Title: Combination therapy for
treating protein deficiencies
United States Patent: 7,446,098
Issued: November 4, 2008
Inventors: Fan; Jian-Qiang
Assignee: Mount Sinai
School of Medicine of New York University (New York, NY)
Appl. No.: 10/781,356
Filed: February 17, 2004
Covidien Pharmaceuticals Outsourcing
This application provides methods of
improving gene therapy by combining gene therapy with active site-specific
chaperones (ASSCs). The ASSC increases the stability and efficiency of the
protein encoded by the recombinant gene that is administered.
Description of the
The present invention improves
the efficiency of gene therapy for protein deficiencies by combining
standard gene therapy approaches with an active site-specific chaperone (ASSC),
i.e., an agent capable of inducing the proper/native folding conformation of
the protein, and stabilizing the protein encoded by the gene. The ASSC
enhances the in vivo expression, efficiency, and stability of the expressed
protein. The invention further provides formulations comprising a
recombinant gene and an ASSC specific for the induction of the proper/native
folding conformation of the protein and stabilization of the protein encoded
by the gene. The invention is based on the discovery that ASSCs can be used
as a combination therapy with gene therapy for the treatment of genetic
disorders and other disorders. Although previous studies have demonstrated
the ability of ASSCs to increase the level of expression of a normal,
wild-type protein in tissue culture, modifying expression levels in
artificial systems does not establish that one can achieve this result for a
wild-type therapeutic protein in vivo. It has now been recognized that,
instead, the in vivo methods for rescuing defective misfolded proteins can
be modified as set forth herein to improve the efficiency of expression of a
therapeutic (wild-type) protein delivered through gene therapy.
ASSCs can be screened and identified using methods known in the art. Once an
ASSC useful for a particular disorder is identified, the chaperone can be
administered to a patient receiving gene therapy. The ASSC can supplement
endogenous molecular chaperones during high level expression of the
therapeutic gene to increase the efficiency of expression by inhibiting
aggregation in the ER. The chaperone can also as a stabilizer to prevent the
degradation of the encoded protein being produced by the administered gene.
Disorders Characterized by Protein Deficiencies
There currently are about 1100 known inherited disorders characterized by
protein deficiency or loss-of-function in a specific tissue. These disorders
may be treatable by gene therapy in theory. The method of the present
invention contemplates co-therapy for proteins currently suited for use in
gene therapy that is available now or will be in the future. In such
disorders, certain cells or all of the cells of an individual lack a
sufficient functional protein, contain an inactive form of the protein or
contain insufficient levels of the protein for biological function.
Further, the list of diseases identified as being conformational disorders,
caused by mutations that alter protein folding and retardation of the mutant
protein in the ER, resulting in protein deficiency, is increasing. These
include cystic fibrosis, .alpha.1-antitrpsin deficiency, familial
hypercholesterolemia, Alzheimer's disease (Selkoe, Annu. Rev. Neurosci.
1994; 17:489-517), osteogenesis imperfecta (Chessler et al., J. Biol. Chem.
1993; 268:18226-18233), carbohydrate-deficient glycoprotein syndrome
(Marquardt et al., Eur. J. Cell. Biol. 1995; 66: 268-273), Maroteaux-Lamy
syndrome (Bradford et al., Biochem. J. 1999; 341:193-201), hereditary
blindness (Kaushal et al., Biochemistry 1994; 33:6121-8), Glanzmann
thrombasthenia (Kato et al., Blood 1992; 79:3212-8), hereditary factor VII
deficiency (Arbini et al., Blood 1996; 87:5085-94), oculocutaneous albinism
(Halaban et al., Proc. Natl. Acad. Sci. USA. 2000; 97:5889-94) and protein C
deficiency (Katsumi, et al., Blood 1996; 87:4164-75). Recently, one mutation
in the X-linked disease adrenoleukodystrophy (ALD) resulted in misfolding of
the defective peroxisome transporter, which could be rescued by
low-temperature cultivation of affected cells (Walter et al., Am. J. Hum.
Genet. 2001;69:35-48). It is generally accepted that mutations take place
evenly over the entire sequence of a gene. Therefore, it is predictable that
the phenotype resulting from protein deficiencies exists in many other
Lysosomal Storage Disorders
Many of the inherited protein deficient disorders are enzyme deficiencies.
