFA-hAGA
[0070] pCFA-hAGA, complexed with the cationic lipid GL-67
(N.sup.4-spermine cholesteryl carbamate), disclosed, e.g., in U.S.
Pat. No. 5,783,565, incorporated herein by reference, was
administered to C57BL/6n mice. .alpha.-gal levels in tissue
homogenates were assayed by an enzyme-linked immunosorbant assay
(ELISA) specific for human .alpha.-galactosidase. Intranasal
instillations were performed using 100 .mu.l of GL-67:
DOPE(1:2):pCFA-hAGA complex at a 0.6 mM:3.6 mM lipid:DNA ratio.
See, for example, International Publication No. WO 96/18372
(Cationic amphiphiles and plasmids for intracellular delivery of
therapeutic molecules, e.g., GL-67); Fasbender, A. J. et al., Am. J
Physiol. 269(1) Pt 1: L45-51 (1995); Zabner, J. et al., J. Biol.
Chem. 270(32):18997-19007 (1995). Animals were sacrificed 2 days
post-instillation. Intravenous injections were performed with 100
.mu.l of GL-67:DOPE:DMPE-PEG (1:2:0.005): pCFA-hAGA complex at a 4
mM:4 mM lipid:DNA ratio into the tail vein. These animals were
sacrificed 2 days post-administration.
[0071] Intramuscular injections of 100 .mu.g of naked pCFA-hAGA in
50 .mu.l were delivered into the right quadriceps muscle group.
These animals were sacrificed 5 days post-administration. Enzyme
was detectable in the tissues primarily transfected by the chosen
lipid/DNA formulations and routes of delivery. (FIG. 5).
Example 5
Tissue Distribution of .alpha.-galactosidase A in Fabry Knockout
Mice after Administration of Ad2/CEH.alpha.-gal
[0072] Virus was injected into the tail vein of female Fabry's
knockout mice at a dose of 5.times.10.sup.9 IU in 260 .mu.l. Mice
were sacrificed after 3 days. The ELISA was used to detect levels
of .alpha.-galactosidase A activity in various organs. Intravenous
injections of virus resulted in high levels of
.alpha.-galactosidase A in all organs tested (10-100 fold). The
wide distribution of enzyme activity makes this a promising therapy
for Fabry's Disease. (FIG. 6A).
[0073] Virus was injected into the right quadriceps muscle group of
female Fabry's knockout mice at a dose of 9.5.times.10.sup.8 IU in
50 .mu.l. These mice were sacrificed after 5 days. An ELISA was
used to detect levels of .alpha.-galactosidase A in various organs.
Intramuscular injections of virus resulted in significant levels of
enzyme at the site of injection, as well as moderate enzyme levels
in liver and spleen, indicating that infected cells at the
injection site secreted enzyme that was taken up by cells in other
tissues. (FIG. 6B).
Example 6
Time Course of .alpha.-galactosidase a Expression after Intravenous
Injection of Ad2/CEH.alpha.-gal into C57BL/6n Mice
[0074] The present experiment showed that significant levels of
active enzyme persisted for some time after administering the
vector. Virus was injected into the tail vein of C57BL/6n mice. The
dose delivered was 5.times.10.sup.9 IU in a volume of 260 .mu.l.
Organs were harvested after 3, 14 and 28 days. An ELISA was used to
detect .alpha.-galactosidase A levels in tissue homogenates. (FIGS.
7A and 7B). By day 28, the levels of enzyme had dropped .about.5-10
fold from day 3 levels, however the levels were still significantly
higher than wild-type levels.
Example 7
Levels of .alpha.-galactosidase A in Whole Blood after Intravenous
Injection of Ad2/CEH.alpha.-Gal into C57BL/6n and
BALB/c(nu/nu)Mice
[0075] Virus was injected into the tail vein of C57BL/6n or
BALB/c(nu/nu) mice. The dose delivered was 5.times.10.sup.9 IU in a
volume of 260 .mu.l. Blood was harvested after 3, 14 and 28 days.
An ELISA was used to detect .alpha.-galactosidase A levels in whole
blood. (FIGS. 8A and 8B). The presence of .alpha.-galactosidase A
in blood indicated secretion of enzyme into the bloodstream from
sites of infection. The levels of enzyme dropped .about.10 fold by
14 days. The similar pattern in nude and normal mice implies that
this decrease is not due to an immune response.
Example 8
Short Term Time Course Showing Reduction of GL3 Levels in Fabry
Mice Intravenously Administered Ad2/CEH.alpha.-gal
[0076] Female Fabry mice between 3 and 8 months of age (n=12, for
each group) were injected via the tail vein with a high dose
(1.65.times.10.sup.11 particles) or a low dose
(1.65.times.10.sup.10) of Ad2/CEH.alpha.-gal in 0.25 ml PBS/5%
sucrose. The mice were sacrificed at 3, 7 or 14 days post injection
(n=4 per time point per dose). Two naive female Fabry mice (3
months and 8 months of age) were sacrificed on day 3 for reference
for GL3 levels in untreated mice. A blood sample was collected at
the time of sacrifice to measure .alpha.-galactosidase A activity.
Upon sacrifice, the animals were perfused with PBS and various
organs collected. The organs were divided into two parts, one to
assay for .alpha.-galactosidase A activity via an ELISA specific
for human .alpha.-galactosidase A and the other extracted and
assayed for GL3 using an ELISA-type assay specific for GL3. The
data were normalized to the weight of the tissue sample.