As indicated above, a large class of inherited enzyme disorders involve
mutations in lysosomal enzymes and are referred to as lysosomal storage
disorders (LSDs). Lysosomal storage disorders are a group of diseases caused
by the accumulation of glycosphingolipids, glycogen, mucopolysaccharides
Examples of lysosomal disorders include Gaucher disease (Beutler et al., The
Metabolic and Molecular Bases of Inherited Disease, 8th ed. 2001 Scriver et
al., ed. pp. 3635-3668, McGraw-Hill, New York), GM1-gangliosidosis (id. at
pp 3775-3810), fucosidosis (The Metabolic and Molecular Bases of Inherited
Disease 1995. Scriver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D., ed
pp. 2529-2561, McGraw-Hill, New York), mucopolysaccharidoses (id. at pp
3421-3452), Pompe disease (id. at pp. 3389-3420), Hurler-Scheie disease (Weismann
et al., Science 1970; 169, 72-74), Niemann-Pick A and B diseases, (The
Metabolic and Molecular Bases of Inherited Disease 8th ed. 2001. Scriver et
al. ed., pp 3589-3610, McGraw-Hill, New York), and Fabry disease (id. at pp.
3733-3774). A list of LSDs and their associated deficient enzymes can be
found in Table 1
(see Original Patent). Two are discussed specifically infra.
Fabry disease is an X-linked inborn error of glycosphingolipid metabolism
caused by deficient lysosomal .alpha.-galactosidase A (.alpha.-Gal A)
activity (Desnick et al., The Metabolic and Molecular Bases of Inherited
Disease, 8.sup.th Edition Scriver et al. ed., pp. 3733-3774, McGraw-Hill,
New York 2001; Brady et al., N. Engl. J. Med. 1967; 276, 1163-1167). This
enzymatic defect leads to the progressive deposition of neutral
glycosphingolipids with .alpha.-galactosyl residues, predominantly
globotriaosylceramide (GL-3), in body fluids and tissue lysosomes. The
frequency of the disease is estimated to be about 1:40,000 in males, and is
reported throughout the world within different ethnic groups. In classically
affected males, the clinical manifestations include angiokeratoma,
acroparesthesias, hypohidrosis, and characteristic corneal and lenticular
opacities (The Metabolic and Molecular Bases of Inherited Disease, 8.sup.th
Edition 2001, Scriver et al., ed., pp. 3733-3774, McGraw-Hill, New York).
The affected male's life expectancy is reduced, and death usually occurs in
the fourth or fifth decade as a result of vascular disease of the heart,
brain, and/or kidneys. In contrast, patients with the milder "cardiac
variant" normally have 5-15% of normal .alpha.-Gal A activity, and present
with left ventricular hypertrophy or a cardiomyopathy. These cardiac variant
patients remain essentially asymptomatic when their classically affected
counterparts are severely compromised. Recently, cardiac variants were found
in 11% of adult male patients with unexplained left ventricular hypertrophic
cardiomyopathy, suggesting that Fabry disease may be more frequent than
previously estimated (Nakao et al., N. Engl. J. Med. 1995; 333, 288-293).
The .alpha.-Gal A gene has been mapped to Xq22, (Bishop et al., Am. J. Hum.
Genet. 1985; 37: A144), and the full-length cDNA and entire 12-kb genomic
sequences encoding .alpha.-Gal A have been reported (Calhoun et al., Proc.
Natl. Acad. Sci. USA 1985; 82; 7364-7368; Bishop et al., Proc. Natl. Acad.
Sci. USA 1986; 83: 4859-4863; Tsuji et al., Eur. J. Biochem. 1987; 165;
275-280; and Kornreich et al., Nucleic Acids Res. 1989; 17: 3301-3302).
There is a marked genetic heterogeneity of mutations that cause Fabry
disease (The Metabolic and Molecular Bases of Inherited Disease, 8.sup.th
Edition 2001, Scriver et al., ed., pp. 3733-3774, McGraw-Hill, New York.;
Eng et al., Am. J. Hum. Genet. 1993; 53: 1186-1197; Eng et al., Mol. Med.
1997; 3: 174-182; and Davies et al., Eur. J. Hum. Genet. 1996; 4: 219-224).
To date, a variety of missense, nonsense, and splicing mutations, in
addition to small deletions and insertions, and larger gene rearrangements
have been reported.
Gaucher disease is a deficiency of the lysosomal enzyme .beta.-glucocerebrosidase
that breaks down fatty glucocerebrosides. The fat then accumulates, mostly
in the liver, spleen and bone marrow. Gaucher disease can result in pain,
fatigue, jaundice, bone damage, anemia and even death. There are three
clinical phenotypes of Gaucher disease. Patients with, Type 1 manifest
either early in life or in young adulthood, bruise easily and experience
fatigue due to anemia, low blood platelets, enlargement of the liver and
spleen, weakening of the skeleton, and in some instances have lung and
kidney impairment. There are no signs of brain involvement. In Type II,
early-onset, liver and spleen enlargement occurs by 3 months of age and
there is extensive brain involvement. There is a high mortality rate by age
2. Type III is characterized by liver and spleen enlargement and brain
seizures. The .beta.-glucocerebrosidase gene is located on the human 1q21
chromosome. Its protein precursor contains 536 amino acids and its mature
protein is 497 amino acids long.
Gaucher disease is considerably more common in the descendants of Jewish
people from Eastern Europe (Ashkenazi), although individuals from any ethnic
group may be affected. Among the Ashkenazi Jewish population, Gaucher
disease is the most common genetic disorder, with an incidence of
approximately 1 in 450 persons. In the general public, Gaucher disease
affects approximately 1 in 100,000 persons. According to the National
Gaucher Foundation, 2,500 Americans suffer from Gaucher disease.
Other Enzyme Deficiency Disorders
Glucose-6-phosphate dehydrogenase (G6PD) deficiency the most common X-linked
human enzyme deficiency. The G6PD enzyme catalyzes an oxidation/reduction
reaction that is essential for the production of ribose, which is an
essential component of both DNA and RNA. G6PD also involved in maintaining
adequate levels of NADPH inside the cell. NADPH is a required cofactor in
many biosynthetic reactions. Individuals with this deficiency have clinical
symptoms including neonatal jaundice, abdominal and/or back pain, dizziness,
headache, dyspnea (irregular breathing), and palpitations.
One form of severe combined immunodeficiency (SCID) is due to lack of the
enzyme adenosine deaminase (ADA), coded for by a gene on chromosome 20. This
means that the substrates for this enzyme accumulate in cells. Immature
lymphoid cells of the immune system are particularly sensitive to the toxic
effects of these unused substrates, so fail to reach maturity. As a result,
the immune system of the afflicted individual is severely compromised or
In addition to inherited disorders, other enzyme deficiencies arise from
damage to a tissue or organ resulting from primary or secondary disorders.
For example, damaged pancreatic tissue, or pancreatitis, is caused by
alcoholism results in a deficiency in pancreatic enzymes necessary for
Other Disorders Treated Using Gene Therapy
There are numerous disorders involving defective genes other than enzymes
involved in metabolic disorders that can be treated using gene therapy. Such
disorder include but are not limed to severe combined immunodeficiency (SCID),
phagocyte disorders such as Wiskott-Aldrich syndrome, bleeding disorders
such as von Willebrand's disease and hemophilia, endocrine disorders such as
growth hormone deficiency and hypothalamic diabetes insipidus, retinal
degradation, cancers caused by inherited genetic defects such as heredetary
non-polyposis colon cancer (HNPCC). Such disorders are listed in Table 2
(see Original Patent).
Treatment of Protein Deficiencies and Other Disorders
By overexpression of wild-type protein in suitable cells (e.g., stem cells
or somatic tissue-specific cells) of an individual, using molecular biology
techniques, the missing or deficient protein is produced in the cells, and
in most cases circulates within the blood stream to the particular tissues.
In order to achieve the therapeutic purpose, it is important to maintain
high expression level of the protein for a sufficient time to confer a
therapeutic benefit. Further, it is important to ensure specific delivery of
the protein to the appropriate tissues.
Co-Therapy Using ASSCs and Gene Therapy
The present invention increases the effectiveness of gene therapy by
increasing the folding and processing of the protein encoded by the gene
administered during synthesis, and increasing the stability of the
newly-synthesized protein in vivo by co-administration of an ASSC for the
protein encoded by the administered gene. Screening for an appropriate ASSC
for the target protein can be achieved by known methods in the art, e.g., as
described in U.S. patent application Ser. No. 10/377,179, filed Feb. 28,
Disorders that can be treated using the method of the present invention
include but are not limited to those mentioned above and those listed in
Table 1. This method can be used in combination with any defective gene
contemplated to be replaced using gene therapy. For example, the method can
be used to provide secreted proteins, membrane proteins, or intracellular
Any of the methods for gene therapy available in the art can be used
according to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al.,
Clinical Pharmacy 1993, 12:488-505; Wu and Wu, Biotherapy 1991, 3:87-95;
Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 1993, 32:573-596; Mulligan,
Science 1993, 260:926-932; and Morgan and Anderson, Ann. Rev. Biochem. 1993,
62:191-217; May, TIBTECH 1993, 11:155-215. Methods commonly known in the art
of recombinant DNA technology that can be used are described in Ausubel et
al., (eds.), 1993, Current Protocols in Molecular Biology, John Wiley &
Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,
Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al., (eds.),
1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colosimo
et al., Biotechniques 2000;29(2):314-8, 320-2, 324.
The gene to be administered for the methods of the present invention can be
isolated and purified using ordinary molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. For example, nucleic
acids encoding the target protein can be isolated using recombinant DNA
expression as described in the literature. See, e.g., Sambrook, Fritsch &
Maniatis, Molecular Cloning. A Laboratory Manual, Second Edition (1989) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein "Sambrook
et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N.
Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic
Acid Hybridization [B. D. Hames & S. J. EHiggins eds. (1985)]; Transcription
And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell
Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL
Press, (1986)]; B. EPerbal, A Practical Guide To Molecular Cloning (1984);
F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, Inc. (1994). The nucleic acid encoding the protein may be
full-length or truncated, so long as the gene encodes a biologically active
protein. For example, a biologically active, truncated form of .alpha.-Gal
A, the defective enzyme associated with Fabry disease, has been described in
U.S. Pat. No. 6,210,666 to Miyamura et al.
The identified and isolated gene can then be inserted into an appropriate
cloning vector. Vectors suitable for gene therapy include viruses, such as
adenoviruses, adeno-associated virus (AAV), vaccinia, herpesviruses,
baculoviruses and retroviruses, parvovirus, lentivirus, 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.
In a preferred embodiment, the vector is a viral vector. Viral vectors,
especially adenoviral vectors can be complexed with a cationic amphiphile,
such as a cationic lipid, polyL-lysine (PLL), and diethylaminoethyldextran (DELAE-dextran),
which provide increased efficiency of viral infection of target cells (See,
e.g., PCT/US97/21496 filed Nov. 20, 1997, incorporated herein by reference).
Preferred 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 Scheie, 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. For a review of viral
vectors in gene therapy, see Mah et al., Clin. Pharmacokinet. 2002;
41(12):901-11; Scott et al., Neuromuscul. Disord. 2002;12 Suppl 1:S23-9. In
addition, see U.S. Pat. No. 5,670,488.
The coding sequences of the gene to be delivered are operably linked to
expression control sequences, e.g., a promoter that directs expression of
the gene. As used herein, the phrase "operatively linked" refers to the
functional relationship of a polynucleotide/gene 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
In one specific embodiment, a vector is used in which the coding sequences
and any other desired sequences are flanked by regions that promote
homologous recombination at a desired site in the genome, thus providing for
expression of the construct from a nucleic acid molecule that has integrated
into the genome (Koller and Smithies, Proc. Natl. Acad. Sci. USA 1989,
86:8932-8935; Zijlstra et al., Nature 1989, 342:435-438; U.S. Pat. No.
6,244,113 to Zarling et al.; and U.S. Pat. No. 6,200,812 to Pati et al.)
Delivery of the vector into a patient may be either direct, in which case
the patient is directly exposed to the vector or a delivery complex, or
indirect, in which case, cells are first transformed with the vector in
vitro, then transplanted into the patient. These two approaches are known,
respectively, as in vivo and ex vivo gene therapy.
Direct transfer. In a specific embodiment, the vector is directly
administered in vivo, where it enters the cells of the organism and mediates
expression of the gene. This can be accomplished by any of numerous methods
known in the art and discussed above, e.g., by constructing it as part of an
appropriate expression vector and administering it so that it becomes
intracellular, e.g., by infection using a defective or attenuated retroviral
or other viral vector (see, U.S. Pat. No. 4,980,286), or by direct injection
of naked DNA, or by use of microparticle bombardment (e.g., a gene gun;
Biolistic, Dupont); or coating with lipids or cell-surface receptors or
transfecting agents, encapsulation in biopolymers (e.g.,
poly-.beta.-1-64-N-acetylglucosamine polysaccharide; see U.S. Pat. No.
5,635,493), encapsulation in liposomes, microparticles, or microcapsules; by
administering it in linkage to a peptide or other ligand known to enter the
nucleus; or by administering it in linkage to a ligand subject to
receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 1987,
62:4429-4432), etc. In another embodiment, a nucleic acid-ligand complex can
be formed in which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal degradation, or
cationic 12-mer peptides, e.g., derived from antennapedia, that can be used
to transfer therapeutic DNA into cells (Mi et al., Mol. Therapy 2000,
2:339-47). In yet another embodiment, the nucleic acid can be targeted in
vivo for cell specific uptake and expression, by targeting a specific
receptor (see, e.g., PCT Publication Nos. WO 92/06180, WO 92/22635, WO
92/20316 and WO 93/14188). Recently, a technique referred to as
magnetofection has been used to deliver vectors to mammals. This technique
associates the vectors with superparamagnetic nanoparticles for delivery
under the influence of magnetic fields. This application reduces the
delivery time and enhances vector efficacy (Scherer et al., Gene Therapy
2002; 9:102-9). Additional targeting and delivery methodologies are
contemplated in the description of the vectors, below.
In a specific embodiment, the nucleic acid can be administered using a lipid
carrier. 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 1989; 337:387). 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. 1989; 298:278). See also, Osaka et
al., J. Pharm. Sci. 1996; 85(6):612-618; San et al., Human Gene Therapy
1993; 4:781-788; Senior et al., Biochemica et Biophysica Acta 1991;
1070:173-179); Kabanov and Kabanov, Bioconjugate Chem. 1995; 6:7-20; Liu et
al., Pharmaceut. Res. 1996; 13; Remy et al., Bioconjugate Chem. 1994;
5:647-654; Behr, J-P., Bioconjugate Chem 1994; 5:382-389; Wyman et al.,
Biochem. 1997; 36:3008-3017; U.S. Pat. No. 5,939,401 to Marshall et al; U.S.