[0077] The time course of .alpha.-galactosidase A activity in
sampled tissue following low dose and high dose administration of
Ad2/CEH.alpha.-gal are shown in FIGS. 9A and 10A, respectively. The
persistence of .alpha.-galactosidase A activity relative to day 3
for each dose is shown in FIGS. 9B and 10B, respectively. This
study showed that the high dose of vector produced a many fold
increase in .alpha.-galactosidase A activity in all tested tissues,
relative to naive mice, that persisted for up to 14 days. There was
a modest increase in .alpha.-galactosidase A activity at the lower
dose.
[0078] Concurrent with the increase in .alpha.-galactosidase A
levels in the tested tissues was a significant decrease in GL3
levels in all tissues at the high doses of vector (FIG. 11). The
lower drop in GL3 levels following the low dose of vector is
believed to be an artifact based on the age of the tested animals.
The low dose studies used younger mice that have lower amounts of
stored GL3 than older mice. For example, studies at Mount Sinai
School of Medicine in New York have shown that Fabry mice
accumulate GL3 in their tissues over time. At 3 months, the GL3
levels are significantly above normal, climbing to about twice the
3 month level in 5 month old mice. Between 5-11 months, the GL3
levels stabilize, with the 5 months GL3 level being about 80% of
the maximum. All of the high dose studies were performed in 5-7
month old mice, so the initial GL3 levels would not vary so much in
this group.
Example 9
Repeat Administration of Adenovirus to Mice Following
Immunosuppression Using Deoxyspergualin (DSG)
[0079] Because repeat administration of an adenoviral vector
containing the .alpha.-galactosidase A gene may be required to
sustain .alpha.-galactosidase A levels in treated individuals,
various immunosuppressants may be used to inhibit an immune
response to the administered adenovirus vector. Such immune
responses can inhibit the effectiveness of readministered virus.
The present experiment shows the effect of the immunosuppressant
agent DSG on repeat adenovirus administration.
[0080] Two groups of four BALB/c mice were treated with
1.times.10.sup.11 particles of Ad2/CFTR-16 (an E1 deleted,
partially E3 deleted vector capable of persistent transgene
expression as disclosed in PCT/US98/07840 filed Apr. 14, 1998, the
disclosure of which is incorporated herein by reference) via tail
vein injection (high dose). Two groups of four mice received
1.times.10.sup.10 particles of the virus (low dose). One group
given each dose received 20 mg/kg of DSG via IP injection on days
-1 through 5 relative to virus administration. This treatment
regime was repeated after 28 days. On day 56 the mice received the
same dose of virus, this time using Ad2/CEH.alpha.-gal. On day 56
two additional groups of mice received 1.times.10.sup.11 or
1.times.10.sup.10 particles of Ad2/CEH.alpha.-gal without any prior
treatment. Blood was collected from these animals on day -1, 27,
and 55 relative to initial virus administration. Three days after
they received the Ad2/CEH.alpha.-gal virus, the animals were
sacrificed and organs collected. Tissue homogenates were analyzed
for .alpha.-galactosidase A expression using an ELISA specific for
human .alpha.-galactosidase A. Antibodies made to adenovirus were
titered from plasma samples. With both dosage levels of virus,
.alpha.-galactosidase A levels were higher in the mice given DSG
then those not receiving DSG (FIG. 12), indicating that DSG was
beneficial in obtaining transgene expression upon repeat viral
administration. Likewise, DSG inhibited anti-adenovirus antibody
titers in mice (FIG. 13).
Example 10
Efficacy of Repeat Adenovirus Administration to Mice Following
Immunosuppression with Anti-CD154 (CD40 Ligand) Antibody (MR1)
[0081] The MR1 antibody, obtained from PharMingen (Catalog No.
090205), reacts with gp39 (CD40 Ligand--CD154), an accessory
molecule expressed on activated T lymphocytes. Noelle et al., Proc.
Natl. Acad. Sci. USA 89:6550 (1992); Roy et al., J. Immunol.
151:2497 (1993). gp39 is required for an immune response to be
mounted; inhibition thereof with MRI inhibits immune responses.
Indeed, antibody to gp39 (CD40 Ligand; CD154) has been shown to
inhibit both human and cellular immune response, facilitating
repeated administration of adenovirus to mouse airway. See Scaria
et al., Gene Therapy 4:611 (1997); WO 98/08541, incorporated herein
by reference.
[0082] The present experiment was designed to show the
effectiveness of MR-1 in inhibiting an immune response to repeat
adenovirus administration in mice.
[0083] Two groups of three BALB/c mice were administered
1.times.10.sup.11 particles of Ad2/CFTR-16 via tail vein injection.
One group of mice received 500 .mu.g of MR1 anti-CD154 antibody via
intraperitoneal injection days -1,1,4,7, and 14 relative to virus
administration. Twenty eight days after the first virus
administration the mice received a second injection of
1.times.10.sup.11 virus particles, this time using
Ad2/CEH.alpha.-gal/CEH.alpha.-gal. A third group of three mice only
received the Ad2/CEH.alpha.-gal injection on day 28. Three days
after the second virus injection animals were sacrificed and organs
harvested. Tissue homogenates were analyzed for
.alpha.-galactosidase A expression using the ELISA. As shown in
FIG. 14, this experiment showed that it was possible to attain high
levels of .alpha.-galactosidase A transgene expression with a
second administration of adenovirus following short term
immunosuppression with MR1 antibody.
* * * * *