Pat. No. 6,331,524 to Scheule et al.
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.sub.4-spermine cholesteryl carbamate (GL-67) disclosed
in U.S. Pat. No. 5,767,099. Additional preferred lipids include
N.sub.4-spermidine cholestryl carbamate (GL-53) and
1-(N.sub.4-spermine)-2,3-dilaurylglycerol carbamate (GL-89)
Preferably, for in vivo administration of viral vectors, an appropriate
immunosuppressive treatment is employed in conjunction with the viral
vector, e.g., adenovirus vector, to avoid immuno-deactivation of the viral
vector and transfected cells. For example, immunosuppressive cytokines, such
as interleukin-12 (IL-12), interferon-.gamma. (IFN-.gamma.), or anti-CD4
antibody, can be administered to block humoral or cellular immune responses
to the viral vectors. In that regard, it is advantageous to employ a viral
vector that is engineered to express a minimal number of antigens.
Indirect transfer. Somatic cells may be engineered ex vivo with a construct
encoding a wild-type protein using any of the methods described above, and
re-implanted into an individual. This method is described generally in WO
93/09222 to Selden et al. In addition, this technology is used in Cell Based
Delivery's proprietary ImPACT technology, described in Payumo et al., Clin.
Orthopaed. and Related Res. 2002; 403S: S228-S242. In such a gene therapy
system, somatic cells (e.g., fibroblasts, hepatocytes, or endothelial cells)
are removed from the patient, cultured in vitro, transfected with the gene(s)
of therapeutic interest, characterized, and reintroduced into the patient.
Both primary cells (derived from an individual or tissue and engineered
prior to passaging), and secondary cells (passaged in vitro prior to
introduction in vivo) can be used, as well as immortalized cell lines known
in the art. Somatic cells useful for the methods of the present invention
include but are not limited to somatic cells, such as fibroblasts,
keratinocytes, epithelial cells, endothelial cells, glial cells, neural
cells, formed elements of the blood, muscle cells, other somatic cells that
can be cultured, and somatic cell precursors. In a preferred embodiment, the
cells are fibroblasts or mesenchymal stem cells.
Nucleic acid constructs, which include the exogenous gene and, optionally,
nucleic acids encoding a selectable marker, along with additional sequences
necessary for expression of the exogenous gene in recipient primary or
secondary cells, are used to transfect primary or secondary cells in which
the encoded product is to be produced. Such constructs include but are not
limited to infectious vectors, such as retroviral, herpes, adenovirus,
adenovirus-associated, mumps and poliovirus vectors, can be used for this
Transdermal delivery is especially suited for indirect transfer using cell
types of the epidermis including keratinocytes, melanocytes, and dendritic
cells (Pfutzner et al., Expert Opin. Investig. Drugs 2000; 9:2069-83).
Mesenchymal stem cells (MSCs) are non-blood-producing stem cells produced in
the bone marrow. MSCs can be made to differentiate and proliferate into
specialized non-blood tissues. Stem cells transfected with retroviruses are
good candidates for the therapy due to their capacity for self-renewal. This
ability precludes repetitive administration of the gene therapy. Another
advantage is that if the injected stem cells reach the target organ and then
differentiate, they can replace the damaged or malformed cells at the organ.
Gene Therapy in Lysosomal Storage Disorders. Recently, recombinant gene
therapy methods are in clinical or pre-clinical development for the
treatment of lysosomal storage disorders, 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; U.S. Pat. No. 6,066,626, issued May 23, 2000 for Compositions and
method for treating lysosomal storage disease; U.S. Pat. No. 6,083,725,
issued Jul. 4, 2000 for Transfected human cells expressing human
alph.alpha.-galactosidase A protein; U.S. Pat. No. 6,335,011, issued Jan. 1,
2002 for Methods for delivering DNA to muscle cells using recombinant adeno-associated
virus virions to treat lysosomal storage disease; Bishop, D. F. et al.,
Proc. Natl. Acad. Sci., USA. 1986; 83:4859-4863; Medin, J. A. et al., Proc.
Natl. Acad. Sci., USA. 1996; 93:7917-7922; Novo, F. J., Gene Therapy 1997;
4:488-492; Ohshima, T. et al., Proc. Natl. Acad. Sci., USA. 1997;
94:2540-2544; Sugimoto Y. et al., Human Gene Therapy 1995; 6:905-915; Sly et
al., Proc. Natl. Acad. Sci. USA. 2002;99(9):5760-2; Raben et al., Curr. Mol.
Med 0.2002; 2(2):145-66; Eto et al., Curr. Mol. Med. 2002; 2(1):83-9; Vogler
et al., Pediatr. Dev. Pathol. 2001; 4(5):421-33; Barranger et al., Expert
Opin. Biol. Ther. 2001; 1(5):857-67; Yew et al., Curr. Opin. Mol. Ther.
2001; 3(4):399-406; Caillaud et al., Biomed. Pharmacother. 2000;
54(10):505-12 and Ioannu et al., J. Am. Soc. Nephrol. 2000; 11(8):1542-7.
In 2002, Brooks et al. demonstrated that gene transfer of .beta.-glucuronidase
into a mouse model of MPS VII using a feline leukemia virus, corrected
associated CNS deficits (PNAS 2002; 99: 6216-6221). Indirect transfer of
encapsulated Madin-Darby canine kidney cells that were genetically modified
to express canine alpha-iduronidase, and implanted into dog brains under
steoreotaxic guidance, was demonstrated to be efficacious in a dog model of
MPS (Barsoum et al., J Lab Clin Med. 2003;142(6):399-413).
Active Site-Specific Chaperones
ASSCs contemplated by the present invention include but are not limited to
small molecules (e.g., organic or inorganic molecules which are less than
about 2 kD in molecular weight, are more preferably less than about 1 kD in
molecular weight), including substrate or binding partner mimetics; small
ligand-derived peptides or mimetics thereof; nucleic acids such as DNA, RNA;
antibodies, including Fv and single chain antibodies, and Fab fragments;
other macromolecules (e.g., molecules greater than about 2 kD in molecular
weight) and members of libraries derived by combinatorial chemistry, such as
molecular libraries of D- and/or L-configuration amino acids;
phosphopeptides, such as members of random or partially degenerate, directed
phosphopeptide libraries (see, e.g., Songyang et al., Cell 1993,
Synthetic libraries (Needels et al., Proc. Natl. Acad. Sci. USA 1993,
90:10700-4; Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 1993,
90:10922-10926; Lam et al., PCT Publication No. WO 92/00252; Kocis et al.,
PCT Publication No. WO 9428028) and the like provide a source of ASSCs
according to the present invention. Synthetic compound libraries are
commercially available from Maybridge Chemical Co. (Trevillet, Cornwall,
UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and
Microsource (New Milford, Conn.). A rare chemical library is available from
Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in
the form of bacterial, fungal, plant and animal extracts are available,
e.g., from Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or are
readily producible. Additionally, natural and synthetically produced
libraries and compounds are readily modified through conventional chemical,
physical, and biochemical means (Blondelle et al., TIBTech 1996, 14:60).
U.S. patent application Ser. No. 10/377,179, filed Feb. 28, 2003 and
incorporated herein by reference, describes screening methods for ASSC's for
In a preferred embodiment, small molecules useful for the present invention
are inhibitors of lysosomal enzymes and include glucose and galactose imino
sugar derivatives as described in Asano et al., J. Med. Chem 1994;
37:3701-06; Dale et al., Biochemistry 1985; 24:3530-39; Goldman et al., J.
Nat. Prod. 1996; 59:1137-42; Legler et al, Carbohydrate Res. 1986;
155:119-29; and Okumiya et al., Biochem. Biophys. Res. Comm. 1995;
214:1219-240. Such derivatives include but are not limited those compound
listed in Table 1.
Other ASSCs can be those mentioned above, e.g., small synthetic compounds,
which were found to stabilize mutant forms of p53 (Foster et al., Science
1999; 286:2507-10); small molecule receptor antagonists and ligands, which
were found to stabilize receptors (Morello et al., J. Clin. Invest. 2000;
105: 887-95; Petaja-Repo et al., EMBO J. 2002; 21:1628-37); and drugs or
substrates, which were found to stabilize channel proteins and transporters
(Rajamani et al., Circulation 2002; 105:2830-5; Zhou et al., J. Biol. Chem.
1999; 274:31123-26; Loo et al., J. Biol. Chem. 1997; 272: 709-12).
In another embodiment, ASSC's useful in the method of the present invention
are activators of cystic fibrosis transmembrane conductance regulator and,
are identified using physical, and biochemical means (Blondelle et al.,
TIBTech 1996, 14:60).
In another preferred embodiment, ASSC's useful for the present invention are
ligands of G protein-coupled receptors, such as .delta. opioid receptor, V2
vasopressin receptor, and photopigment rhodopsin, as described in
Petaja-Repo et al., EMBO J. 2002; 21: 1628-37; Morello et al., J. Clin.
Invest.2000; 105: 887-95; Saliba et al., J. Cell Sci. 2002; 115: 2907-18.
In yet another preferred embodiment, ASSC's useful for the present invention
are blockers of ion channel proteins, such as HERG potassium channel in
human Long QT syndrome, pancereatic ATP-sensitive potassium (KATP) channel
in familial hyperinsulinism, as described in Zhou et al., J. Biol. Chem.
1999; 274: 31123-26; Taschenberger et al., J. Biol. Chem. 2002; 277:
Formulations and Administration
ASSCs. The ASSCs to be administered to an individual with a recombinant gene
may be formulated for administration by, e.g., oral, parenteral, transdermal,
or transmucosal routes, depending on whether the chaperone is a small
molecule, synthetic compound, or protein or peptide.
For oral administration, e.g., for small molecules, the pharmaceutical
compositions may take the form of tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such as
binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline
cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium
stearate, talc or silica); disintegrants (e.g., potato starch or sodium
starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The
tablets may be coated by methods well known in the art. Liquid preparations
for oral administration may take the form of, for example, solutions, syrups
or suspensions, or they may be presented as a dry product for constitution
with water or other suitable vehicle before use. Such liquid preparations
may be prepared by conventional means with pharmaceutically acceptable
additives such as suspending agents (e.g., sorbitol syrup, cellulose
derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin
or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl
alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain
buffer salts, flavoring, coloring and sweetening agents as appropriate.
Preparations for oral administration may be suitably formulated to give
controlled release of the active compound. For buccal administration the
compositions may take the form of tablets or lozenges formulated in
conventional manner. For administration by inhalation, the chaperones for
use according to the present invention are conveniently delivered in the
form of an aerosol spray presentation from pressurized packs or a nebulizer,
with the use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other
suitable gas. In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g., gelatin for use in an inhaler or insufflator may be
formulated containing a powder mix of the compound and a suitable powder
base such as lactose or starch.
The ASSCs may be formulated for parenteral administration by injection,
e.g., by bolus injection or continuous infusion. Formulations for injection
may be presented in unit dosage form, e.g., in ampoules or in multi-dose
containers, with an added preservative. The compositions may take such forms
as suspensions, solutions or emulsions in oily or aqueous vehicles, and may
contain formulatory agents such as suspending, stabilizing and/or dispersing
agents. Alternatively, the active ingredient may be in powder form for
constitution with a suitable vehicle, e.g., sterile pyrogen-free water,
The compounds may also be formulated in rectal compositions such as
suppositories or retention enemas, e.g., containing conventional suppository
bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the ASSCs may also be
formulated as a depot preparation. Such long acting formulations may be
administered by implantation (for example subcutaneously or intramuscularly)
or by intramuscular injection. Thus, for example, the compounds may be
formulated with suitable polymeric or hydrophobic materials (for example as
an emulsion in an acceptable oil) or ion exchange resins, or as sparingly
soluble derivatives, for example, as a sparingly soluble salt.
Recombinant gene. As described above, there are several methods known in the
art for delivering naked DNA to individuals, including direct injection into
the target tissue, e.g., intramuscular, use of cationic lipid carriers, by
intravenous infusion or inhalation. See the gene therapy disclosure above.
For administration of somatic cells engineered to overexpress the
recombinant gene product, the cells may be introduced into an individual,
through various standardized routes of administration, so that they will
reside in, for example, the renal subcapsule, a subcutaneous compartment,
the central nervous system, the intrathecal space, the liver, the
intraperiotoneal cavity, or within a muscle. The cells may also be injected
intravenously or intra-arterially so that they circulate within the
The cells may alternatively be embedded in a matrix or gel material, such as
described in U.S. Pat. No. 5,965,125 to Mineau-Hanschke, which describes the
use of hybrid matrix implants, or in Jain et al. (PCT application WO
95/19430), which describes macroencapsulation of secretory cells in a
hydrophilic gel material (each of which is hereby incorporated by
The number of genetically modified cells will depend on the individual's
weight, age, and clinical status, and can be routinely determined by those
skilled in the art. In one embodiment, about 1.times.10.sup.6 and
1.times.10.sup.9 cells/day will be used.
Timing. Administration of the ASSC according to the present invention will
generally follow delivery of the gene, to allow for expression of the
recombinant protein by the target cells/tissue. Since the expression of the
gene will be sustained for a period of time, for as long as the gene is
expressible, the ASSC will be remained effective as a chaperone and
stabilizer for the recombinant protein. Therefore, administration of ASSC
will be necessary for the same period as the gene is expressed.
In an embodiment where the ASSC has a short circulating half-life (e.g., a
small molecule), the ASSC will be orally administered continuously, such as
daily, in order to maintain a constant level in the circulation. Such a
constant level will be one that has been determined to be non-toxic to the
patient, and optimal regarding interaction with the protein, which will be
continuously produced, to confer a non-inhibitory, therapeutic effect.
In the event that the therapeutic gene supplements inadequate activity of an
endogenous mutant gene, the timing of chaperone delivery becomes less
significant since the effective amount can enhance the activity of the
endogenous mutant as well as increase the efficiency of the therapeutic
In vivo stability. The presence of an ASSC for the protein encoded by the
administered gene will have the benefit of improving the efficiency of
protein processing during synthesis in the ER (i.e. by preventing
aggregation), and prolonging in the circulation and tissue the half-life of
the protein, thereby maintaining effective protein levels over longer time
periods. This will result in increased expression in clinically affected
tissues. This confers such beneficial effects to the patient as enhanced
relief, reduction in the frequency of treatment, and/or reduction in the
amount of gene administered. This will also reduce the cost of treatment.
In addition to stabilizing the expressed protein, the ASSC will also
stabilize and enhance expression of any endogenous mutant proteins that are
deficient as a result of mutations that prevent proper folding and
processing in the ER, as in conformational disorders such as the LSDs.
The effective amount of ASSC to be administered with the recombinant gene
will depend, in part, on the method of delivery, specific amount and typical
expression level of the recombinant gene administered. The specific
effective amount can be determined on a case-by-case basis, depending on the
protein and corresponding ASSC, by those skilled in the art. The variation
depends, for example, on the patient and the recombinant gene and ASSC used.
Other factors to consider in determining doses are the individual's age,
weight, sex, and clinical status. Pharmacokinetic and pharmacodynamics such
as half-life (t.sub.1/2), peak plasma concentration (c.sub.max) time to peak
plasma concentration (t.sub.max), exposure as measured by area under the
curve (AUC) and tissue distribution for both the protein and the ASSC, as
well as data for ASSC-replacement protein binding (affinity constants,
association and dissociation constants, and valency), can be obtained using
ordinary methods known in the art to determine compatible amounts required
in a dosage form to confer a therapeutic effect.
Data obtained from cell culture assay or animal studies may be used to
formulate a range of dosages for use in humans. The dosage of compounds used
in therapeutic methods of the present invention preferably lie within a
range of circulating concentrations that includes the ED.sub.50
concentration (effective for 50% of the tested population) but with little
or no toxicity (e.g., below the LD.sub.50 concentration). The particular
dosage used in any application may vary within this range, depending upon
factors such as the particular dosage form employed, the route of
administration utilized, the conditions of the individual (e.g., patient),
and so forth.
A therapeutically effective dose may be initially estimated from cell
culture assays and formulated in animal models to achieve a circulating
concentration range that includes the IC.sub.50. The IC.sub.50 concentration
of a compound is the concentration that achieves a half-maximal inhibition
of symptoms (e.g., as determined from the cell culture assays). Appropriate
dosages for use in a particular individual, for example in human patients,
may then be more accurately determined using such information.
Measures of compounds in plasma may be routinely measured in an individual
such as a patient by techniques such as high performance liquid
chromatography (HPLC) or gas chromatography.
Toxicity and therapeutic efficacy of the composition can be determined by
standard pharmaceutical procedures, for example in cell culture assays or
using experimental animals to determine the LD.sub.50 and the ED.sub.50. The
parameters LD.sub.50 and ED.sub.50 are well known in the art, and refer to
the doses of a compound that is lethal to 50% of a population and
therapeutically effective in 50% of a population, respectively. The dose
ratio between toxic and therapeutic effects is referred to as the
therapeutic index and may be expressed as the ratio: LD.sub.50/ED.sub.50.
Chaperone compounds that exhibit large therapeutic indices are preferred.
The concentrations of the ASSC will be determined according to the amount
required to stabilize the protein in vivo, in tissue or circulation, without
preventing its activity. For example, where the ASSC is an enzyme inhibitor,
the concentration of the inhibitor can be determined by calculating the
IC.sub.50 value of the ASSC for the enzyme. Concentrations below the
IC.sub.50 value can then be evaluated based on effects on enzyme activity,
e.g., the amount of inhibitor needed to increase the amount of enzyme
activity or prolong enzyme activity of the administered enzyme. The
IC.sub.50 value of the compound deoxygalactonojiromycin (DGJ) for the
.alpha.-Gal A enzyme is 0.04 .mu.M, indicating that DGJ is a potent
inhibitor. Accordingly, it is expected that the concentration of .alpha.-Gal
A would be much lower than that of the .alpha.-Gal A administered.
Claim 1 of 40 Claims
1. A method of improving gene therapy by
increasing the level of expression of a recombinant protein corresponding
to an individual's endogenous protein in vivo in cells of an individual,
wherein the recombinant protein is expressed from an expression vector
which has been introduced into the cells, which method comprises
administering to the individual an active site-specific chaperone of the
protein, with the proviso that the individual's endogenous protein is not
a mutant protein that is deficient due to defective folding or processing
in the endoplasmic reticulum.
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